Program and Abstract Book

Agenda

Friday December 9: DAY 1 8.00-8.50 Conference registration Auditorium Foyer 8.50-8.55 Welcome address: Claire Fraser, President of TIGR Auditorium 8.55-9.00 From Incredulity to Reality: Malaria Genomics 1995-2005 Stephen Hoffman 9.00-9.30 Keynote Address:

Drugs Against Plasmodium vivax: A Portrait of Neglect J. Kevin Baird Session 1: The Genome Projects Chair: Jane Carlton 9.30-10.00 The Plasmodium vivax Genome Sequence and Comparative Analysis Jane Carlton 10.00-10.30 The Plasmodium knowlesi Genome Sequence Matt Berriman 10.30-11.00 Coffee break Session 2: Whole Genome Analyses and Data Mining Chair: Neil Hall 11.00-11.15 Protein-Protein Interactions in Plasmodium vivax Doug LaCount 11.15-11.30 Host Gene Expression in Response to P. cynomolgi, a Simian Model

of Human P. vivax Frank Cogswell 11.30-11.45 Inferring Population History of Malaria Parasites Austin Hughes 11.45-12.00 PlasmoDB 5.0 Provides a New Look at Plasmodium vivax Chris Stoeckert 12.00-12.15 MR4: A Central Repository for Malaria and Vector Research Tim Stedman 12.15-2.00 Lunch Pavilion

Viewing of odd-numbered posters Pavilion Tour of J. Craig Venter Sequencing Center for max. 75 attendees (meet at bus parking lot no later than 12.45)

Session 3: Epidemiology and Diversity Chair: John Reeder 2.00-2.15 Challenges of Malaria in the Americas Keith Carter 2.15-2.30 The Epidemiology of P. vivax in Papua New Guinea: An Update on

Recent Studies Ivo Mueller 2.30-2.45 Vivax Malaria Re-Emerged in Central Part of China and DPR Korea Gao Qi 2.45-3.00 Malaria in India: Epidemiology, Parasite and Vector Diversity A.P. Dash 3.00-3.15 Mixed Species Infections Nick White 3.15-3.30 Malaria, Man, Monkeys and Malaysia Balbir Singh 3.30-4.00 Tea break Session 4: Vectors and Transmission Chair: Jetsumon Sattabongkot 4.00-4.15 Estimating the Efficiency of Plasmodium vivax Sporogony

in Western Thailand Jefferson Vaughan 4.15-4.30 Epidemiology of Plasmodium vivax Transmission and Vector

Interaction in Southern Mexico Lilia Gonzalez-Cerón

Session 5: Drug Treatment, Drug Development and Drug Resistance Chair: Kevin Baird 4.30-4.45 Trials of New Antimalarials for Resistant Vivax Malaria Ric Price 4.45-5.00 Tafenoquine, a Novel 8-Aminoquinoline for Vivax Malaria Colin Ohrt 5.00-5.15 Antifolates Can Have a Role in Vivax Chemotherapy Carol Sibley 5.15-5.30 Studying P. vivax Dihydrofolate Reductase in a P. falciparum

Transfection System Mike O’Neil

Day 1 conference close: list of nearby restaurants provided for dinner

5.30-9.30 PlasmoDB Bioinformatics Workshop Training Classroom

Approx. 40 students have been invited to attend a hands-on bioinformatics workshop to gain experience with downloading, searching and manipulating the P. vivax and other Plasmodium species genome sequence data. Computer hardware, software, manuals and pizza provided. Workshop coordinators: Aaron Mackey, Chris Stoeckert, David Roos

Saturday December 10: DAY 2 8.30-9.00 Conference registration Auditorium Foyer 9.00-9.30 Keynote Address: Observations on the Biology of

Plasmodium vivax and Plasmodium knowlesi Bill Collins Session 1: Relapse, Pathology and Morbidity Chair: Nick White 9.30-9.45 Genetic Diversity of P. vivax Parasites in Relapsing Patients Qin Cheng 9.45-10.00 Vivax Malaria: Severe Disease and Pathophysiology Nick Anstey 10.00-10.15 Primate Model of Coincident AIDS and Malaria Robert Garry 10.15-10.45 Coffee break Session 2: Vaccines & Immunity Chair: Stephen Hoffman 10.45-11.00 Progress on P.vivax Vaccine Development: Current Preclinical and Clinical Studies Socrates Herrera 11.00-11.15 Immune Responses to Plasmodium vivax Preerythrocytic Stage

Antigens in Naturally Exposed Duffy Negative Humans, and Identification of Liver Stage Antigens Ruobing Wang

11.15-11.30 Development of a Plasmodium vivax Circumsporozoite Protein Vaccine Kim Lee Sim 11.30-11.45 Merozoite Surface Protein-1 of Plasmodium vivax Induces A Protective Response Against Plasmodium cynomolgi Challenge in Rhesus Monkeys Sheetij Dutta 11.45-12.00 Aiming for Sustained High Level Antibody Responses: Development

of Pvs25-based Transmission Blocking Vaccine against Vivax Malaria Yimin Wu 12.00-12.15 Plasmodium knowlesi in Rhesus Monkeys: a Model for

Pre-Erythrocytic Malaria Vaccines Walter Weiss 12.15-2.00 Lunch Pavilion

Viewing of even-numbered posters Pavilion Tour of J. Craig Venter Sequencing Center for max. 75 attendees (meet at bus parking lot no later than 12.45) PLEASE TAKE DOWN ALL POSTERS PRIOR TO SESSION 3

Session 3: Antigens & Immunity Chair: John Barnwell 2.00-2.15 Plasmodium vivax and P. knowlesi Erythrocyte Membrane Antigens Mary Galinski 2.15-2.30 Large-scale Study of the Multigene vir Superfamily

of Plasmodium vivax, a Major Human Malaria Parasite Hernando del Portillo 2.30-2.45 Targeting the P. vivax Duffy Binding protein Chetan Chitnis 2.45-3.00 Elements in Malaria Parasite Duffy Binding-like Domains Necessary

for Erythrocyte Invasion Rachna Hora 3.00-3.15 P. vivax-Duffy Binding Protein-specific Antibodies Inhibits

P. vivax Erythrocyte Infection Christopher King 3.15-3.30 Reduced P. vivax Erythrocyte Infection in Papua New Guinean

Duffy-negative Heterozygotes Peter Zimmerman 3.30-4.00 Tea break

Session 4: Developing Technologies Chair: Barbara Sina 4.00-4.15 In Search of a Small Animal Model for Human Malaria Study:

Development of a Human Duffy Transgenic Mouse Strain Asok Chaudhuri 4.15-4.30 Application of Erythropoetic Stem Cells in the Establishment

of Continuous Culture of Plasmodium vivax Rachanee Udomsangpetch 4.30-4.45 In vitro and Molecular Genotyping Studies of Chloroquine Resistant

and Sensitive Plasmodium vivax Rossarin Suwanarusk 4.45-5.00 Transgenic P. knowlesi to Study Malaria Drug Targets and Vaccine

Candidates Clemens Kocken 5.00-5.15 A Comparison of Protein Expression from Plasmodium Organisms Ray Hui 5.15-5.30 A New Tool for Genetic Analysis of Malaria Parasites John Adams

5.30-7.00 Recommendations to Enhance P. vivax Research, Resources and Training

Please join in the discussion at four focus groups convened to draft recommendations to specifically enhance P. vivax research in the areas of Vaccines, Antigens and Immunity; Drug Treatment, Resistance and Development; Transmission, Epidemiology, Pathology and Morbidity; and Genomics, Genetics and Population Studies. Each group will be chaired by two moderators, who have been tasked to produce a list of priorities at the conclusion of the discussions.

5.30-6.15 Auditorium: Genomics, Genetics & Population Studies

Moderators: Dyann Wirth and Jane Carlton

Admin Conference Room: Vaccines, Antigens and Immunity Moderators: Socrates Herrera and Chetan Chitnis

6.15-7.00 Auditorium: Drug Treatment, Drug Resistance and Drug Development Moderators: Kevin Baird and Qin Cheng Admin Conference Room: Transmission, Epidemiology, Pathology and Morbidity Moderators: Karen Day and Nick Anstey

Questions to be considered and responses prioritized:

1. What research topics unique to P. vivax malaria need to be addressed? 2. What neglected research topics specific to P. vivax malaria need to be addressed? 3. What can be done to enhance P. vivax research collaborations? 4. What P. vivax research resources, standardized reagents and protocols are needed?

6.30-7.30 Wine and snack reception Auditorium Foyer 7.30-9.30 Conference dinner & announcement of poster prizes Pavilion

Program & Abstract Book for the conference:

Vivax Malaria Research: 2005 and Beyond

The Institute for Genomic Research

December 9-10, 2005

In 2002, a landmark in the fight against malaria was reached with the simultaneous

publication of the Plasmodium falciparum genome sequence and that of the mosquito vector

Anopheles gambiae . Three years later and the genome sequence of a second human malaria

species, Plasmodium vivax, is close to completion. “Vivax Malaria Research: 2005 and Beyond”

has several goals: to mark the completion of the P. vivax genome sequence and a closely related

monkey species Plasmodium knowlesi ; to continue the momentum generated from the first vivax

malaria research conference held in Bangkok, Thailand in 2002 which identified priorities for P. vivax research; to bring together P. vivax and P. knowlesi researchers from a multitude of

disciplines and from developed and endemic countries to discuss future priorities; and to

provide hands-on experience of bioinformatics techniques that can be used to manipulate the P. vivax genome sequence data.

Sponsors: The Institute for Genomic Research would like to thank the following

organizations for their sponsorship of the meeting:

and the National Institute of Allergies and Infectious Diseases, NIH.

Conference Organizers and Committee:

Organizer and co-chair: Jane Carlton, Ph.D. Associate Investigator, The Institute for Genomic Research, Rockville, USA

Organizer and co-chair: Barbara Sina, Ph.D. Program Officer, Fogarty International Center, National Institutes of Health, Bethesda, USA

Karen Day, Ph.D. Professor and Chair, Department of Medical Parasitology, New York University School of Medicine, New York, USA

John Adams, Ph.D. Associate Professor, Center for Tropical Disease Research and Training, Notre Dame, USA

John Reeder, Ph.D. Director, Papua New Guinea Institute of Medical Research, Papua New Guinea

Hernando del Portillo, Ph.D. Associate Professor, Institute of Biomedical Science, Sao Paolo, Brazil

John Barnwell, Ph.D. Chief, Malaria Research and Development Laboratories Unit, Centers for Disease Control and Prevention, Atlanta, USA

Jetsumon Prachumsri, Ph.D. Chief, Laboratory Sciences Section, Entomology Department, AFRIMS, Bangkok, Thailand

Antoniana Kretteli, Ph.D. Professor and Senior Researcher, Malaria Laboratory, Oswaldo Cruz Foundation, Belo Horizonte, Brazil

Matthew Berriman, Ph.D. Project Manager, The Wellcome Trust Sanger Institute, Cambridge, United Kingdom

Alan Thomas , Ph.D. Chair, Department of Parasitology, Biomedical Primate Research Centre, Rijswijk , The Netherlands

Qin Cheng, Ph.D. Head, Department of Drug Resistance and Diagnosis, Australian Army Malaria Insitute , Queensland , Australia

Acknowledgments Special thanks to Conference Manager Amy Rabin, Conference helpers Trina

Eacho, Tinu Akinyemi, John Nixon, Hanbang Zhang, Joana Silva and Hernan

Lorenzi, and to Lis Caler for the conference logo design and Connie Shiau for

design of the conference website.

POSTER ABSTRACTS P-01 Identification of the Optimal Third Generation Antifolate Against P. falciparum and P. vivax Carol Hopkins Sibley, Sonia, Y. Hunt, David P. Jacobus, Guy A. Schiehser, Carsten Detering, Gabriele Varani; University of Washington, Seattle, WA, USA; Jacobus Pharmaceutical Company, Princeton, NJ, USA Antifolates have not traditionally been recommended for treatment of vivax malaria. However, recent studies have suggested that a third-generation antifolate, WR99210, is remarkably effective even against highly pyrimethamine-resistant parasites from both species. Two methods were used to identify a compound that is effective against quadruple mutant alleles from P. falciparum (N51I/C59R/S108N/I164L) and from P. vivax (57L/111L/117T/173F). The first was simple yeast system used to screen a panel of WR99210 analogs. The biguanide prodrug, JPC-2056, of the 2-chloro-4-trifluoromethoxy analog of WR99210 was effective against both the P. falciparum and P. vivax enzymes, and has been selected for further development. The second method compared the analogs in silico by docking them in the known structure of the P. falciparum DHFR-thymidylate synthase. The program reproduced well the position of the triazine ring, but the calculated energies of ligand binding were very similar for different compounds and therefore did not reproduce the observed trends in biological activity. During docking, multiple conformations were observed for the benzene ring part of the molecules in the DHFR active site, making computer-based predictions of binding energy less informative than for more rigid ligands. This flexibility is a key factor in their effectiveness against the highly mutant forms of DHFR.

P-02 Selective Pressures and Intragenic Recombination in the Genes Encoding Merozoite Surface Proteins 4 and 5 of Plasmodium vivax Chaturong Putaporntip1, Somchai Jongwutiwes1, Marcelo U Ferreira4, Rachanee Udomsangpetch3, Liwang Cui2, Hiroji Kanbara5; 1Department of Parasitology, Faculty of Medicine, Chulalongkorn University; 2Department of Entomology, The Pennsylvania State University, PA, USA; 3Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok, Thailand; 4Departamento de Parasitologia, Instituto de Ciencias Biomedicas da Universidade de Sao Paulo, Brazil; 5Department of Protozoology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan

The merozoite surface proteins-4 and -5 of Plasmodium vivax (PvMsp-4 and PvMsp-5) are potential vaccine candidates. To investigate whether the extent of sequence variation in both PvMsp4 and PvMsp-5 akin to those found in their ortholoques in P. falciparum, we examined the complete nucleotide sequences of both genes from 45 isolates from Thailand, Brazil, Indonesia, Papua New Guinea, Solomon Island and India. Results showed that PvMsp-4 contained limited sequence variation among isolates and both exons contained repeats with sequence and size variation. In contrast, extensive polymorphism occurred in exon 1 of PvMsp-5 but no repeats were identified. The rate of nonsynonymous substitutions per nonsynonymous site (dN) significantly outnumbered the rate of synonymous substitutions per synonymous site (dS) in exon 1 of PvMsp-5, suggesting positive selection on this region. Furthermore, Tajima’s D test revealed significant positive deviation from neutrality in this exon. Unlike PvMsp-5, PvMsp4 was under negative selection based on neutrality tests. Meanwhile, a rapid decline in linkage disequilibrium between pairs of informative loci over molecular distance was observed in PvMsp-5, suggesting that intragenic recombination further enhances sequence polymorphism in this gene. Therefore, polymorphism in these genes seems to be under different selective pressures.

P-03 P.vivax MSP3 Alpha: Highly Polymorphic Marker in Indian Field Isolates Anju Verma, Hema Joshi; Department of Genetics, Malaria Research Centre, Delhi, India In India P. vivax is the commonest species and accounts for 60-65% of total malaria cases (2.5 to 3.0 million cases per year). Considerable variations have been reported in P.vivax such as relapse patterns (Li et al. 2001), antigenic variations (Udagama et al. 1987) and drug response (Hasting et al. 2005). Even among Indian isolates polymorophic nature of isolates in respect of isozyme pattern, relapse pattern and drug response has been reported. No reports are available using DNA markers. Therefore we report the findings on the polymorphism of PvMSP3 alpha a highly polymorphic marker. MSP3 alpha has been shown to be a good epidemiologic marker among PNG isolates. Therefore, with an objective to determine the extent of genetic diversity existing among the Indian isolates, studies on analysis of PvMSP3 alpha was taken up. A total of 136 samples from Delhi, Tamil Nadu, Goa, Orrisa, Maharashtra and Car Nicobar were analyzed and polymorphism was observed in both size and sequence of PvMSP3 alpha gene. PCR products were approximately 1.2, 1.4 and 1.8 Kb sizes. Distribution of PCR polymorphism was similar in all study sites. 1.8 Kb variant was commonest with proportion being 60%. During our study it was observed that genotype H1 of Hha I digestion occurred with very high frequency with 1.2 and 1.4 variant while it was totally absent in the 1.8 variant which is more common. Sequencing of some of the isolates was also carried out. P-04 P. vivax: Genetic Complexity Among Field Isolates in India Hema Joshi; Malaria Research Centre (ICMR), Delhi, India

Malaria is one of the most serious public health problems in India and is prevalent in whole of the country except in areas 1800 meters above sea level. Malaria control is hampered by the development of resistance to antimalarials in pathogen and to insecticides in vector along with

operational problems. Therefore, there is an urgent need to develop newer technologies/tools to overcome this situation. Understanding genetic diversity existing among the malaria parasite is important to plan appropriate control strategy and to develop and test newer vaccines and drugs. With an aim to get information on the genetic structure of Indian Plasmodia species, a study has been carried out to analyze field isolates of P. vivax from patients for polymorphism in merozoite surface proteins

gene (MSP3), housekeeping genes, sub-types of P. vivax as well as drug resistant markers. Identical length variations were observed in different geographical regions with quantitative differences in respect of space. However, observations revealed a high degree of complexity among the field isolates with a number of genetically different parasite types prevailing in the population as well as co-existence of many genotypes in an isolates. P-05 Transcriptome Analysis of Anopheles albimanus Immune Response to Infection with Bacteria and Plasmodium vivax Rosa E. Gómez, Carlos A Machado, Fidel de la Cruz Hernandez, Lilia González Cerón, Mario H Rodríguez; CISEI. INSP, Cuernavaca, México We are investigating genes involved in the Anophles albimanus response to Plasmodium vivax and other pathogens infection and comparing this response with available data in An. gambiae and Drosophila . To do so, we are sequencing full length enriched cDNA libraries from An. albimanus salivary gland, midgut and whole mosquito. Transcriptome analysis will be further enriched by sequencing subtractive cDNA libraries derived from P. vivax and bacteria challenged vs non-challanged midgut, dorsal vessel, fat body and salivary gland libraries. Libraries will lead to characterization of an “immunity transcriptome”. Then, cDNA microarrays will be printed and hybridized to analyze the transcriptional profile upon bacterial and P. vivax challenge in different anatomic locations. Preliminary analysis of An. albimanus transcriptome analysis will be presented and discussed. Integration of structural and functional data will enhance our understanding of the molecular and anatomical basis underlying parasite transmission; provide clues regarding the extent of conservation in the mechanisms implicated in parasite transmission among vector

species; offer potential gene targets for malaria control and potential markers of susceptibility and resistance useful for population monitoring; and will build capacity in genomics and bioinformatics in Mexico, a malaria endemic country. P-06 The Influence of Population Structure on Mosquito Infectivity in Plasmodium vivax From Southern Mexico Deirdre A. Joy, Lilia Gonzalez-Ceron, Thomas F. McCutchan, Xin-zhuan Su; National Institutes of Health, NIAID, LMVR, Rockville, MD, USA

Previous studies have shown that in southern Mexico Anopheles albimanus is more susceptible to Plasmodium vivax parasites with the CS protein VK210 phenotype and the reverse is true for Anopheles pseudopunctipennis. However, observations of VK247 ookinetes and oocyst destruction in An. albimanus suggest that CS protein may not play a direct role in this host specificity, as it is expressed after oocyst maturation. Nonetheless, the VK210/VK247 phenotype is one of only a few observed differences among these parasites. To investigate the molecular basis of this host-specificity, and to better characterize the underlying parasite population structure, we typed 160 P. vivax isolates using 12 variable tandem repeat markers spanning 80 Kb and flanking the csp gene. Using a model-based structuring method, we found support for five parasite populations; one of which was found across all geographical regions, the others being restricted to either foothill or coastal areas. Our results clarify the contribution of parasite populations to mosquito infectivity and indicate that the association between the VK210/VK247 phenotype and infectivity is dependent upon the genetic background of the parasites. In addition, one VK210 phenotype population that showed significantly lower infectivity for An. albimanus may be a recent introduction to the region. P-07 Bioinformatic Definition and Analysis of the Exportome of Plasmodium vivax Tobias J. Sargeant, Matthias Marti, Elisabet Caler, Jane M. Carlton, Terence P. Speed, Alan F. Cowman; The Walter and Eliza Hall Institute of

Medical Research, Melbourne, Victoria, Australia A major mechanism of protein export to the host erythrocyte in Plasmodium is mediated by the presence of an ER targeting signal and a short motif with the consensus RxLx[EDQ] (the PEXEL motif). We have constructed generalized hidden Markov model for the prediction of protein export in P.falciparum, and here we apply this predictor to the newly available genome sequence and annotation of P.vivax. Predictions based solely upon annotated gene models tend to underestimate the full exportome due to misprediction of initial exons. In order to augment our P.vivax predictions, we have attempted to utilise extra information and bioinformatic approaches including synteny with P.falciparum and targeted gene finding approaches. Our results provide evidence of significant differences between the exported protein complements of P.falciparum and P.vivax, as well as demonstrating some commonalities between the two. In P.vivax we have identified several members of the PHIST family and a subgroup of VIRs that appear to be exported via PEXEL mediated pathway, and may represent the link between Plasmodium interspersed repeats and the P.falciparum RIF gene family. The presence of a conserved “exportome” in the genus Plasmodium has important implications for our understanding of both common mechanisms and species-specific differences in host-parasite interactions. P-08 Functional Analysis of Plasmodium MAEBL Fabian E. Saenz, Bharath Balu, Jun Fu, John Adams; Center for Tropical Disease Research & Training, Department of Biological Sciences, University of Notre Dame, Notre Dame, IN, USA Malaria is a leading cause of death and clinical illness in the tropical regions of the world. The transmission of the parasite relies on the passage through the Anopheles mosquito and the biology of the parasite in the vector is poorly understood. We are studying the molecular mechanisms that the parasite uses to invade the mosquito salivary glands. One of the proteins considered essential for the invasion of salivary glands is MAEBL, which is a conserved protein expressed abundantly in midgut sporozoites of different Plasmodium species. MAEBL is proteolitically processed and different potential isoforms are

formed by alternative splicing of the 3’ end. In order to understand the function of MAEBL in the sporozoite invasion of salivary glands, we created a series of constructs by allelic replacement of the 3’ end to disrupt P. falciparum maebl or to express the different molecular forms of MAEBL. Our results for the disruption of maebl confirm that MAEBL is not essential in the erythrocytic stage of the parasite. The understanding of the biological function of MAEBL in invasion of salivary glands will allow us to validate the use of MAEBL as a target for vector–based strategies for transmission blocking. P-09 Identification of Conserved Putatitve Cis-Elements in Plasmodium vivax Kobby Essien1, 2Jane Carlton, 1Christian Stoeckert, Jr.; 1Penn Center for Bioinformatics, University of Pennsylvania, Philadelphia, PA, USA; 2The Institute for Genomic Research, Rockville, MD, USA Little is known about gene regulation in apicomplexa. Few cis-elements have been characterized and members of the phylum lack many of the well-studied transcription factors present in other eukaryotes suggesting that these parasites might have unique sets of transcription factors. In order to shed more light on gene regulation in the phylum, we used a comparative genomics approach to identify putative cis-elements in Plasmodium vivax. Specifically, upstream regions of P. vivax gene clusters orthologous to groups of Plasmodium falciparum coexpressed genes were searched for motifs. To strengthen the case for predicted motifs being functional, each motif was assessed for its specificity to the cluster of genes used in its discovery with the Kolmogorov-Smirnov test. In parallel, the upstream regions of the orthologous gene clusters in P. falciparum were examined for enriched motifs and the resulting motifs were compared to functionally specific motifs identified in the corresponding clusters in P. vivax. Via this approach we identified functionally specific putative P. vivax cis-elements that are conserved in P. falciparum.

P-10 Plasmodium vivax and Plasmodium falciparum Single and Mixed Infections in Anopheles albimanus Mosquitoes: Transmission Dynamics and Immune Response LF Lopes1, PX Marques1, AC Mendes1, MJ Leandro1, F Zamora2, L Rocha2, M Arevalo2, S Herrera2, VE do Rosário1, H Silveira1, AP Arez1; 1IHMT/CMDT, Lisbon, Portugal; 2MVDC, Cali, Colombia A protocol for the production of mixed Plasmodium falciparum and Plasmodium vivax

infections in Anopheles albimanus mosquitoes was established in Buenaventura (Colombia). These experimental infections were used to access reciprocal interference of these Plasmodium species during the sporogonic development. We analyzed the infection dynamics in the mosquito by characterizing parasite composition in bloodmeals, infection rates, overall oocysts and sporozoite numbers in single or mixed-infections and proportion of species present in midguts or salivary glands. Detection and identification of parasites in individual mosquitoes were carried out both by optical microscopy and species specific PCR. Real time quantitative PCR was performed for the relative quantification of each species during sporogonic development. Vector-parasite interactions are being studied through the analysis of the mosquito genes transcription involved in immune response to each parasite and to parasite mixtures. As most of the immune related genes are not described for A. albimanus we are using comparative genomics with Anopheles gambiae to obtain partial sequences of some of these genes. These studies could gather some insights to how the development of one species can affect the development of the other and how the mosquito immune system reacts to each species together or separated.

P-11 Pv200L, A Potential Vaccine Candidate to Vivax Malaria Augusto Valderrama-Aguirre1, Evelin Zúñiga1, David Narum2, Myriam Arévalo-Herrera1, and Sócrates Herrera1; 1Malaria Vaccine and Drug Development Center, Cali, Colombia; 2Malaria Vaccine Development Branch, NIAID, NIH, Rockville, MD, USA The Pv200L is a subunit located toward the N-

terminal fragment of PvMSP-1. It comprises a fragment of >40% sequence homology to Pf190L and most of the HBR-I region. This subunit was expressed as a recombinant protein in E. coli and used to test its relevance as a vaccine candidate. We found that >70% of P. vivax-infected and >50% of P. vivax-exposed individuals have IgG antibodies to Pv200L. Anti-Pv200L IgG antibodies induced in BALB/c mice and Aotus monkeys are able to recognize the antigen onto the surface of P. vivax blood schizonts. Immunized Aotus monkeys are able to reduce parasitemia peaks as well as to induce self-cure from a challenge with P. vivax (Sal I) blood stages. More recently, a polymorphism study in the Colombian pacific coast showed that the nucleotide diversity of this fragment is lower than for other P.vivax vaccine candidates with no geographical relationship between the identified alleles. Currently we are developing a purification process to obtain a non-GMP recombinant product compatible with clinical grade standards in order to asses its immunogenicity and protective efficacy in Aotus monkeys when administrated mixed or alone in adjuvants allowed for humans. P-12 Promoter Regions of Plasmodium vivax Are Unable to Effieciently Recruit the Transcriptional Machinery of P.falciparum MF. Azevedo, HA del Portillo; Departamento de Parasitologia, Instituto de Ciências Biomédicas - Universidade de Sao Paulo, Sao Paulo, SP, Brazil Promoters of malaria parasites contain conserved regulatory elements as promoters from different Plasmodium species can drive expression of reporter genes in heterologous transfection systems. Here we describe transient transfections in P. falciparum with luciferase reporter plasmids containing intergenic regions of the msp1, dhfr, vir3, vir24 and ef1-a genes of P. vivax. Only the ef1-a and the vir24 promoters produced detectable luciferase activity. Moreover, these luc activities were two orders of magnitude lower than the ones of the ef1-a intergenic regions of P. falciparum and P. berghei. A minimal promoter based on the Pb ef1-a promoter was cloned between the Pv ef1-a promoter and the luciferase gene. This construction increased about three times the strength of the Pv ef1-a promoter. Sequence analysis of the ef1-a intergenic regions of six Plasmodium species identified a motif of six nucleotides totally conserved in all of them,

but P. vivax. Present efforts are now guided to clone this motif inside the Pv ef1-a promoter. Together, these data indicate that promoter regions of P. vivax cannot efficiently recruit the transcription initiation complex of P. falciparum and that both the basal promoter and upstream elements are probably involved in this process. P-13 Identifying Binding Residues in the Plasmodium vivax Duffy Binding Protein Ligand Domain Amy M. McHenry, Kelley M. VanBuskirk, Elitza S. Sevova, John H. Adams; University of Notre Dame, Department of Biological Sciences, Notre Dame, IN, USA Plasmodium vivax is the second leading cause of malaria worldwide. Merozoite invasion of erythrocytes is a vital step in the clinical progression of the human stage of infection and a possible target of therapeutic intervention. The invasion process requires molecules of the erythrocyte-binding like (ebl) protein family which contain conserved cysteine-rich DBL (Duffy-binding like) domains. Plasmodium vivax has only one known member of the ebl family, the Duffy binding protein (DBP). Erythrocyte recognition is therefore dependent on binding between the DBP and the Duffy blood group receptor. In the P. vivax DBP, region II of the DBL domain is responsible for receptor recognition and the residues between cysteines 4 and 6 have been implicated as the minimal region necessary for binding. We have previously analyzed this region of the DBL domain of the P. vivax DBP using site-directed mutagenesis to identify specific residues associated with binding. Combining this data with bioinformatics analysis of sequence data from several species, we are characterizing the importance of residues for binding specific erythrocyte receptors using a phenotype conversion approach. Deeper understanding of the residues in the P. vivax DBP and their role will provide important data for the development of a subunit vaccine.

P-14 Genetic Diversity of Plasmodium vivax Isolates From China, Korea, and Thailand Benjawan Khuntirat, Bousaraporn Tippayachai, Panadda Krairojananan, Sommai Promstaporn, Qi Gao, Won Ja Lee, James W. Jones, Jetsumon Sattabongkot; US Army Medical Component (USAMC), Armed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok, Thailand It has been shown that two types of Plasmodium vivax exist in China and Korea: one with a short and one with a long incubation period, whereas only the short incubation type has been recorded in Thailand. In this study, genetic diversity of P. vivax isolates from China, Korea, and Thailand during May through October 2005 were investigated using three polymorphic genetic markers, the circumsporozoite protein (CSP), the merozoite surface protein-1 (MSP-1), the merozoite surface protein-3 alpha (MSP-3). Preliminary results indicated that a) for the dimorphic CSP gene, the majority of the parasites from all locations were the VK210 type, b) based on the length of polymerase chain reaction (PCR) product, two types of the MSP-1 locus were observed from isolates from every location, and c) based on the length of PCR product, three types of the MSP-3 locus were distinguished; however, only one type was found in common in all locations. These results suggest that P. vivax populations are highly diverse and further study is needed to determine the importance of biological differences among P. vivax isolates. P-15 Using Ancient DNA to Detect Changes in the Populaton Structure of Plasmodium vivax Christie Pinello, Terry Brown; University of Manchester, Faculty of Life Sciences, Manchester, UK The objective of this project is to determine the extent to which it is possible to use ancient DNA sequencing to study changes in the molecular diversity of Plasmodium vivax, as well as to gather information on changes in parasite structure and virulence in medieval and post-medieval England. Historical records and literature from the time suggest a change in parasite virulence through the centuries, with a peak occuring in the 1600's. This study will investigate these claims and draw upon both archaeological and

biological evidence to discern a more complete picture of a changing parasite. A silica-based method was used to extract ancient DNA from bone samples collected from notoriously marshy areas of south-east England. PCR amplification targeted the 18S rRNA gene of P. vivax and the human mtDNA hypervariable region 1 to asses the survival of DNA in the archaeological samples. Ancient DNA was extracted and successfully amplified from several samples thus far, and research is ongoing to confirm these sequences and detect additional ancient P. vivax DNA in the remaining samples. P-16 A Framework for the Analysis of Antigen Gene Repertoires in Malaria Parasites Alyssa E. Barry, Gilean A. V. McVean, Karen P. Day; New York University School of Medicine, New York, NY, USA Malaria parasites contain subtelomeric multigene families implicated in virulence and antigenic variation. The most studied sub-telomeric gene family of Plasmodium vivax is known as vir. These comprise a 600-1000 genes per parasite genome and can be subdivided into 6 sub-families (A-F). The vir genes are highly immunogenic and thus thought to be important in persistence and re-infection, similar to the var genes of P. falciparum. We have developed a framework for the population genetic analysis of the var genes of P. falciparum. High throughput sequencing from field isolates, coupled with population genetic and bioinformatics analysis allowed us to describe the diversity of these important antigen genes. We describe this framework and how it may be used to accelerate the discovery and understanding of vir genes in natural infections. P-17 High Throughput Identification of the Predominant Malaria Parasite Clone in Complex Blood Stage Infections Using an Oligonucleotide Ligation Assay J.L. Cole-Tobian, P.A. Zimmerman, W.E. Collins, and C.L. King; Case Western Reserve University, Center for Global Health and Disease, Cleveland, OH, USA Individuals living in endemic areas are often infected with multiple clones of malaria

parasites. These complex infections can complicate accurate DNA sequencing-based strain identification within individual blood samples. Here we describe an oligonucleotide ligation assay (OLA) to distinguish single nucleotide polymorphisms (SNPs) in the Plasmodium vivax Duffy binding protein gene (Pvdbp) at 14 polymorphic residues simultaneously. This post-PCR assay is based on ligation of allele-specific and biotin-labeled common oligonucleotides to target DNA. The 5’ end of the ligation product is then hybridized to a unique fluorescent classification microsphere while the 3’ end is labeled with a reporter fluorochrome through binding of streptavidin-phycoerythrin. Flow cytometric analysis is then used to sort reporter fluorescent signals in association with sequence-specific classification fluorescence. Allele abundance is determined by the highest mean fluorescent intensity of each SNP. Using plasmids encoding known sequences of the Pvdbp, the correct allele was identified 100% of the time. In experiments where plasmids encoding different Pvdbp alleles were mixed at different ratios, the predominant SNP was identified 93% of the time at a 2:1 ratio, 97% of time in 5:1 ratio, and 97% of the time in a 10:1 ratio. We always identified the most abundant clones in similar mixing experiments using Aotus monkey blood with single clones of P. vivax parasites. Furthermore, using mixed vivax infections from field samples from Papua New Guinea, we were able to identify the predominant Pvdbp genotype with similar accuracy. The dominant genotype from these samples was determined by sequencing 10-19 clones from each sample, and the genotype with the largest number of clones was considered the most abundant genotype. Thus the OLA can accurately, reproducibly and rapidly determine the predominant parasite clone in complex blood stage infections. This has important implications since only the dominant strain in complex infections may contribute to disease. P-18 The Changing Dynamics of Plasmodium vivax and P.falciparum in Central India

Neeru Singh, A.C. Nagpal, Ajay Saxena, M.P. Singh, Om Kataria and A.P. Dash; Marlaria Research Centre (IMCR), Delhi, India An increase in the number of Plasmodium falciparum infections in central India (Madhya Pradesh and Chhattisgarh) has been noticed,

which is the second highly malarious state in India. A field station of Malaria Research Centre (ICMR) Jabalpur (Madhya Pradesh) is conducting a multidisciplinary study on malaria since 1986 to define epidemiological characteristics of the infection with each Plasmodium species in different seasons of the year to record the extent and timing of changes in P.vivax and P.falciparum infections to determine trends. The only two Plasmodium species encountered were P.vivax and P.falciparum. The study revealed summer peak due to P.vivax and autumn peak due to P.falciparum. There was a significant falling trend in P.vivax from 1988 onwards. Between 1986 to 1990 malaria was mainly due to P.vivax (60-75%). During 1991 the infection caused by two species almost equal and from 1992 onwards P.falciparum became dominant infection (65-90%). Record showed (1986-2004) that transmission of P.falciparum has extended from post monsoon, to extreme winter to spring to summer months. Retrospective analysis of Chhattisgarh (State Malaria Programme Data, 1975-2004) also revealed a steady upward trend in P.falciparum from 1979 onwards. Therefore, more effective measures (chemotherapy and vector control) are required to control P.falciparum malaria. P-19 Genetic Diversity of P. vivax in Different Endemic Zones of Ethiopia Netsanet Gizaw1,2, Beyene Petros1, Howard Engers2, Lawrence K.Yamuah2, Abraham Aseffa2; 1Addis Ababa University, Addis Ababa, Ethiopia; 2Armauer Hansen Research Institute, Addis Ababa, Ethiopia The present study is aimed at describing the prevalent parasite population dynamics of P. vivax isolates in Ethiopia in different geographical settings. For this purpose, finger prick blood samples were collected on filter paper and slides from 55 microscopically confirmed vivax malaria patients (age > 3 months) attending health clinics in Arbaminch, Burie, Derra, and Zeway. Then DNA was extracted by chelex extraction method and used for PCR amplification: Nested-PCR followed by restriction digestion by Alu I of MSP-3α gene was used to genotype P.vivax. The findings of the study showed that three major genotypes ~ 1.9Kb size, 1.5Kb size, and 1.1Kb were detected after PCR amplification. 1.9 Kb type was the most dominant allele accounting for 78.2% of all types.

The other two types, 1.5 Kb and 1.1 Kb, accounted for 7.3% and 14.5%, respectively and fourteen different genotypes of P. vivax were found depending on their restriction pattern and 7.2% of the isolates were multiclonal. Based on the results of the study it was concluded that Ethiopian isolates P. vivax are highly diverse as the countries with the same level of transmission. Further nation wide investigation is recommended to better understand polymorphism of malaria parasites in relation to acquired immune response and pathogenicity. P-20 Functional Analysis of the Multidrug Resistance Gene mdr1 of Plasmodium vivax Through Heterologous Over-Expression in P. falciparum Pamela Orjuela, Hernando del Portillo; Parasitology Department, Institute of Biomedical Sciences, University of São Paulo (USP), Brazil Chloroquine resistance (CQR) in Plasmodium vivax, the most widely distributed human malaria parasite, is threating to render this cheap and widely used first-line antimalarial drug, ineffective. Moreover, resistance to primaquine has also been reported for P. vivax, strongly indicating that this human malaria parasite is developing multidrug resistance (MDR). CQR and MDR in P. falciparum, the most deadly species, have been associated with the pfmdr1 gene. This gene codes for an energy-dependent membrane transport protein and mutations as well as expression and gene amplifications have been strongly, though not completely, associated with the CQR phenotype. To initiate studies on MDR in P. vivax, we previously cloned and characterized the orthologous gene, pvmdr1, in P. vivax. Strikingly, there were no mutations associated with CQR suggesting that a different mechanism should be involved in this resistance. Functional studies through reverse genetics of pvmdr1 are difficult to conduct because P. vivax cannot be continuously grown in vitro. To overcome this difficulty, we have generated a stable transgenic line of the P.falciparum CQ sensitive clone 3D7 over-expressing pvmdr1 in order to verify drug phenotype changes.

P-21 Polymorphism in Merozoite Surface Protein 1 of Plasmodium vivax Population in Thailand Correlates with Malaria Endemicity Chaturong Putaporntip1, Liwang Cui2, Rachanee Udomsangpetch3, Hiroji Kanbara4, Somchai Jongwutiwes1; 1Department of Parasitology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand; 2Department of Entomology, The Pennsylvania State University, University Park, PA, USA; 3Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok, Thailand; 4Department of Protozoology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan To test if intensity of malaria transmission influences the extent of diversity in PvMsp-1, we collected 169 Plasmodium vivax isolates from northern (n=66), eastern (n=49) and southern (n=54) Thailand where annual parasite incidence per 1,000 (API) during the period of sample collection was >20, 5-20 and <2, respectively. Of these, 133 isolates containing single PvMsp-1 alleles were used for analysis of variable blocks 2 and 12. Results revealed that block 2 contained 14 sequence types. The variable block 12 displayed 10 sequence types. It is noteworthy that certain alleles of blocks 2 and 12 predominated in Thai P. vivax populations. Interestingly, the magnitudes of haplotype diversity, nucleotide diversity and average number of nucleotide differences in both blocks 2 and 12 of PvMsp-1 were highest in the P. vivax population from northern Thailand. Analysis of inter-population variance in allele frequencies (Wright’s Fst) revealed no significant values of both blocks (p> 0.2), suggesting that these loci were under balancing selection. Likewise, the mean number of nonsynonymous substitutions per nonsynonymous site (dN) significantly outnumbered that of synonymous substitutions per synonymous site (dS) (p<0.001) in block 12 for each population, indicating that positive selection has diversified sequences in this region. P-22 Establishment of Erythropoietic Cells in vitro for Continuous Culture of Plasmodium vivax Tasanee Panichakul1, Jetsumon Sattabongkot2, Kesinee Chotivanich1, Jeeraphat Sirichaisinthop3, Qi Gao4, Liwang Cui5, Rachanee Udomsangpetch6; 1Department of Clinical

Tropical Medicine, Faculty of Tropical Medicine and 6Department of Pathobiology Faculty of Science, Mahidol University, Bangkok, Thailand; 2Department of Entomology, AFRIMS, Bangkok, Thailand; 3 Center of Malaria Research and Training, Ministry of Public Health, Saraburi, Thailand; 4Jiangsu Institute of Parasitic Diseases, Jiangsu, People’s Republic of China; 5Department of Entomology, Pennsylvania State University, USA Plasmodium vivax cannot be maintained in a continuous culture. Evidently, reticulocytes are the main targets for merozoite interaction and subsequent invasion to carry on the blood stage cycles. In this study, we produced newly differentiated red blood cells (RBC) in vitro from freshly isolated cord blood stem cells. Normal umbilical cord blood (UCB) was used as a source of hematopoietic stem cells. CD133+ hematopoietic stem cells were isolated from cord blood by using super-magnetic microbead (Miltenyi Biotech). To obtain the RBC in vitro, these cells were cultured in serum free Stemline II medium (Sigma-Aldrich) supplemented with stem cell factor, IL-3, transferrin, insulin, hydrocortisone and erythropoietin (EPO). Proliferation of erythroid progenitors was determined by burst forming unit-erythroid (BFU-E) and colony forming unit-erythroid (CFU-E). Cell morphology was confirmed by Giemsa’s staining and microscopy. After 8 days of culture, number of cells were increased up to 100-fold. Majority of the cells were in erythroid linage and erythroblastic cells. To obtain maturation of the RBC, co-culture with mesenchymal cells (MSC) in medium supplemented with EPO was done for 3 days and then without EPO for another 10 days. Flow cytometric analysis showed that expression of CD133+ and CD34+, markers of hematopoietic stem cells, slowly decreased and not detectable after 11 days of culture. Additionally CD71+, known to be highly expressed in erythroblasts, was detected in 90% of the cells obtained from 5 days old culture. The CD71+ became negative and glycophorin-A was positive in mature RBCs. P.vivax-infected RBC from fresh parasite isolate concentrated with percoll was added into the RBC culture containing ≥10% of enucleated RBCs. Co-cultures of parasite and established RBCs (eRBCs) were darily determined the invasion and propagation of parasite in eRBCs by Immunofluorescence assay and Giemsa staining. Results showed that merozoites of P. vivax could invade eRBCs which were nucleated and enucleated RBCs, and develop to schizont stage

in these RBCs. Recently P.vivax culture is being maintained by the in vitro produced human RBCs to establish a laboratory line of the parasite. The in vitro production of RBC could be an alternative method providing reticulocytes essential for development of P.vivax in continuous culture. P-23 Malariometric Indices in Bharia Primitive Tribe Central India C K Dolla, R Yadav, D Kumar, V G Rao; Regional Medical Research Center for Tribals, ICMR, Madhya Pradesh, India Malariometric indices are one of the important measures in epidemiological surveillance and in planning the control strategy for malaria; The classical malaorimetric measures are spleen rate, average enlarged spleen, parasite rate etc. the importance of malaria transmission in tribal areas lead to increasing morbidity and mortality. The research has shown change in climate, lack of prompt recognition and treatment are reasons for transmission of this devastating parasitic disease. The aboriginal communities show an increasing interest in the use of traditional treatment (guniya) as a first-line for the disease. The malariometric indices provide needed information about the trend of the disease. The present study was carried out in 12 Bharia villages located in the “Patalkot Valley” a deep valley of Tamia block, district Chhindwara of south-eastern Madhya Pradesh. The study period is in the month of December 2000. Populations of 451 were clinically examined for spleen enlargement and 120 blood smear examination done for the cases of fever based on probable diagnosis. The results shows: proportionate case rate-26.6, spleen rate-29.4, average enlarged spleen , Gr.I 13.7, Gr.II 86.0, Gr.III 0.3, Parasite Rate-21.7, Infant parasite rate-20.7, slide positivity rate-46.1, slide falciparum rate-29.2, Pf percentage-62.5, Pv percentage-28.6. On conclusion the area is mesoendemic based on parasite rate and spleen rate.