title: Profiling the humoral immune responses to Plasmodium vivax infection and identification of candidate immunogenic rhoptry-associated membrane antigen (RAMA).
abstract: Completion of sequencing of the Plasmodium vivax genome and transcriptome offers the chance to identify antigens among >5000 candidate proteins. To identify those P. vivax proteins that are immunogenic, a total of 152 candidate proteins (160 fragments) were expressed using a wheat germ cell-free system. The results of Western blot analysis showed that 92.5% (148/160) of the targets were expressed, and 96.6% (143/148) were in a soluble form with 67.7% of solubility rate. The proteins were screened by protein arrays with sera from 22 vivax malaria patients and 10 healthy individuals to confirm their immune profile, and 44 (27.5%, 44/160) highly reactive P. vivax antigens were identified. Overall, 5 candidates (rhoptry-associated membrane antigen [RAMA], Pv-fam-a and -b, EXP-1 and hypothetical protein PVX_084775) showed a positive reaction with >80% of patient sera, and 21 candidates with 50% to 80%. More than 23% of the highly immunoreactive proteins were hypothetical proteins, described for the first time in this study. One of the top immunogenic proteins, RAMA, was characterized and confirmed to be a serological marker of recent exposure to P. vivax infection. These novel immunoproteomes should greatly facilitate the identification of promising novel malaria antigens and may warrant further study.
  
  
  CONCLUSIONS
  The establishment of high-throughput cloning and expression systems has permitted the construction of protein arrays for proteome-wide study of Plasmodium vivax. In this study, high-throughput screening assays have been applied to investigate blood stage-specific immune proteomes from P. vivax. We identified 44 antigenic proteins from the 152 putative candidates, more than 23% of which were hypothetical proteins described for the first time in this study. In addition, PvRAMA was characterized further and confirmed to be a serological marker of exposure to infections. The expression of one-third of the selected antigenic genes were shifted between P. vivax and Plasmodium falciparum, suggesting that these genes may represent important factors associated with P. vivax selectivity for young erythrocytes and/or with immune evasion. These novel immune proteomes of the P. vivax blood stage provide a baseline for further prospective serological marker studies in malaria. These methods could be used to determine immunodominant candidate antigens from the P. vivax genome.
authors.affiliation: Department of Medical Environmental Biology and Tropical Medicine, School of Medicine, Kangwon National University, Chunchon, Gangwon-do, Republic of Korea; Jiangsu Institute of Parasitic Diseases, Key Laboratory on Technology for Parasitic Disease Prevention and Control, Ministry of Health, Wuxi, Jiangsu, People's Republic of China.
authors.forename: Feng
authors.initials: F
authors.lastname: Lu
authors.affiliation: Department of Medical Environmental Biology and Tropical Medicine, School of Medicine, Kangwon National University, Chunchon, Gangwon-do, Republic of Korea; Jiangsu Institute of Parasitic Diseases, Key Laboratory on Technology for Parasitic Disease Prevention and Control, Ministry of Health, Wuxi, Jiangsu, People's Republic of China.
authors.forename: Jian
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Medical Environmental Biology and Tropical Medicine, School of Medicine, Kangwon National University, Chunchon, Gangwon-do, Republic of Korea.
authors.forename: Bo
authors.initials: B
authors.lastname: Wang
authors.affiliation: Department of Medical Environmental Biology and Tropical Medicine, School of Medicine, Kangwon National University, Chunchon, Gangwon-do, Republic of Korea.
authors.forename: Yang
authors.initials: Y
authors.lastname: Cheng
authors.affiliation: Department of Molecular and Cellular Biochemistry, School of Medicine, Kangwon National University, Chunchon, Gangwon-do, Republic of Korea.
authors.forename: Deok-Hoon
authors.initials: DH
authors.lastname: Kong
authors.affiliation: Department of Entomology, The Pennsylvania State University, University Park, PA, USA.
authors.forename: Liwang
authors.initials: L
authors.lastname: Cui
authors.affiliation: Department of Molecular and Cellular Biochemistry, School of Medicine, Kangwon National University, Chunchon, Gangwon-do, Republic of Korea.
authors.forename: Kwon-Soo
authors.initials: KS
authors.lastname: Ha
authors.affiliation: Mahidol Vivax Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand.
authors.forename: Jetsumon
authors.initials: J
authors.lastname: Sattabongkot
authors.affiliation: Division of Malaria Research, Proteo-Science Center, Ehime University, Matsuyama, Ehime, Japan. Electronic address: tsuboi.takafumi.mb@ehime-u.ac.jp.
authors.forename: Takafumi
authors.initials: T
authors.lastname: Tsuboi
authors.affiliation: Department of Medical Environmental Biology and Tropical Medicine, School of Medicine, Kangwon National University, Chunchon, Gangwon-do, Republic of Korea. Electronic address: ethan@kangwon.ac.kr.
authors.forename: Eun-Taek
authors.initials: ET
authors.lastname: Han
mesh_terms: D000328:Adult; D000595:Amino Acid Sequence; D000913:Antibodies, Protozoan / Q000097:blood; D000953:Antigens, Protozoan / Q000378:metabolism; D002474:Cell-Free System; D003001:Cloning, Molecular; D004912:Erythrocytes / Q000469:parasitology; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D018503:Genome, Protozoan; D006801:Humans; D056724:Immunity, Humoral; D016780:Malaria, Vivax / Q000276:immunology*; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D010963:Plasmodium falciparum / Q000378:metabolism; D010966:Plasmodium vivax; D015800:Protozoan Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012995:Solubility; D055815:Young Adult
chemical_list: D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C065352:rhoptry associated protein, Plasmodium
keywords: High-throughput protein expression; Immunogenic protein; Plasmodium vivax; Protein array; RAMA; Serological marker