Immunoproteomics Profiling of Blood Stage Plasmodium vivax Infection by High-Throughput Screening Assays

Abstract

Completed genome sequences and stage-specific transcriptomes of the intraerythrocytic developmental cycle of Plasmodium vivax offers the opportunity to profile immune responses against P. vivax infection using innovative screening approaches. To detect the immune responses to blood stage-specific proteins, we applied a protein array technology to screen the sera of vivax malaria patients. Herein, a set of genes from the P. vivax blood stage was cloned using the In-Fusion cloning method and expressed by a wheat germ cell-free system. A total of 94 open reading frames (ORFs) were cloned and 89 (95%, 89/94) proteins were expressed, which were screened with sera from P. vivax-infected patients and healthy individuals using protein arrays. A total of 18 (19.1%, 18/94) highly immunoreactive proteins were identified, including 7 well-characterized vivax vaccine candidates. The remaining 11 ORFs have not been previously described as immunologically reactive. These novel immunoproteomes of the vivax malaria blood stage will be further studied as potential vaccine candidates. In this first report, high-throughput screening assays have been applied to investigate blood stage-specific immunoproteomes from vivax malaria. These methods may be used to determine immunodominant candidate antigens from the P. vivax genome.

The Plasmodium vivax Merozoite Surface Protein 1 Paralog Is a Novel Erythrocyte-Binding Ligand of P. vivax

“…PvMSPs with GPI anchors (PvMSP1, PvMSP5, and PvMSP8) are immunogenic in humans in areas where malaria is endemic (14,15,18,27,32). Acquired immune responses to PvMSP1 suggest that antibodies and cellular immune responses to PvMSP1 are strongly induced during vivax malaria (15,38).…”

“…The study was approved by the Institutional Review Board at Kangwon National University Hospital. Human sera from 150 patients with vivax malaria and 100 healthy individuals were screened for immunoreactivity using a protein array method (27). We used sera from 50 patients with vivax malaria and 40 malaria-naïve individuals and eight fragments of PvMSP1P for primary humoral immune response screening by protein array.…”

“…Amine-coated slides were prepared as described previously (27,30). To develop the protein arrays, sera from patients with vivax malaria and malaria-naïve individuals were used to analyze the humoral immune response by well-type amine arrays.…”

“…The chips were then probed with sera from the malaria patients or healthy individuals (1:10 dilution) that had been preabsorbed against wheat germ lysate (1:100 dilution) to block anti-wheat germ antibodies. Alexa Fluor 546-conjugated goat anti-human IgG (10 ng/l; Invitrogen) in PBST was used as the detection antibody, and the fluorescent signals were scanned in a fluorescence scanner (ScanArray Express, PerkinElmer, Boston, MA) and quantified as described previously (27). The cutoff value was defined as two standard deviations (SDs) above the mean fluorescence intensity of the negative-control samples.…”

Plasmodium vivax Pre-Erythrocytic–Stage Antigen Discovery: Exploiting Naturally Acquired Humoral Responses

“…MSP-8 (PVA205) was previously characterized as a glycosylphosphatidylinisotol-anchored protein, and it was recognized by P. vivax-infected patients in Columbia 53 as well as P. vivax-infected patients from Korea. 54 Proteins recognized by both Fy+ and Fy− donors were expressed highly in the sporozoite; however, five of six were also expressed in the blood stages of the parasite. These responses could be caused by antigens expressed by hepatic merozoites.…”