Selection and identification of malaria vaccine target molecule using bioinformatics and DNA vaccination.
Following a genome-wide search for a blood stage malaria DNA-based vaccine using web-based bioinformatic tools, 29 genes from the annotated Plasmodium yoelii genome sequence (www.PlasmoDB.org and www.tigr.org) were identified as encoding GPI-anchored proteins. Target genes were those with orthologues in P. falciparum, containing an N-terminal signal sequence containing hydrophobic amino acid stretch and signal P criteria, a transmembrane-like domain and GPI anchor motif. Focusing on the blood stage, we extracted mRNA from pRBCs, PCR-amplified 22 out of the 29 selected genes, and eventually cloned nine of these into a DNA vaccine plasmid, pVAX 200-DEST. Biojector-mediated delivery of the nine DNA vaccines was conducted using ShimaJET to C57BL/6 mice at a dose of 4 μg/mouse three times at an interval of 3 weeks. Two weeks after the second booster, immunized mice were challenged with P. y. yoelii 17XL-parasitized RBCs and the level of parasitaemia, protection and survival was assessed. Immunization with one gene (PY03470) resulted in 2-4 days of delayed onset and level of parasitaemia and was associated with increased survival compared to non-immunized mice. Antibody production was, however, low following DNA vaccination, as determined by immunofluorescence assay. Recombinant protein from this gene, GPI8p transamidase-related protein (rPyTAM) in PBS or emulsified with GERBU adjuvant was also used to immunize another set of C57BL/6 mice with 10-20 μg/mouse three times at 3-week interval. Higher antibody response was obtained as determined by ELISA with similar protective effects as observed after DNA vaccination.
Shuaibu
M N
MN
Department of Immunogenetics, Institute of Tropical Medicine (NEKKEN), Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan.
Kikuchi
M
M
Cherif
M S
MS
Helegbe
G K
GK
Yanagi
T
T
Hirayama
K
K
D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood; D000953:Antigens, Protozoan / Q000235:genetics* / Q000276:immunology; D003001:Cloning, Molecular; D019295:Computational Biology; D005260:Female; D017125:Genes, Protozoan; D017261:Glycosylphosphatidylinositols / Q000235:genetics; D007117:Immunization, Secondary; D008288:Malaria / Q000276:immunology / Q000517:prevention & control*; D017780:Malaria Vaccines / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008810:Mice, Inbred C57BL; D010957:Plasmids; D010967:Plasmodium yoelii / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D019444:Vaccines, DNA / Q000235:genetics / Q000276:immunology*
D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D017261:Glycosylphosphatidylinositols; D017780:Malaria Vaccines; D011994:Recombinant Proteins; D019444:Vaccines, DNA