posttranslational modification of recombinant Plasmodium falciparum apical membrane antigen 1: impact on functional immune responses to a malaria vaccine candidate.
Recombinant apical membrane antigen 1 (AMA1) is a leading vaccine candidate for Plasmodium falciparum malaria, as antibodies against recombinant P. falciparum AMA1 (PfAMA1) interrupt merozoite invasion into erythrocytes. In order to investigate the role of posttranslational modification in modulating the functional immune response to recombinant AMA1, two separate alleles of PfAMA1 (FVO and 3D7), in which native N-glycosylation sites have been mutated, were produced using Escherichia coli and a Pichia pastoris expression system. Recombinant Pichia pastoris AMA1-FVO (PpAMA1-FVO) and PpAMA1-3D7 are O-linked glycosylated, and 45% of PpAMA1-3D7 is nicked, though all four recombinant molecules react with conformation-specific monoclonal antibodies. To address the immunological effect of O-linked glycosylation, we compared the immunogenicity of E. coli AMA1-FVO (EcAMA1-FVO) and PpAMA1-FVO antigens, since both molecules are intact. The effect of antigen nicking was then investigated by comparing the immunogenicity of EcAMA1-3D7 and PpAMA1-3D7. Our data demonstrate that there is no significant difference in the rabbit antibody titer elicited towards EcAMA1-FVO and PpAMA1-FVO or to EcAMA1-3D7 and PpAMA1-3D7. Furthermore, we have demonstrated that recombinant AMA1 (FVO or 3D7), whether expressed and refolded from E. coli or produced from the Pichia expression system, is equivalent and mimics the functionality of the native protein in in vitro growth inhibition assay experiments. We conclude that in the case of recombinant AMA1, the E. coli- and P. pastoris-derived antigens are immunologically and functionally equivalent and are unaffected by the posttranslational modification resulting from expression in these two systems.
Giersing
Birgitte
B
Malaria Vaccine Development Branch, National Institute of Allergy and Infectious Diseases, Twinbrook I, Room 1210A, 5640 Fisher Lane, Rockville, Maryland 20852, USA.
Miura
Kazutoyo
K
Shimp
Richard
R
Wang
Jin
J
Zhou
Hong
H
Orcutt
Andrew
A
Stowers
Anthony
A
Saul
Allan
A
Miller
Louis H
LH
Long
Carole
C
Singh
Sanjay
S
D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics; D017780:Malaria Vaccines / Q000276:immunology*; D008565:Membrane Proteins / Q000276:immunology*; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D010963:Plasmodium falciparum / Q000276:immunology*; D011499:Protein Processing, Post-Translational; D015800:Protozoan Proteins / Q000276:immunology*; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology; D014614:Vaccines, Synthetic / Q000276:immunology*
D000953:Antigens, Protozoan; D017780:Malaria Vaccines; D008565:Membrane Proteins; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D014614:Vaccines, Synthetic; C090077:apical membrane antigen I, Plasmodium