scholarly journals Posttranslational Modification of Recombinant Plasmodium falciparum Apical Membrane Antigen 1: Impact on Functional Immune Responses to a Malaria Vaccine Candidate

2005 ◽  
Vol 73 (7) ◽  
pp. 3963-3970 ◽  
Author(s):  
Birgitte Giersing ◽  
Kazutoyo Miura ◽  
Richard Shimp ◽  
Jin Wang ◽  
Hong Zhou ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Pichia Pastoris ◽  
Posttranslational Modification ◽  
Vaccine Candidate ◽  
Apical Membrane ◽  
Expression System ◽  
Apical Membrane Antigen 1 ◽  
E Coli ◽  
Apical Membrane Antigen ◽  
Significant Difference

ABSTRACT 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.

scholarly journals Phase 1 Clinical Trial of Apical Membrane Antigen 1: an Asexual Blood-Stage Vaccine for Plasmodium falciparum Malaria

2005 ◽  
Vol 73 (6) ◽  
pp. 3677-3685 ◽  
Author(s):  
Elissa M. Malkin ◽  
David J. Diemert ◽  
Julie H. McArthur ◽  
John R. Perreault ◽  
Aaron P. Miles ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Malaria Vaccine ◽  
Vaccine Candidate ◽  
Apical Membrane ◽  
Phase 1 ◽  
Blood Stage ◽  
Enzyme Linked Immunosorbent Assay ◽  
Apical Membrane Antigen 1 ◽  
Apical Membrane Antigen ◽  
Microscopic Evaluation

ABSTRACT Apical membrane antigen 1 (AMA1), a polymorphic merozoite surface protein, is a leading blood-stage malaria vaccine candidate. A phase 1 trial was conducted with 30 malaria-naïve volunteers to assess the safety and immunogenicity of the AMA1-C1 malaria vaccine. AMA1-C1 contains an equal mixture of recombinant proteins based on sequences from the FVO and 3D7 clones of Plasmodium falciparum. The proteins were expressed in Pichia pastoris and adsorbed on Alhydrogel. Ten volunteers in each of three dose groups (5 μg, 20 μg, and 80 μg) were vaccinated in an open-label study at 0, 28, and 180 days. The vaccine was well tolerated, with pain at the injection site being the most commonly observed reaction. Anti-AMA1 immunoglobulin G (IgG) was detected by enzyme-linked immunosorbent assay (ELISA) in 15/28 (54%) volunteers after the second immunization and in 23/25 (92%) after the third immunization, with equal reactivity to both AMA1-FVO and AMA1-3D7 vaccine components. A significant dose-response relationship between antigen dose and antibody response by ELISA was observed, and the antibodies were predominantly of the IgG1 isotype. Confocal microscopic evaluation of sera from vaccinated volunteers demonstrated reactivity with P. falciparum schizonts in a pattern similar to native parasite AMA1. Antigen-specific in vitro inhibition of both FVO and 3D7 parasites was achieved with IgG purified from sera of vaccinees, demonstrating biological activity of the antibodies. To our knowledge, this is the first AMA1 vaccine candidate to elicit functional immune responses in malaria-naïve humans, and our results support the further development of this vaccine.

scholarly journals High-Level Expression of the Malaria Blood-Stage Vaccine Candidate Plasmodium falciparum Apical Membrane Antigen 1 and Induction of Antibodies That Inhibit Erythrocyte Invasion

2002 ◽  
Vol 70 (8) ◽  
pp. 4471-4476 ◽  
Author(s):  
Clemens H. M. Kocken ◽  
Chrislaine Withers-Martinez ◽  
Martin A. Dubbeld ◽  
Annemarie van der Wel ◽  
Fiona Hackett ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Vaccine Candidate ◽  
Apical Membrane ◽  
High Titer ◽  
Scale Up ◽  
Blood Stage ◽  
Apical Membrane Antigen 1 ◽  
Apical Membrane Antigen ◽  
Antigenic Properties ◽  
High Level

ABSTRACT Apical membrane antigen 1 (AMA-1) is a highly promising malaria blood-stage vaccine candidate that has induced protection in rodent and nonhuman primate models of malaria. Authentic conformation of the protein appears to be essential for the induction of parasite-inhibitory antibody responses. Here we have developed a synthetic gene with adapted codon usage to allow expression of Plasmodium falciparum FVO strain AMA-1 (PfAMA-1) in Pichia pastoris. In addition, potential N-glycosylation sites were changed, exploiting the lack of conservation of these sites in Plasmodium, to obtain high-level secretion of a homogeneous product, suitable for scale-up according to current good manufacturing procedures. Purified PfAMA-1 displayed authentic antigenic properties, indicating that the amino acid changes had no deleterious effect on the conformation of the protein. High-titer antibodies, raised in rabbits, reacted strongly with homologous and heterologous P. falciparum by immunofluorescence. In addition, purified immunoglobulin G from immunized animals strongly inhibited invasion of red blood cells by homologous and, to a somewhat lesser extent, heterologous P. falciparum.

scholarly journals Use of Immunodampening To Overcome Diversity in the Malarial Vaccine Candidate Apical Membrane Antigen 1

2014 ◽  
Vol 82 (11) ◽  
pp. 4707-4717 ◽  
Author(s):  
Karen S. Harris ◽  
Christopher G. Adda ◽  
Madhavi Khore ◽  
Damien R. Drew ◽  
Antonina Valentini-Gatt ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Antibody Response ◽  
Vaccine Candidate ◽  
Apical Membrane ◽  
Targeted Mutagenesis ◽  
Parasite Line ◽  
Content Type ◽  
Apical Membrane Antigen 1 ◽  
Apical Membrane Antigen ◽  
Mutation Strategy

ABSTRACTApical membrane antigen 1 (AMA1) is a leading malarial vaccine candidate; however, its polymorphic nature may limit its success in the field. This study aimed to circumvent AMA1 diversity by dampening the antibody response to the highly polymorphic loop Id, previously identified as a major target of strain-specific, invasion-inhibitory antibodies. To achieve this, five polymorphic residues within this loop were mutated to alanine, glycine, or serine in AMA1 of the 3D7 and FVOPlasmodium falciparumstrains. Initially, the corresponding antigens were displayed on the surface of bacteriophage, where the alanine and serine but not glycine mutants folded correctly. The alanine and serine AMA1 mutants were expressed inEscherichia coli, refoldedin vitro, and used to immunize rabbits. Serological analyses indicated that immunization with a single mutated form of 3D7 AMA1 was sufficient to increase the cross-reactive antibody response. Targeting the corresponding residues in an FVO backbone did not achieve this outcome. The inclusion of at least one engineered form of AMA1 in a biallelic formulation resulted in an antibody response with broader reactivity against different AMA1 alleles than combining the wild-type forms of 3D7 and FVO AMA1 alleles. For one combination, this extended to an enhanced relative growth inhibition of a heterologous parasite line, although this was at the cost of reduced overall inhibitory activity. These results suggest that targeted mutagenesis of AMA1 is a promising strategy for overcoming antigenic diversity in AMA1 and reducing the number of variants required to induce an antibody response that protects against a broad range ofPlasmodium falciparumAMA1 genotypes. However, optimization of the immunization regime and mutation strategy will be required for this potential to be realized.

Sign in / Sign up

Export Citation Format

Share Document