The Most Polymorphic Residue on Plasmodium falciparum Apical Membrane Antigen 1 Determines Binding of an Invasion-Inhibitory Antibody

ABSTRACT Apical membrane antigen 1 (AMA1) is currently one of the leading malarial vaccine candidates. Anti-AMA1 antibodies can inhibit the invasion of erythrocytes by Plasmodium merozoites and prevent the multiplication of blood-stage parasites. Here we describe an anti-AMA1 monoclonal antibody (MAb 1F9) that inhibits the invasion of Plasmodium falciparum parasites in vitro. We show that both reactivity of MAb 1F9 with AMA1 and MAb 1F9-mediated invasion inhibition were strain specific. Site-directed mutagenesis of a fragment of AMA1 displayed on M13 bacteriophage identified a single polymorphic residue in domain I of AMA1 that is critical for MAb 1F9 binding. The identities of all other polymorphic residues investigated in this domain had little effect on the binding of the antibody. Examination of the P. falciparum AMA1 crystal structure localized this residue to a surface-exposed α-helix at the apex of the polypeptide. This description of a polymorphic inhibitory epitope on AMA1 adds supporting evidence to the hypothesis that immune pressure is responsible for the polymorphisms seen in this molecule.

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Immunization with the Malaria Diversity-Covering Blood-Stage Vaccine Candidate Plasmodium falciparum Apical Membrane Antigen 1 DiCo in Complex with Its Natural Ligand PfRon2 Does Not Improve the In Vitro Efficacy

Frontiers in Immunology ◽

10.3389/fimmu.2017.00743 ◽

2017 ◽

Vol 8 ◽

Cited By ~ 1

Author(s):

Holger Spiegel ◽

Alexander Boes ◽

Rolf Fendel ◽

Andreas Reimann ◽

Stefan Schillberg ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Vaccine Candidate ◽

Apical Membrane ◽

Blood Stage ◽

Apical Membrane Antigen 1 ◽

Apical Membrane Antigen ◽

Natural Ligand

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A Diversity-Covering Approach to Immunization with Plasmodium falciparum Apical Membrane Antigen 1 Induces Broader Allelic Recognition and Growth Inhibition Responses in Rabbits

Infection and Immunity ◽

10.1128/iai.00170-08 ◽

2008 ◽

Vol 76 (6) ◽

pp. 2660-2670 ◽

Cited By ~ 80

Author(s):

Edmond J. Remarque ◽

Bart W. Faber ◽

Clemens H. M. Kocken ◽

Alan W. Thomas

Keyword(s):

Plasmodium Falciparum ◽

Amino Acid ◽

Growth Inhibition ◽

Apical Membrane ◽

Recombinant Expression ◽

Methylotrophic Yeast ◽

Apical Membrane Antigen 1 ◽

Apical Membrane Antigen ◽

Amino Acid Variability

ABSTRACT Plasmodium falciparum apical membrane antigen 1 (PfAMA1), a candidate malaria vaccine, is polymorphic. This polymorphism is believed to be generated predominantly under immune selection pressure and, as a result, may compromise attempts at vaccination. Alignment of 355 PfAMA1 sequences shows that around 10% of the 622 amino acid residues can vary between alleles and that linkages between polymorphic residues occur. Using this analysis, we have designed three diversity-covering (DiCo) PfAMA1 sequences that take account of these linkages and, when taken together, on average incorporate 97% of amino acid variability observed. For each of the three DiCo sequences, a synthetic gene was constructed and used to transform the methylotrophic yeast Pichia pastoris, allowing recombinant expression. All three DiCo proteins were reactive with the reduction-sensitive monoclonal antibody 4G2, suggesting the DiCo sequences had conformations similar to those of naturally occurring PfAMA1. Rabbits were immunized with FVO strain PfAMA1 or with the DiCo proteins either individually or as a mixture. Antibody titers and the ability to inhibit parasite growth in vitro were determined. Animals immunized with the DiCo mix performed similarly to animals immunized with FVO AMA1 when measured against FCR3 strain parasites but outperformed animals immunized with FVO AMA1 when assessed against other strains. The levels of growth inhibition (∼70%) induced by the mix of three DiCo proteins were comparable for FVO, 3D7, and HB3, suggesting that a considerable degree of diversity in AMA1 is adequately covered. This suggests that vaccines based upon the DiCo mix approach provide a broader functional immunity than immunization with a single allele.

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In Immunization with Plasmodium falciparum Apical Membrane Antigen 1, the Specificity of Antibodies Depends on the Species Immunized

Infection and Immunity ◽

10.1128/iai.00593-07 ◽

2007 ◽

Vol 75 (12) ◽

pp. 5827-5836 ◽

Cited By ~ 28

Author(s):

Kazutoyo Miura ◽

Hong Zhou ◽

Olga V. Muratova ◽

Andrew C. Orcutt ◽

Birgitte Giersing ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Growth Inhibition ◽

Vaccine Development ◽

Apical Membrane ◽

Rabbit Model ◽

Enzyme Linked Immunosorbent Assay ◽

Allelic Polymorphism ◽

Apical Membrane Antigen 1 ◽

Apical Membrane Antigen

ABSTRACT At least a million people, mainly African children under 5 years old, still die yearly from malaria, and the burden of disease and death has increased. Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is one of the most promising blood-stage malarial vaccine candidates. However, the allelic polymorphism observed in this protein is a potential stumbling block for vaccine development. To overcome the polymorphism- and strain-specific growth inhibition in vitro, we previously showed in a rabbit model that vaccination with a mixture of two allelic forms of PfAMA1 induced parasite growth-inhibitory antisera against both strains of P. falciparum parasites in vitro. In the present study, we have established that, in contrast to a single-allele protein, the antigen mixture elicits primarily antibodies recognizing antigenic determinants common to the two antigens, as judged by an antigen reversal growth inhibition assay (GIA). We also show that a similar reactivity pattern occurs after immunization of mice. By contrast, sera from rhesus monkeys do not distinguish the two alleles when tested by an enzyme-linked immunosorbent assay or by GIA, regardless of whether the immunogen is a single AMA1 protein or the mixture. This is the first report that a malarial vaccine candidate induced different specificities of functional antibodies depending on the animal species immunized. These observations, as well as data available on human immune responses in areas of endemicity, suggest that polymorphism in the AMA1 protein may not be as formidable a problem for vaccine development as anticipated from studies with rabbits and mice.

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Production of the Subdomains of the Plasmodium falciparum Apical Membrane Antigen 1 Ectodomain and Analysis of the Immune Response

Infection and Immunity ◽

10.1128/iai.72.8.4464-4470.2004 ◽

2004 ◽

Vol 72 (8) ◽

pp. 4464-4470 ◽

Cited By ~ 27

Author(s):

P. V. Lalitha ◽

Lisa A. Ware ◽

Arnoldo Barbosa ◽

Sheetij Dutta ◽

J. Kathleen Moch ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Apical Membrane ◽

Transmembrane Protein ◽

Invasion Assay ◽

Apical Membrane Antigen 1 ◽

Apical Membrane Antigen ◽

Growth Inhibitory ◽

Inhibitory Antibodies ◽

The Individual

ABSTRACT The apical membrane antigen 1 of Plasmodium falciparum is one of the leading candidate antigens being developed as a vaccine to prevent malaria. This merozoite transmembrane protein has an ectodomain that can be divided into three subdomains (D I, D II, and D III). We have previously expressed a major portion of this ectodomain and have shown that it can induce antibodies that prevent merozoite invasion into red blood cells in an in vitro growth and invasion assay. To analyze the antibody responses directed against the individual subdomains, we constructed six different genes that express each of the domains separately (D I, D II, or D III) or in combination with another domain (D I+II, D II+III, or D I+III). These proteins were purified and used to immunize rabbits to raise construct-specific antibodies. We demonstrated that D I+II induced a significant amount of the growth-inhibitory antibodies active in the growth and invasion assay.

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In Vitro Studies with Recombinant Plasmodium falciparum Apical Membrane Antigen 1 (AMA1): Production and Activity of an AMA1 Vaccine and Generation of a Multiallelic Response

Infection and Immunity ◽

10.1128/iai.70.12.6948-6960.2002 ◽

2002 ◽

Vol 70 (12) ◽

pp. 6948-6960 ◽

Cited By ~ 186

Author(s):

Michael C. Kennedy ◽

Jin Wang ◽

Yanling Zhang ◽

Aaron P. Miles ◽

Farideh Chitsaz ◽

...

Keyword(s):

Apical Membrane ◽

Allelic Diversity ◽

Cross Reactivity ◽

Human Infection ◽

Apical Membrane Antigen 1 ◽

Economy Of Scale ◽

Apical Membrane Antigen ◽

Immune Pressure ◽

Single Antigen

ABSTRACT Apical membrane antigen 1 (AMA1) is regarded as a leading malaria blood-stage vaccine candidate. While the overall structure of AMA1 is conserved in Plasmodium spp., numerous AMA1 allelic variants of P. falciparum have been described. The effect of AMA1 allelic diversity on the ability of a recombinant AMA1 vaccine to protect against human infection by different P. falciparum strains is unknown. We characterize two allelic forms of AMA1 that were both produced in Pichia pastoris at a sufficient economy of scale to be usable for clinical vaccine studies. Both proteins were used to immunize rabbits, singly and in combination, in order to evaluate their immunogenicity and the ability of elicited antibodies to block the growth of different P. falciparum clones. Both antigens, when used alone, elicited high homologous anti-AMA1 titers, with reduced strain cross-reactivity. Similarly, sera from rabbits immunized with a single antigen were capable of blocking the growth of homologous parasite strains at levels theoretically sufficient to clear parasite infections. However, heterologous inhibition was significantly reduced, providing experimental evidence that AMA1 allelic diversity is a result of immune pressure. Encouragingly, rabbits immunized with a combination of both antigens exhibited titers and levels of parasite inhibition as good as those of the single-antigen-immunized rabbits for each of the homologous parasite lines, and consequently exhibited a broadening of allelic diversity coverage.

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