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

2004 ◽  
Vol 72 (8) ◽  
pp. 4464-4470 ◽  
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.

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

2006 ◽  
Vol 74 (5) ◽  
pp. 2628-2636 ◽  
Author(s):  
A. M. Coley ◽  
K. Parisi ◽  
R. Masciantonio ◽  
J. Hoeck ◽  
J. L. Casey ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Apical Membrane ◽  
Site Directed Mutagenesis ◽  
Supporting Evidence ◽  
Inhibitory Antibody ◽  
Apical Membrane Antigen 1 ◽  
Apical Membrane Antigen ◽  
Immune Pressure ◽  
Polymorphic Residue

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.

scholarly journals Specificity of the Protective Antibody Response to Apical Membrane Antigen 1

2001 ◽  
Vol 69 (5) ◽  
pp. 3286-3294 ◽  
Author(s):  
Anthony N. Hodder ◽  
Pauline E. Crewther ◽  
Robin F. Anders
Keyword(s):  
Apical Membrane ◽  
Affinity Purification ◽  
Point Mutations ◽  
Apical Membrane Antigen 1 ◽  
Apical Membrane Antigen ◽  
Potential Vaccine ◽  
Inhibitory Antibodies ◽  
Domain I ◽  
The Impact

ABSTRACT Apical membrane antigen 1 (AMA1) is considered one of the leading candidates for inclusion in a vaccine against blood stages ofPlasmodium falciparum. Although the ama1 gene is relatively conserved compared to those for some other potential vaccine components, numerous point mutations have resulted in amino acid substitutions at many sites in the polypeptide. The polymorphisms in AMA1 have been attributed to the diversifying selection pressure of the protective immune responses. It was therefore of interest to investigate the impact of sequence diversity in P. falciparum AMA1 on the ability of anti-AMA1 antibodies to inhibit the invasion of erythrocytes in vitro by P. falciparummerozoites. For these studies, we used antibodies to recombinantP. falciparum 3D7 AMA1 ectodomain, which was prepared for testing in early clinical trials. Antibodies were raised in rabbits to the antigen formulated in Montanide ISA720, and human antibodies to AMA1 were isolated by affinity purification from the plasma of adults living in regions of Papua New Guinea where malaria is endemic. Both rabbit and human anti-AMA1 antibodies were found to be strongly inhibitory to the invasion of erythrocytes by merozoites from both the homologous and two heterologous lines of P. falciparum. The inhibitory antibodies targeted both conserved and strain-specific epitopes within the ectodomain of AMA1; however, it appears that the majority of these antibodies reacted with strain-specific epitopes in domain I, the N-terminal disulfide-bonded domain, which is the most polymorphic region of AMA1.

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

2008 ◽  
Vol 76 (6) ◽  
pp. 2660-2670 ◽  
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.

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

2007 ◽  
Vol 75 (12) ◽  
pp. 5827-5836 ◽  
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.

scholarly journals Induction of Parasite Growth-Inhibitory Antibodies by a Virosomal Formulation of a Peptidomimetic of Loop I from Domain III of Plasmodium falciparum Apical Membrane Antigen 1

2003 ◽  
Vol 71 (8) ◽  
pp. 4749-4758 ◽  
Author(s):  
Markus S. Mueller ◽  
Annabelle Renard ◽  
Francesca Boato ◽  
Denise Vogel ◽  
Martin Naegeli ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Monoclonal Antibodies ◽  
Apical Membrane ◽  
Parasite Growth ◽  
Vaccine Formulation ◽  
Peptide Antigen ◽  
Apical Membrane Antigen 1 ◽  
Growth Inhibitory ◽  
Inhibitory Antibodies ◽  
Domain Iii

ABSTRACT Apical membrane antigen 1 (AMA-1) of Plasmodium falciparum is a leading candidate antigen for inclusion in a malaria subunit vaccine. Its ectodomain can be divided into three subdomains, each with disulfide bond-stabilized structures. Since the majority of antibodies raised against the ectodomain appear to recognize strain-specific epitopes in domain I, we attempted to develop a vaccine formulation which directs the immune response to a region that contains more conserved epitopes. Here we demonstrate that a virosomal formulation of a peptide that mimics the semiconserved loop I of domain III elicits parasite growth-inhibitory antibodies. A synthetic peptide comprising residues 446 to 490 of AMA-1 (AMA-1446-490) was conjugated through the N terminus to a derivative of phosphatidylethanolamine and the phosphatidylethanolamine-peptide conjugate was incorporated into immunopotentiating reconstituted influenza virosomes as a human-compatible antigen delivery system. Both cyclized and linear versions of the peptide antigen elicited antibodies which specifically bound to parasite-expressed AMA-1 in Western blotting with parasite lysates as well as in immunofluorescence assays with blood stage parasites. All 11 peptidomimetic-specific monoclonal antibodies generated were cross-reactive with parasite-expressed AMA-1. Antigen binding assays with a library of overlapping cyclic peptides covering the target sequence revealed differences in the fine specificity of these monoclonal antibodies and provided evidence that at least some of them recognized discontinuous epitopes. The two immunodominant epitopes comprised the conserved linear sequences K459RIKLN464 and D467DEGNKKII475. A key feature of the synthetic vaccine formulation proposed here is the display of the peptide antigen in a native-like state on the surface of the virosome.

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