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 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 Overcoming Allelic Specificity by Immunization with Five Allelic Forms of Plasmodium falciparum Apical Membrane Antigen 1

2013 ◽  
Vol 81 (5) ◽  
pp. 1491-1501 ◽  
Author(s):  
Kazutoyo Miura ◽  
Raul Herrera ◽  
Ababacar Diouf ◽  
Hong Zhou ◽  
Jianbing Mu ◽  
...  
Keyword(s):  
Rational Design ◽  
Vaccine Development ◽  
Apical Membrane ◽  
Enzyme Linked Immunosorbent Assay ◽  
Allelic Polymorphism ◽  
Apical Membrane Antigen 1 ◽  
Percent Inhibition ◽  
Apical Membrane Antigen ◽  
Single Allele ◽  
Mixture Group

ABSTRACTApical membrane antigen 1 (AMA1) is a leading vaccine candidate, but the allelic polymorphism is a stumbling block for vaccine development. We previously showed that a global set of AMA1 haplotypes could be grouped into six genetic populations. Using this information, six recombinant AMA1 proteins representing each population were produced. Rabbits were immunized with either a single recombinant AMA1 protein or mixtures of recombinant AMA1 proteins (mixtures of 4, 5, or 6 AMA1 proteins). Antibody levels were measured by enzyme-linked immunosorbent assay (ELISA), and purified IgG from each rabbit was used for growth inhibition assay (GIA) with 12 different clones of parasites (a total of 108 immunogen-parasite combinations). Levels of antibodies to all six AMA1 proteins were similar when the antibodies were tested against homologous antigens. When the percent inhibitions in GIA were plotted against the number of ELISA units measured with homologous AMA1, all data points followed a sigmoid curve, regardless of the immunogen. In homologous combinations, there were no differences in the percent inhibition between the single-allele and allele mixture groups. However, all allele mixture groups showed significantly higher percent inhibition than the single-allele groups in heterologous combinations. The 5-allele-mixture group showed significantly higher inhibition to heterologous parasites than the 4-allele-mixture group. On the other hand, there was no difference between the 5- and 6-allele-mixture groups. These data indicate that mixtures with a limited number of alleles may cover a majority of the parasite population. In addition, using the data from 72 immunogen-parasite combinations, we mathematically identified 13 amino acid polymorphic sites which significantly impact GIA activities. These results could be a foundation for the rational design of a future AMA1 vaccine.

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 Structural and immunological characterization of an epitope within the PAN motif of ectodomain I in Babesia bovis apical membrane antigen 1 for vaccine development

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11765
Author(s):  
Amarin Rittipornlertrak ◽  
Boondarika Nambooppha ◽  
Anucha Muenthaisong ◽  
Veerasak Punyapornwithaya ◽  
Saruda Tiwananthagorn ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Apical Membrane ◽  
Rabbit Antiserum ◽  
Babesia Bovis ◽  
Inhibitory Effects ◽  
Bovine Babesiosis ◽  
Apical Membrane Antigen 1 ◽  
Apical Membrane Antigen ◽  
Domain I

Background Bovine babesiosis caused by Babesia bovis (B. bovis) has had a significant effect on the mobility and mortality rates of the cattle industry worldwide. Live-attenuated vaccines are currently being used in many endemic countries, but their wide use has been limited for a number of reasons. Although recombinant vaccines have been proposed as an alternative to live vaccines, such vaccines are not commercially available to date. Apical membrane antigen-1 (AMA-1) is one of the leading candidates in the development of a vaccine against diseases caused by apicomplexan parasite species. In Plasmodium falciparum (P. falciparum) AMA-1 (PfAMA-1), several antibodies against epitopes in the plasminogen, apple, and nematode (PAN) motif of PfAMA-1 domain I significantly inhibited parasite growth. Therefore, the purpose of this study was to predict an epitope from the PAN motif of domain I in the B. bovis AMA-1 (BbAMA-1) using a combination of linear and conformational B-cell epitope prediction software. The selected epitope was then bioinformatically analyzed, synthesized as a peptide (sBbAMA-1), and then used to immunize a rabbit. Subsequently, in vitro growth- and the invasion-inhibitory effects of the rabbit antiserum were immunologically characterized. Results Our results demonstrated that the predicted BbAMA-1 epitope was located on the surface-exposed α-helix of the PAN motif in domain I at the apex area between residues 181 and 230 with six polymorphic sites. Subsequently, sBbAMA-1 elicited antibodies capable of recognizing the native BbAMA-1 in immunoassays. Furthermore, anti-serum against sBbAMA-1 was immunologically evaluated for its growth- and invasion-inhibitory effects on B. bovis merozoites in vitro. Our results demonstrated that the rabbit anti-sBbAMA-1 serum at a dilution of 1:5 significantly inhibited (p < 0.05) the growth of B. bovis merozoites by approximately 50–70% on days 3 and 4 of cultivation, along with the invasion of merozoites by approximately 60% within 4 h of incubation when compared to the control groups. Conclusion Our results indicate that the epitope predicted from the PAN motif of BbAMA-1 domain I is neutralization-sensitive and may serve as a target antigen for vaccine development against bovine babesiosis caused by B. bovis.

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 Structural and immunological characterization of an epitope within the PAN motif of ectodomain I in Babesia bovis apical membrane antigen 1

2020 ◽  
Author(s):  
Amarin Rittipornlertrak ◽  
Boondarika Nambooppha ◽  
Anucha Muenthaisong ◽  
Veerasak Punyapornwithaya ◽  
Saruda Tiwananthagorn ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Apical Membrane ◽  
Cell Epitope ◽  
Babesia Bovis ◽  
Target Antigen ◽  
Bovine Babesiosis ◽  
Apical Membrane Antigen 1 ◽  
Apical Membrane Antigen ◽  
Domain I

Abstract Background Bovine babesiosis caused by Babesia bovis (B. bovis) affects the cattle industry worldwide with high mobility and mortality. Live-attenuated vaccines are currently used in some of the endemic countries, but their wide use is limited due to various reasons. Although recombinant vaccines have been proposed as an alternative to the live vaccines, such vaccines are not commercially available to date. Apical membrane antigen-1 (AMA-1) is one of the leading candidates for vaccine development against diseases caused by apicomplexan parasite species. In this study, we predicted an epitope from the plasminogen, apple and nematode (PAN) motif of domain I in the B. bovis AMA-1 (BbAMA-1) using a combination of linear and conformational B-cell epitope prediction software. The selected epitope was bioinformatically analyzed, synthesized as a peptide (sBbAMA-1), and then used to immunize a rabbit. Results The anti-sBbAMA-1 serum obtained was evaluated for its growth- and invasion-inhibitory effects on B. bovis merozoites in vitro. Our results demonstrated that the predicted BbAMA-1 epitope, which is located on surface-exposed α-helix of PAN motif in domain I at the apex area, elicits antibodies capable of recognizing the native BbAMA-1 in immunoassays. Importantly, as compared to the control groups, the rabbit anti-sBbAMA-1 serum at dilution of 1:5 significantly inhibited (p < 0.05) the growth of B. bovis merozoites by approximately 50–70% on day 3 and 4 of cultivation and the invasion of merozoites by approximately 60% within 4 h of incubation. Conclusion Our results indicate the epitope predicted from the PAN motif of BbAMA-1 domain I is neutralization-sensitive and may serve as a target antigen for vaccine development against bovine babesiosis caused by B. bovis.

scholarly journals Vaccination of Monkeys with Recombinant Plasmodium falciparum Apical Membrane Antigen 1 Confers Protection against Blood-Stage Malaria

2002 ◽  
Vol 70 (12) ◽  
pp. 6961-6967 ◽  
Author(s):  
Anthony W. Stowers ◽  
Michael C. Kennedy ◽  
Brian P. Keegan ◽  
Allan Saul ◽  
Carole A. Long ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Malaria Vaccine ◽  
Vaccine Development ◽  
Apical Membrane ◽  
Surface Protein ◽  
Merozoite Surface Protein ◽  
Lethal Challenge ◽  
Apical Membrane Antigen 1 ◽  
Apical Membrane Antigen

ABSTRACT A major challenge facing malaria vaccine development programs is identifying efficacious combinations of antigens. To date, merozoite surface protein 1 (MSP1) is regarded as the leading asexual vaccine candidate. Apical membrane antigen 1 (AMA1) has been identified as another leading candidate for an asexual malaria vaccine, but without any direct in vivo evidence that a recombinant form of Plasmodium falciparum AMA1 would have efficacy. We evaluated the efficacy of a form of P. falciparum AMA1, produced in Pichia pastoris, by vaccinating Aotus vociferans monkeys and then challenging them with P. falciparum parasites. Significant protection from this otherwise lethal challenge with P. falciparum was observed. Five of six animals had delayed patency; two of these remained subpatent for the course of the infection, and two controlled parasite growth at <0.75% of red blood cells parasitized. The protection induced by AMA1 was superior to that obtained with a form of MSP1 used in the same trial. The protection induced by a combination vaccine of AMA1 and MSP1 was not superior to the protection obtained with AMA1 alone, although the immunity generated appeared to operate against both vaccine components.

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.

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