scholarly journals Invasion-Inhibitory Antibodies Elicited by Immunization with Plasmodium vivax Apical Membrane Antigen-1 Expressed in Pichia pastoris Yeast

2013 ◽  
Vol 82 (3) ◽  
pp. 1296-1307 ◽  
Author(s):  
Elaine C. Vicentin ◽  
Kátia S. Françoso ◽  
Mariana V. Rocha ◽  
Dmitri Iourtov ◽  
Fernanda L. dos Santos ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Recombinant Protein ◽  
Plasmodium Vivax ◽  
Apical Membrane ◽  
Content Type ◽  
Apical Membrane Antigen 1 ◽  
Apical Membrane Antigen ◽  
Monophosphoryl Lipid A ◽  
Antibody Titers ◽  
Inhibitory Antibodies

ABSTRACTIn a recent vaccine trial performed with African children, immunization with a recombinant protein based onPlasmodium falciparumapical membrane antigen 1 (AMA-1) conferred a significant degree of strain-specific resistance against malaria. To contribute to the efforts of generating a vaccine againstPlasmodium vivaxmalaria, we expressed the ectodomain ofP. vivaxAMA-1 (PvAMA-1) as a secreted soluble protein in the methylotrophic yeastPichia pastoris. Recognized by a high percentage of sera from individuals infected byP. vivax, this recombinant protein was found to have maintained its antigenicity. The immunogenicity of this protein was evaluated in mice using immunization protocols that included homologous and heterologous prime-boost strategies with plasmid DNA and recombinant protein. We used the following formulations containing different adjuvants: aluminum salts (Alum),Bordetella pertussismonophosphoryl lipid A (MPLA), flagellin FliC fromSalmonella entericaserovar Typhimurium, saponin Quil A, or incomplete Freund's adjuvant (IFA). The formulations containing the adjuvants Quil A or IFA elicited the highest IgG antibody titers. Significant antibody titers were also obtained using a formulation developed for human use containing MPLA or Alum plus MPLA. Recombinant PvAMA-1 produced under “conditions of good laboratory practice” provided a good yield, high purity, low endotoxin levels, and no microbial contaminants and reproduced the experimental immunizations. Most relevant for vaccine development was the fact that immunization with PvAMA-1 elicited invasion-inhibitory antibodies against different Asian isolates ofP. vivax. Our results show that AMA-1 expressed inP. pastorisis a promising antigen for use in future preclinical and clinical studies.

scholarly journals High-Level Expression of Plasmodium vivax Apical Membrane Antigen 1 (AMA-1) in Pichia pastoris: Strong Immunogenicity in Macaca mulatta Immunized with P. vivax AMA-1 and Adjuvant SBAS2

1999 ◽  
Vol 67 (1) ◽  
pp. 43-49 ◽  
Author(s):  
Clemens H. M. Kocken ◽  
Martin A. Dubbeld ◽  
Annemarie Van Der Wel ◽  
Jack T. Pronk ◽  
Andrew P. Waters ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Plasmodium Vivax ◽  
Apical Membrane ◽  
Gel Filtration ◽  
Methylotrophic Yeast ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
Apical Membrane Antigen 1 ◽  
Apical Membrane Antigen ◽  
High Level

ABSTRACT The apical membrane antigen 1 (AMA-1) family is a promising family of malaria blood-stage vaccine candidates that have induced protection in rodent and nonhuman primate models of malaria. Correct conformation of the protein appears to be essential for the induction of parasite-inhibitory responses, and these responses appear to be primarily antibody mediated. Here we describe for the first time high-level secreted expression (over 50 mg/liter) of thePlasmodium vivax AMA-1 (PV66/AMA-1) ectodomain by using the methylotrophic yeast Pichia pastoris. To prevent nonnative glycosylation, a conservatively mutagenized PV66/AMA-1 gene (PV66Δglyc) lacking N-glycosylation sites was also developed. Expression of the PV66Δglyc ectodomain yielded similar levels of a homogeneous product that was nonglycosylated and was readily purified by ion-exchange and gel filtration chromatographies. Recombinant PV66Δglyc43–487 was reactive with conformation-dependent monoclonal antibodies. With the SBAS2 adjuvant,Pichia-expressed PV66Δglyc43–487 was highly immunogenic in five rhesus monkeys, inducing immunoglobulin G enzyme-linked immunosorbent assay titers in excess of 1:200,000. This group of monkeys had a weak trend showing lower cumulative parasite loads following a Plasmodium cynomolgi infection than in the control group.

scholarly journals Long-Term Humoral and Cellular Immune Responses Elicited by a Heterologous Plasmodium vivax Apical Membrane Antigen 1 Protein Prime/Adenovirus Boost Immunization Protocol

2011 ◽  
Vol 79 (9) ◽  
pp. 3642-3652 ◽  
Author(s):  
Leoneide Érica Maduro Bouillet ◽  
Mariana Oliveira Dias ◽  
Natália Alves Dorigo ◽  
Andrew Douglas Moura ◽  
Bruce Russell ◽  
...  
Keyword(s):  
Immune Responses ◽  
Plasmodium Vivax ◽  
Apical Membrane ◽  
Proliferating Cells ◽  
Content Type ◽  
Apical Membrane Antigen 1 ◽  
Cellular Immune Responses ◽  
Apical Membrane Antigen ◽  
Boost Immunization ◽  
Cellular Immune

ABSTRACTApical membrane antigen 1 (AMA-1) is an invasion-relatedPlasmodiumantigen that is expressed during both intracellular and extracellular asexual stages of the parasite's life cycle, making it an ideal target for induction of humoral and cellular immune responses that can protect against malaria. We show here that when it is administered as a recombinant protein (P) in Montanide ISA720 adjuvant, followed by a recombinant human type 5 adenovirus (Ad), intense and long-lastingPlasmodium vivaxAMA-1-specific antibody responses (including both IgG1 and IgG2a), as well as proliferative memory T cell responses, can be detected in immunized mice. Memory T cells displayed both central (CD44hiCD62Lhi) and effector (CD44hiCD62Llo) phenotypes, with the central memory phenotype prevailing (56% of AMA-1-specific proliferating cells). Considering the main traits of the memory immune responses induced against AMA-1, this particular sequence of immunogens (P followed by Ad), but no others (Ad/Ad, Ad/P, or P/P), displayed an optimal synergistic effect. These results give further support to the need for preclinical studies ofP. vivaxvaccine candidate AMA-1 administered in prime/boost protocols that include recombinant proteins and adenoviral vectors.

scholarly journals Expression, Purification, and Biological Characterization of Babesia microti Apical Membrane Antigen 1

2015 ◽  
Vol 83 (10) ◽  
pp. 3890-3901 ◽  
Author(s):  
Prasun Moitra ◽  
Hong Zheng ◽  
Vivek Anantharaman ◽  
Rajdeep Banerjee ◽  
Kazuyo Takeda ◽  
...  
Keyword(s):  
Apical Membrane ◽  
The United States ◽  
Host Cells ◽  
Health Concern ◽  
Babesia Microti ◽  
Type I ◽  
Content Type ◽  
Apical Membrane Antigen 1 ◽  
Apical Membrane Antigen ◽  
Extracellular Region

The intraerythrocytic apicomplexanBabesia microti, the primary causative agent of human babesiosis, is a major public health concern in the United States and elsewhere. Apicomplexans utilize a multiprotein complex that includes a type I membrane protein called apical membrane antigen 1 (AMA1) to invade host cells. We have isolated the full-lengthB. microtiAMA1 (BmAMA1) gene and determined its nucleotide sequence, as well as the amino acid sequence of the AMA1 protein. This protein contains an N-terminal signal sequence, an extracellular region, a transmembrane region, and a short conserved cytoplasmic tail. It shows the same domain organization as the AMA1 orthologs from piroplasm, coccidian, and haemosporidian apicomplexans but differs from all other currently known piroplasmida, including otherBabesiaandTheileriaspecies, in lacking two conserved cysteines in highly variable domain III of the extracellular region. Minimal polymorphism was detected in BmAMA1 gene sequences of parasite isolates from six babesiosis patients from Nantucket. Immunofluorescence microscopy studies showed that BmAMA1 is localized on the cell surface and cytoplasm near the apical end of the parasite. Native BmAMA1 from parasite lysate and refolded recombinant BmAMA1 (rBmAMA1) expressed inEscherichia colireacted with a mouse anti-BmAMA1 antibody using Western blotting.In vitrobinding studies showed that both native BmAMA1 and rBmAMA1 bind to human red blood cells (RBCs). This binding is trypsin and chymotrypsin treatment sensitive but neuraminidase independent. Incubation ofB. microtiparasites in human RBCs with a mouse anti-BmAMA1 antibody inhibited parasite growth by 80% in a 24-h assay. Based on its antigenically conserved nature and potential role in RBC invasion, BmAMA1 should be evaluated as a vaccine candidate.

Analysis of the Plasmodium vivax apical membrane antigen-1 gene from re-emerging Korean isolates

2003 ◽  
Vol 90 (4) ◽  
pp. 325-329 ◽  
Author(s):  
Joon-Yong Chung ◽  
Eui-Hyun Chun ◽  
Jin-Ho Chun ◽  
Weon-Gyu Kho
Keyword(s):  
Plasmodium Vivax ◽  
Apical Membrane ◽  
Apical Membrane Antigen 1 ◽  
Apical Membrane Antigen

scholarly journals Natural Human Antibody Responses to Plasmodium vivax Apical Membrane Antigen 1 under Low Transmission and Unstable Malaria Conditions in Sri Lanka

2006 ◽  
Vol 74 (1) ◽  
pp. 798-801 ◽  
Author(s):  
Thilan Wickramarachchi ◽  
Prasad H. Premaratne ◽  
K. L. R. Lakshman Perera ◽  
Sumith Bandara ◽  
Clemens H. M. Kocken ◽  
...  
Keyword(s):  
Sri Lanka ◽  
Plasmodium Vivax ◽  
Malaria Vaccine ◽  
Apical Membrane ◽  
Human Antibody ◽  
Antibody Prevalence ◽  
Low Transmission ◽  
Apical Membrane Antigen 1 ◽  
Apical Membrane Antigen ◽  
Malaria Vaccine Candidate

ABSTRACT Plasmodium vivax apical membrane antigen 1, an important malaria vaccine candidate, was immunogenic during natural malaria infections in Sri Lanka, where low transmission and unstable malaria conditions prevail. Antibody prevalence increased with exposure in areas where malaria was or was not endemic. A marked isotype switch to cytophilic (immunoglobulin G1 [IgG1]/IgG3) antibodies was evident with increasing exposure exclusively in residents from areas of endemicity.

scholarly journals Identification of a Highly Antigenic Linear B Cell Epitope within Plasmodium vivax Apical Membrane Antigen 1 (AMA-1)

PLoS ONE ◽  
2011 ◽  
Vol 6 (6) ◽  
pp. e21289 ◽  
Author(s):  
Lilian Lacerda Bueno ◽  
Francisco Pereira Lobo ◽  
Cristiane Guimarães Morais ◽  
Luíza Carvalho Mourão ◽  
Ricardo Andrez Machado de Ávila ◽  
...  
Keyword(s):  
B Cell ◽  
Plasmodium Vivax ◽  
Apical Membrane ◽  
Cell Epitope ◽  
Apical Membrane Antigen 1 ◽  
Apical Membrane Antigen ◽  
B Cell Epitope ◽  
Linear B

Evaluation of the genetic diversity of domain II of Plasmodium vivax Apical Membrane Antigen 1 (PvAMA-1) and the ensuing strain-specific immune responses in patients from Sri Lanka

Vaccine ◽  
2011 ◽  
Vol 29 (43) ◽  
pp. 7491-7504 ◽  
Author(s):  
Sajani Dias ◽  
Maheshika Somarathna ◽  
Aresha Manamperi ◽  
Ananias A. Escalante ◽  
Anusha M. Gunasekera ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Sri Lanka ◽  
Immune Responses ◽  
Plasmodium Vivax ◽  
Apical Membrane ◽  
Apical Membrane Antigen 1 ◽  
Apical Membrane Antigen

scholarly journals Genetic Diversity and Selection at the Plasmodium vivax Apical Membrane Antigen-1 (PvAMA-1) Locus in a Sri Lankan Population

2007 ◽  
Vol 24 (4) ◽  
pp. 939-947 ◽  
Author(s):  
A. M. Gunasekera ◽  
T. Wickramarachchi ◽  
D. E. Neafsey ◽  
I. Ganguli ◽  
L. Perera ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Plasmodium Vivax ◽  
Apical Membrane ◽  
Sri Lankan ◽  
Apical Membrane Antigen 1 ◽  
Apical Membrane Antigen

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.

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