Vivax Apical Membrane Antigen-1 Research Articles

A recombinant vaccine based on domain II of Plasmodium vivax Apical Membrane Antigen 1 induces high antibody titres in mice

The Apical Membrane Antigen 1 (AMA-1) is considered a promising candidate for development of a malaria vaccine against asexual stages of Plasmodium. We recently identified domain II (DII) of Plasmodium vivax AMA-1 (PvAMA-1) as a highly immunogenic region recognised by IgG antibodies present in many individuals during patent infection with P. vivax. The present study was designed to evaluate the immunogenic properties of a bacterial recombinant protein containing PvAMA-1 DII. To accomplish this, the recombinant protein was administered to mice in the presence of each of the following six adjuvants: Complete/Incomplete Freund's Adjuvant (CFA/IFA), aluminium hydroxide (Alum), Quil A, QS21 saponin, CpG-ODN 1826 and TiterMax. We found that recombinant DII was highly immunogenic in BALB/c mice when administered in the presence of any of the tested adjuvants. Importantly, we show that DII-specific antibodies recognised the native AMA-1 protein expressed on the surface of P. vivax merozoites isolated from the blood of infected patients. These results demonstrate that a recombinant protein containing PvAMA-1 DII is immunogenic when administered in different adjuvant formulations, and indicate that this region of the AMA-1 protein should continue to be evaluated as part of a subunit vaccine against vivax malaria.

Structural Characterization of Apical Membrane Antigen 1 (AMA1) from Toxoplasma gondii

Apical membrane antigen 1 (AMA1) is an essential component of the moving junction complex used by Apicomplexan parasites to invade host cells. We report the 2.0 A resolution x-ray crystal structure of the full ectodomain (domains I, II, and III) of AMA1 from the pervasive protozoan parasite Toxoplasma gondii. The structure of T. gondii AMA1 (TgAMA1) is the most complete of any AMA1 structure to date, with more than 97.5% of the ectodomain unambiguously modeled. Comparative sequence analysis reveals discrete segments of divergence in TgAMA1 that map to areas of established functional importance in AMA1 from Plasmodium vivax (PvAMA1) and Plasmodium falciparum (PfAMA1). Inspection of the TgAMA1 structure reveals a network of apical surface loops, reorganized in both size and chemistry relative to PvAMA1/PfAMA1, that appear to serve as structural filters restricting access to a central hydrophobic groove. The terminal portion of this groove is formed by an extended loop from DII that is 14 residues shorter in TgAMA1. A pair of tryptophan residues (Trp(353) and Trp(354)) anchor the DII loop in the hydrophobic groove and frame a conserved tyrosine (Tyr(230)), forming a contiguous surface that may be critical for moving junction assembly. The minimalist DIII structure folds into a cystine knot that probably stabilizes and orients the bulk of the ectodmain without providing excess surface area to which invasion-inhibitory antibodies can be generated. The detailed structural characterization of TgAMA1 provides valuable insight into the mechanism of host cell invasion by T. gondii.

Plasmodium vivax apical membrane antigen-1: comparative recognition of different domains by antibodies induced during natural human infection

The Apical Membrane Antigen-1 (AMA-1) of Plasmodium sp. has been suggested as a vaccine candidate against malaria. This protein seems to be involved in merozoite invasion and its extra-cellular portion contains three distinct domains: DI, DII, and DIII. Previously, we described that Plasmodium vivax AMA-1 (PvAMA-1) ectodomain is highly immunogenic in natural human infections. Here, we expressed each domain, separately or in combination (DI-II or DII-III), as bacterial recombinant proteins to map immunodominant epitopes within the PvAMA-1 ectodomain. IgG recognition was assessed by ELISA using sera of P. vivax-infected individuals collected from endemic regions of Brazil or antibodies raised in immunized mice. The frequencies of responders to recombinant proteins containing the DII were higher than the others and similar to the ones observed against the PvAMA-1 ectodomain. Moreover, ELISA inhibition assays using the PvAMA-1 ectodomain as substrate revealed the presence of many common epitopes within DI-II that are recognized by human immune antibodies. Finally, immunization of mice with the PvAMA-1 ectodomain induced high levels of antibodies predominantly to DI-II. Together, our results indicate that DII is particularly immunogenic during natural human infections, thus indicating that this region could be used as part of an experimental sub-unit vaccine to prevent vivax malaria.

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

Plasmodium vivax apical membrane antigen 1 (PvAMA-1) is an important malaria vaccine candidate. We present the first comprehensive analysis of nucleotide diversity across the entire PvAMA-1 gene using a single population sample from Sri Lanka. In contrast to what has been observed at the AMA-1 locus of Plasmodium falciparum, the signature of diversifying selection is seen most strongly in Domain II of PvAMA-1, indicating that the different domains in each species may be subject to varying selective pressures and functional constraints. We also find that recombination plays an important role in generating haplotype diversity at this locus, even in a region of low endemicity such as Sri Lanka. Mapping of diversity and recombination hotspots onto a 3-dimensional structural model of the protein indicates that one surface of the molecule may be particularly likely to bear epitopes for antibody recognition. Regions of this surface that show constrained variability may prove to be promising vaccine targets.

Plasmodium vivax: Genetic diversity of the apical membrane antigen-1 (AMA-1) in isolates from India

Malaria parasites exhibit sequence diversity for a number of stage specific antigens. Several studies have proved that apical membrane antigen-1 (AMA-1) is an effective target for eliciting a protective immune response in humans and other experimental animals. We have investigated the sequence variation in Plasmodium vivax AMA-1 (Pv AMA-1) from different Indian isolates. This is the first study of its kind for the nearly full length Pv AMA-1 from India. Our analysis reveals greater degree of genetic diversity in Pv AMA-1 than reported so far and identifies five novel haplotypes. This is significant to establish the antigenic repertoire of isolates in a malaria endemic country like India.

Antibody response of naturally infected individuals to recombinant Plasmodium vivax apical membrane antigen-1

In the present study, we evaluate the naturally acquired antibody response to the Plasmodium vivax apical membrane antigen 1 (PvAMA-1), a leading vaccine candidate against malaria. The gene encoding the PvAMA-1 ectodomain region (amino acids 43–487) was cloned by PCR using genomic DNA from a Brazilian individual with patent P. vivax infection. The predicted amino acid sequence displayed a high degree of identity (97.3%) with a previously published sequence from the P. vivax Salvador strain. A recombinant protein representing the PvAMA-1 ectodomain was expressed in Escherichia coli and refolded. By ELISA, this recombinant protein reacted with 85 and 48.5% of the IgG or IgM antibodies, respectively, from Brazilian individuals with patent P. vivax malaria. IgG1 was the predominant subclass of IgG. The frequency of response increased according to the number of malaria episodes, reaching 100% in individuals in their fourth malaria episode. The high degree of recognition of PvAMA-1 by human antibodies was confirmed using a second recombinant protein expressed in Pichia pastoris (PV66/AMA-1). The observation that recognition of the bacterial recombinant PvAMA-1 was only slightly lower than that of the highly immunogenic 19 kDa C-terminal domain of the P. vivax Merozoite Surface Protein-1 was also important. DNA sequencing of the PvAMA-1 variable domain from 20 Brazilian isolates confirmed the limited polymorphism of PvAMA-1 suggested by serological analysis. In conclusion, we provide evidence that PvAMA-1 is highly immunogenic during natural infection in humans and displays limited polymorphism in Brazil. Based on these observations, we conclude that PvAMA-1 merits further immunological studies as a vaccine candidate against P. vivax malaria.

Plasmodium vivax synonymous substitution frequencies, evolution and population structure deduced from diversity in AMA 1 and MSP 1 genes

Polymorphic regions of the genes encoding Plasmodium vivax apical membrane antigen 1 ( PvAMA1) and P. vivax merozoite surface protein 1 ( PvMSP1) were sequenced to examine population diversity both within and between geographical areas. Sequences were obtained for 219 isolates for PvAMA1 and for 175 isolates for PvMSP1 from Africa, China, India, Indonesia, Philippines, Papua New Guinea, Solomon Islands and Thailand. Over half of the isolates were obtained from different regions within the Philippines, and this was used to look at the diversity within a country. Sixty nine haplotypes and 22 polymorphic sites in a 414-bp region of PvAMA1 and 41 haplotypes and 34 polymorphic sites in a 249-bp fragment of PvMSP1 were detected. For both PvAMA1 and PvMSP1, four previously unreported polymorphic nucleotide positions were identified. Population analysis indicated that there were significant differences in allele frequencies between different regions but these differences were small compared to the diversity within populations (Fixation index, F ST, of 0.126 and 0.078 for PvAMA1 and PvMSP1, respectively). PvAMA1 and PvMSP1 had similar nonsynonymous substitution frequencies but surprisingly, the synonymous substitution frequency for PvMSP1 was eight times the frequency for PvAMA1 suggesting that synonymous substitutions in at least PvAMA1 are not neutral.