Tresa S. Fraser

Naturally Acquired and Vaccine-Elicited Antibodies Block Erythrocyte Cytoadherence of the Plasmodium vivax Duffy Binding Protein

ABSTRACT Malaria merozoites require the presence of specific surface receptors on the red blood cell for invasion. Plasmodium vivax, requires the Duffy blood group antigen as an obligate receptor for invasion. The parasite Duffy binding protein (DBP) is the ligand involved in this process, making the DBP a potential vaccine candidate. A preliminary objective was to study whether people exposed to vivax malaria acquire antibodies that have the ability to block erythrocyte cytoadherence to the PvDBP. In comparison, we studied the immunogenicity of various recombinant DBP vaccines and investigated their potential to induct antifunctional antibodies. In order to do so, recombinant proteins to different regions of the putative ectodomain of the DBP and a DNA vaccine were used to immunize laboratory animals. An in vitro cytoadherence assay was used to investigate the presence of antifunctional antibodies in plasmas from people naturally exposed to vivax malaria, as well as in antisera obtained by animal vaccination. Our results showed that human plasma from populations naturally exposed to vivax malaria, as well as antisera obtained by vaccination using recombinant proteins, a DNA vaccine, and a synthetic peptide to DBP, inhibited in vitro binding of human erythrocytes to the DBP ligand domain (DBPII) in correlation to their previously measured antibody titer. Our results provide further evidence for the vaccine potential of this essential parasite adhesion molecule.

Erythrocyte-binding activity of Plasmodium yoelii apical membrane antigen-1 expressed on the surface of transfected COS-7 cells.

Malaria merozoite surface and apical organellar molecules facilitate invasion into the host erythrocyte. The underlying molecular mechanisms of invasion are poorly understood, and there are few data to delineate roles for individual merozoite proteins. Apical membrane antigen-1 (AMA-1) is a conserved apicomplexan protein present in the apical organelle complex and at times on the surface of Plasmodium and Toxoplasma zoites. AMA-1 domains 1/2 are conserved between Plasmodium and Toxoplasma and have similarity to the defined ligand domains of MAEBL, an erythrocyte-binding protein identified from Plasmodium yoelii. We expressed selected portions of the AMA-1 extracellular domain on the surface of COS-7 cells to assay for erythrocyte-binding activity. The P. yoelii AMA-1 domains 1/2 mediated adhesion to mouse and rat erythrocytes, but not to human erythrocytes. Adhesion to rodent erythrocytes was sensitive to trypsin and chymotrypsin, but not to neuraminidase. Other parts of the AMA-1 ectodomain, including the full-length extracellular domain, mediated significantly less erythrocyte adhesion activity than the contiguous domains 1/2. The results support the role of AMA-1 as an adhesion molecule during merozoite invasion of erythrocytes and identify highly conserved domains 1/2 as the principal ligand of the Plasmodium AMA-1 and possibly the Toxoplasma AMA-1. Identification of the AMA-1 ligand domains involved in interaction between the parasite and host cell should help target the development of new therapies to block growth of the blood-stage malaria parasites.

Mapping Epitopes of the Plasmodium vivax Duffy Binding Protein with Naturally Acquired Inhibitory Antibodies

ABSTRACT Plasmodium vivax Duffy binding protein (DBP) is a merozoite microneme ligand vital for blood-stage infection, which makes it an important candidate vaccine for antibody-mediated immunity against vivax malaria. A differential screen with a linear peptide array compared the reactivities of noninhibitory and inhibitory high-titer human immune sera to identify target epitopes associated with protective immunity. Naturally acquired anti-DBP-specific serologic responses observed in the residents of a region of Papua New Guinea where P. vivax is highly endemic exhibited significant changes in DBP-specific titers over time. The anti-DBP functional inhibition for each serum ranged from complete inhibition to no inhibition even for high-titer responders to the DBP, indicating that epitope specificity is important. Inhibitory immune human antibodies identified specific B-cell linear epitopes on the DBP (SalI) ligand domain that showed significant correlations with inhibitory responses. Affinity-purified naturally acquired antibodies on these epitopes inhibited the DBP erythrocyte binding function greatly, confirming the protective value of specific epitopes. These results represent an important advance in our understanding of part of blood-stage immunity to P. vivax and some of the specific targets for vaccine-elicited antibody protection.

Interplasmid transposition demonstrates piggyBac mobility in vertebrate species.

The piggyBac transposon is an extremely versatile helper-dependent vector for gene transfer and germ line transformation in a wide range of invertebrate species. Analyses of genome sequencing databases have identified piggyBac homologues among several sequenced animal genomes, including the human genome. In this report we demonstrate that this insect transposon is capable of transposition in primate cells and embryos of the zebrafish, Danio rerio. piggyBac mobility was demonstrated using an interplasmid transposition assay that has consistently predicted the germ line transformation capabilities of this mobile element in several other species. Both transfected COS-7 primate cells and injected zebrafish embryos supported the helper-dependent movement of tagged piggyBac element between plasmids in the characteristic cut-and-paste, TTAA target-site specific manner. These results validate piggyBac as a valuable tool for genetic analysis of vertebrates.

Effective suppression of Dengue fever virus in mosquito cell cultures using retroviral transduction of hammerhead ribozymes targeting the viral genome

Outbreaks of Dengue impose a heavy economic burden on developing countries in terms of vector control and human morbidity. Effective vaccines against all four serotypes of Dengue are in development, but population replacement with transgenic vectors unable to transmit the virus might ultimately prove to be an effective approach to disease suppression, or even eradication. A key element of the refractory transgenic vector approach is the development of transgenes that effectively prohibit viral transmission. In this report we test the effectiveness of several hammerhead ribozymes for suppressing DENV in lentivirus-transduced mosquito cells in an attempt to mimic the transgenic use of these effector molecules in mosquitoes. A lentivirus vector that expresses these ribozymes as a fusion RNA molecule using an Ae. aegypti tRNAval promoter and terminating with a 60A tail insures optimal expression, localization, and activity of the hammerhead ribozyme against the DENV genome. Among the 14 hammerhead ribozymes we designed to attack the DENV-2 NGC genome, several appear to be relatively effective in reducing virus production from transduced cells by as much as 2 logs. Among the sequences targeted are 10 that are conserved among all DENV serotype 2 strains. Our results confirm that hammerhead ribozymes can be effective in suppressing DENV in a transgenic approach, and provide an alternative or supplementary approach to proposed siRNA strategies for DENV suppression in transgenic mosquitoes.

Analysis of the piggyBac transposase reveals a functional nuclear targeting signal in the 94 c-terminal residues

BackgroundThe piggyBac transposable element is a popular tool for germ-line transgenesis of eukaryotes. Despite this, little is known about the mechanism of transposition or the transposase (TPase) itself. A thorough understanding of just how piggyBac works may lead to more effective use of this important mobile element. A PSORTII analysis of the TPase amino acid sequence predicts a bipartite nuclear localization signal (NLS) near the c-terminus, just upstream of a putative ZnF (ZnF).ResultsWe fused the piggyBac TPase upstream of and in-frame with the enhanced yellow fluorescent protein (EYFP) in the Drosophila melanogaster inducible metallothionein protein. Using Drosophila Schneider 2 (S2) cells and the deep red fluorescent nuclear stain Draq5, we were able to track the pattern of piggyBac localization with a scanning confocal microscope 48 hours after induction with copper sulphate.ConclusionThrough n and c-terminal truncations, targeted internal deletions, and specific amino acid mutations of the piggyBac TPase open reading frame, we found that not only is the PSORTII-predicted NLS required for the TPase to enter the nucleus of S2 cells, but there are additional requirements for negatively charged amino acids a short length upstream of this region for nuclear localization.

A novel dengue virus detection method that couples DNAzyme and gold nanoparticle approaches

BackgroundRecent epidemics of dengue viruses (DENV) coupled with new outbreaks on the horizon have renewed the demand for novel detection methods that have the ability to identify this viral pathogen prior to the manifestation of symptoms. The ability to detect DENV in a timely manner is essential for rapid recovery from disease symptoms. A modified lab-derived 10-23 DNAzyme tethered to gold nanoparticles provides a powerful tool for the detection of viruses, such as DENV.ResultsWe examined the effectiveness of coupling DNAzyme (DDZ) activation to the salt-induced aggregation of gold nanoparticles (AuNP) to detect dengue virus (DENV) progeny in mosquito cells. A DNAzyme was designed to recognize the 5’ cyclization sequence (5’ CS) that is conserved among all DENV, and conjugated to AuNPs. DDZ-AuNP has demonstrated the ability to detect the genomic RNA of our model dengue strain, DENV-2 NGC, isolated from infected Aedes albopictus C6/36 cells. These targeting events lead to the rapid aggregation of AuNPs, resulting in a red to clear color transition of the reaction mixes, and thus positive detection of the DENV RNA genome. The inclusion of SDS in the reaction mixture permitted the detection of DENV directly from cell culture supernatants without additional sample processing. Specificity assays demonstrated detection is DENV-specific, while sensitivity assays confirm detection at levels of 1 × 101 TCID50 units. These results demonstrate DDZ-AuNP effectively detects DENV genomes in a sequence specific manner and at concentrations that are practical for field use.ConclusionsWe have developed an effective detection assay using DNAzyme catalysis coupled with AuNP aggregation for the detection of DENV genomes in a sequence specific manner. Full development of our novel DDZ-AuNP detection method will provide a practical, rapid, and low cost alternative for the detection of DENV in mosquito cells and tissues, and possibly infected patient serum, in a matter of minutes with little to no specialized training required.

Effective suppression of Dengue virus using a novel group-I intron that induces apoptotic cell death upon infection through conditional expression of the Bax C-terminal domain

IntroductionApproximately 100 million confirmed infections and 20,000 deaths are caused by Dengue virus (DENV) outbreaks annually. Global warming and rapid dispersal have resulted in DENV epidemics in formally non-endemic regions. Currently no consistently effective preventive measures for DENV exist, prompting development of transgenic and paratransgenic vector control approaches. Production of transgenic mosquitoes refractory for virus infection and/or transmission is contingent upon defining antiviral genes that have low probability for allowing escape mutations, and are equally effective against multiple serotypes. Previously we demonstrated the effectiveness of an anti-viral group I intron targeting U143 of the DENV genome in mediating trans-splicing and expression of a marker gene with the capsid coding domain. In this report we examine the effectiveness of coupling expression of ΔN Bax to trans-splicing U143 intron activity as a means of suppressing DENV infection of mosquito cells.ResultsTargeting the conserved DENV circularization sequence (CS) by U143 intron trans-splicing activity appends a 3’ exon RNA encoding ΔN Bax to the capsid coding region of the genomic RNA, resulting in a chimeric protein that induces premature cell death upon infection. TCID50-IFA analyses demonstrate an enhancement of DENV suppression for all DENV serotypes tested over the identical group I intron coupled with the non-apoptotic inducing firefly luciferase as the 3’ exon. These cumulative results confirm the increased effectiveness of this αDENV-U143-ΔN Bax group I intron as a sequence specific antiviral that should be useful for suppression of DENV in transgenic mosquitoes. Annexin V staining, caspase 3 assays, and DNA ladder observations confirm DCA-ΔN Bax fusion protein expression induces apoptotic cell death.ConclusionThis report confirms the relative effectiveness of an anti-DENV group I intron coupled to an apoptosis-inducing ΔN Bax 3’ exon that trans-splices conserved sequences of the 5’ CS region of all DENV serotypes and induces apoptotic cell death upon infection. Our results confirm coupling the targeted ribozyme capabilities of the group I intron with the generation of an apoptosis-inducing transcript increases the effectiveness of infection suppression, improving the prospects of this unique approach as a means of inducing transgenic refractoriness in mosquitoes for all serotypes of this important disease.