Development of effective malaria vaccines is hampered by the problem of producing correctly folded Plasmodium proteins for use as vaccine components. We have investigated the use of a novel ciliate ...expression system, Tetrahymena thermophila, as a P. falciparum vaccine antigen platform. A synthetic vaccine antigen composed of N-terminal and C-terminal regions of merozoite surface protein-1 (MSP-1) was expressed in Tetrahymena thermophila. The recombinant antigen was secreted into the culture medium and purified by monoclonal antibody (mAb) affinity chromatography. The vaccine was immunogenic in MF1 mice, eliciting high antibody titers against both N- and C-terminal components. Sera from immunized animals reacted strongly with P. falciparum parasites from three antigenically different strains by immunofluorescence assays, confirming that the antibodies produced are able to recognize parasite antigens in their native form. Epitope mapping of serum reactivity with a peptide library derived from all three MSP-1 Block 2 serotypes confirmed that the MSP-1 Block 2 hybrid component of the vaccine had effectively targeted all three serotypes of this polymorphic region of MSP-1. This study has successfully demonstrated the use of Tetrahymena thermophila as a recombinant protein expression platform for the production of malaria vaccine antigens.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Polymorphic parasite antigens are known targets of protective immunity to malaria, but this antigenic variation poses challenges to vaccine development. A synthetic MSP-1 Block 2 construct, based on ...all polymorphic variants found in natural Plasmodium falciparum isolates has been designed, combined with the relatively conserved Block 1 sequence of MSP-1 and expressed in E.coli. The MSP-1 Hybrid antigen has been produced with high yield by fed-batch fermentation and purified without the aid of affinity tags resulting in a pure and extremely thermostable antigen preparation. MSP-1 hybrid is immunogenic in experimental animals using adjuvants suitable for human use, eliciting antibodies against epitopes from all three Block 2 serotypes. Human serum antibodies from Africans naturally exposed to malaria reacted to the MSP-1 hybrid as strongly as, or better than the same serum reactivities to individual MSP-1 Block 2 antigens, and these antibody responses showed clear associations with reduced incidence of malaria episodes. The MSP-1 hybrid is designed to induce a protective antibody response to the highly polymorphic Block 2 region of MSP-1, enhancing the repertoire of MSP-1 Block 2 antibody responses found among immune and semi-immune individuals in malaria endemic areas. The target population for such a vaccine is young children and vulnerable adults, to accelerate the acquisition of a full range of malaria protective antibodies against this polymorphic parasite antigen.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The ecology of
Anopheles arabiensis and its relationship to malaria transmission was investigated in two villages in eastern Sudan. Seasonal malaria case incidence was compared with the number of ...vectors detected and with climatic variables. Following the end of the short rainy season in October the number of
A. arabiensis detected dropped gradually until February when neither outdoor human bait trapping nor indoor spray catches revealed any mosquitoes. Vectors re-appeared in June as humidity rose with the onset of rain. Despite the apparent absence of the vector at the height of the long, hot dry season between February and May, sporadic asymptomatic malaria infections were detected in the two villages. The low endemicity of malaria in the area was reflected by the relatively low total September–December parasite and sporozoite rates (15 and 1.4%, respectively) measured in the villages. The entomological inoculation rate (EIR) was estimated to be around two to three infective bites per person per year, although heterogeneity in the transmission indices of malaria between the two villages was observed. The implications of these patterns of anopheline population dynamics for the epidemiology and control of malaria in eastern Sudan are considered.
Improved
Plasmodium falciparum cDNA expression libraries were constructed by combining mRNA oligo-capping with in vitro recombination and directional cloning of cDNA inserts into a plasmid vector ...that expresses sequences as thioredoxin fusion proteins. A novel procedure has also been developed for the rapid identification of seropositive clones on high-density filters, using direct labelling of
P. falciparum immune immunoglobulin with fluorescein isothiocynate (FITC). This approach combines the advantages of recombination-assisted cDNA cloning with high throughput, non-radioactive serological screening of expression libraries. Production of replicate colony matrices allows the identification of antigens recognised by different pools with different specificities from residents of a malaria endemic region. Analyses of DNA sequences derived from sero-reactive colonies indicate that this is an effective method for producing recombinant proteins that react with antibodies from malaria-exposed individuals. This approach permits the systematic construction of a database of antigenic proteins recognised by sera from malaria-exposed individuals.
Development of effective malaria vaccines is hampered by the problem of producing correctly folded Plasmodium proteins for use as vaccine components. We have investigated the use of a novel ciliate ...expression system, Tetrahymena thermophila, as a P. falciparum vaccine antigen platform. A synthetic vaccine antigen composed of N-terminal and C-terminal regions of merozoite surface protein-1 (MSP-1) was expressed in Tetrahymena thermophila. The recombinant antigen was secreted into the culture medium and purified by monoclonal antibody (mAb) affinity chromatography. The vaccine was immunogenic in MF1 mice, eliciting high antibody titers against both N- and C-terminal components. Sera from immunized animals reacted strongly with P. falciparum parasites from three antigenically different strains by immunofluorescence assays, confirming that the antibodies produced are able to recognize parasite antigens in their native form. Epitope mapping of serum reactivity with a peptide library derived from all three MSP-1 Block 2 serotypes confirmed that the MSP-1 Block 2 hybrid component of the vaccine had effectively targeted all three serotypes of this polymorphic region of MSP-1. This study has successfully demonstrated the use of Tetrahymena thermophila as a recombinant protein expression platform for the production of malaria vaccine antigens.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The inheritance of an extrachromosomal 6-kb element has been examined in the human malaria parasite Plasmodium falciparum. A single base pair difference in the cytochrome b gene from the 6-kb element ...of two different cloned lines of the parasite was identified, and used as a marker in a cross in the mosquito stage of the life cycle. Analysis of 59 individual hybrid oocysts resulting from this cross clearly demonstrated that inheritance of the cytochrome b gene was uniparental. This observation makes it possible to investigate the inheritance and evolution of cytoplasmic traits, including certain forms of drug resistance, in natural populations of this parasite.
Twenty-nine plasmodium falciparum isolates from patients in Asar village, eastern Sudan, were characterized for variation in 18 different genetically controlled characters, including iso-enzymes, ...proteins detected by two-dimensional polyacrylamide gel electrophoresis and blood-stage antigens. Considerable allelic diversity in the genes determining these characters was detected. Each isolate contained genetically distinct parasites. Fifteen individuals were infected with more than one parasite genotype. The diversity of parasite types is most probably generated by recombination during mosquito transmission of mixed parasite clones.
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