Dataset Information

Cloning, expression and transmission-blocking activity of anti-PvWARP, malaria vaccine candidate, in Anopheles stephensi mysorensis.

ABSTRACT:

Background

Notwithstanding progress in recent years, a safe, an effective and affordable malaria vaccine is not available yet. Ookinete-secreted protein, Plasmodium vivax von Willebrand factor A domain-related protein (PvWARP), is a candidate for malaria transmission-blocking vaccines (TBVs).

Methods

The PvWARP was expressed in Escherichia coli BL21 using the pET-23a vector and was purified using Ni-NTA affinity chromatography from a soluble fraction. Polyclonal antibody was raised against rPvWARP and transmission blocking activity was carried out in an Anopheles stephensi-P. vivax model.

Results

Expression of full length of PvWARP (minus signal peptide) expression showed a 35-kDa protein. The purified protein was recognized by mouse polyclonal antibody directed against rPvWARP. Sera from the animals displayed significantly a blocking activity in the membrane feeding assay of An. stephensi mysorensis.

Conclusions

This is the first report on P. vivax WARP expression in E. coli that provides an essential base for development of the malaria TBV against P. vivax. This may greatly assist in malaria elimination, especially in the oriental corner of WHO Eastern Mediterranean Regional Office (WHO/EMRO) including Afghanistan, Iran and Pakistan.

SUBMITTER: Gholizadeh S

PROVIDER: S-EPMC2904785 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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Publications

Cloning, expression and transmission-blocking activity of anti-PvWARP, malaria vaccine candidate, in Anopheles stephensi mysorensis.

Gholizadeh Saber S Basseri Hamid Reza HR Zakeri Sedigheh S Ladoni Hossein H Djadid Navid Dinparast ND

Malaria journal 20100611

<h4>Background</h4>Notwithstanding progress in recent years, a safe, an effective and affordable malaria vaccine is not available yet. Ookinete-secreted protein, Plasmodium vivax von Willebrand factor A domain-related protein (PvWARP), is a candidate for malaria transmission-blocking vaccines (TBVs).<h4>Methods</h4>The PvWARP was expressed in Escherichia coli BL21 using the pET-23a vector and was purified using Ni-NTA affinity chromatography from a soluble fraction. Polyclonal antibody was raise ...[more]

PMID: 20537198

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Project description:BackgroundsMalaria, a preventive and treatable disease, is still responsible for annual deaths reported in most tropical regions, principally in sub-Saharan Africa. Subunit recombinant transmission-blocking vaccines (TBVs) have been proposed as promising vaccines to succeed in malaria elimination and eradication. Here, a provisional study was designed to assess the immunogenicity and functional activity of alanyl aminopeptidase N (APN1) of Anopheles stephensi, as a TBV candidate, administered with MPL, CpG, and QS21 adjuvants in the murine model.Methodology/principal findingsThe mouse groups were immunized with recombinant APN1 (rAPN1) alone or formulated with CpG, MPL, QS-21, or a combination of adjuvants (CMQ), and the elicited immune responses were evaluated after the third immunization. The standard membrane feeding assay (SMFA) measured the functional activity of antibodies against bacterial-expressed APN1 protein in adjuvanted vaccine groups on transmission of P. falciparum (NF54) to An. stephensi mosquitoes. Evaluation of mice vaccinated with rAPN1 formulated with distinct adjuvants manifested a significant increase in the high-avidity level of anti-APN1 IgG and IgG subclasses; however, rAPN1 induced the highest level of high-avidity anti-APN1 IgG1, IgG2a, and IgG2b antibodies in the immunized vaccine group 5 (APN1/CMQ). In addition, vaccine group 5 (receiving APN1/CMQ), had still the highest level of anti-APN1 IgG antibodies relative to other immunized groups after six months, on day 180. The SMFA data indicates a trend towards higher transmission-reducing activity in groups 2 and 5, which received the antigen formulated with CpG or a combination of three adjuvants.Conclusions/significanceThe results have shown the capability of admixture to stimulate high-affinity and long-lasting antibodies against the target antigen to hinder Plasmodium parasite development in the mid-gut of An. stephensi. The attained results authenticated APN1/CMQ and APN1/CpG as a potent APN1-based TBV formulation which will be helpful in designing a vaccine in the future.

Project description:Extrinsic environmental factors influence the spatiotemporal dynamics of many organisms, including insects that transmit the pathogens responsible for vector-borne diseases (VBDs). Temperature is an especially important constraint on the fitness of a wide variety of ectothermic insects. A mechanistic understanding of how temperature impacts traits of ectotherms, and thus the distribution of ectotherms and vector-borne infections, is key to predicting the consequences of climate change on transmission of VBDs like malaria. However, the response of transmission to temperature and other drivers is complex, as thermal traits of ectotherms are typically nonlinear, and they interact to determine transmission constraints. In this study, we assess and compare the effect of temperature on the transmission of two malaria parasites, Plasmodium falciparum and Plasmodium vivax, by two malaria vector species, Anopheles gambiae and Anopheles stephensi. We model the nonlinear responses of temperature dependent mosquito and parasite traits (mosquito development rate, bite rate, fecundity, proportion of eggs surviving to adulthood, vector competence, mortality rate, and parasite development rate) and incorporate these traits into a suitability metric based on a model for the basic reproductive number across temperatures. Our model predicts that the optimum temperature for transmission suitability is similar for the four mosquito-parasite combinations assessed in this study, but may differ at the thermal limits. More specifically, we found significant differences in the upper thermal limit between parasites spread by the same mosquito (A. stephensi) and between mosquitoes carrying P. falciparum. In contrast, at the lower thermal limit the significant differences were primarily between the mosquito species that both carried the same pathogen (e.g., A. stephensi and A. gambiae both with P. falciparum). Using prevalence data, we show that the transmission suitability metric ST$$ S(T) $$ calculated from our mechanistic model is consistent with observed P. falciparum prevalence in Africa and Asia but is equivocal for P. vivax prevalence in Asia, and inconsistent with P. vivax prevalence in Africa. We mapped risk to illustrate the number of months various areas in Africa and Asia predicted to be suitable for malaria transmission based on this suitability metric. This mapping provides spatially explicit predictions for suitability and transmission risk.

Project description:BackgroundmicroRNAs (miRNAs) are non-coding RNAs that are now recognized as a major class of gene-regulating molecules widely distributed in metozoans and plants. miRNAs have been found to play important roles in apoptosis, cancer, development, differentiation, inflammation, longevity, and viral infection. There are a few reports describing miRNAs in the African malaria mosquito, Anopheles gambiae, on the basis of similarity to known miRNAs from other species. An. stephensi is the most important malaria vector in Asia and it is becoming a model Anopheline species for physiological and genetics studies.ResultsWe report the cloning and characterization of 27 distinct miRNAs from 17-day old An. stephensi female mosquitoes. Seventeen of the 27 miRNAs matched previously predicted An. gambiae miRNAs, offering the first experimental verification of miRNAs from mosquito species. Ten of the 27 are miRNAs previously unknown to mosquitoes, four of which did not match any known miRNAs in any organism. Twenty-five of the 27 Anopheles miRNAs had conserved sequences in the genome of a divergent relative, the yellow fever mosquito Aedes aegypti. Two clusters of miRNAs were found within introns of orthologous genes in An. gambiae, Ae. aegypti, and Drosophila melanogaster. Mature miRNAs were detected in An. stephensi for all of the nine selected miRNAs, including the four novel miRNAs (miR-x1- miR-x4), either by northern blot or by Ribonuclease Protection Assay. Expression profile analysis of eight of these miRNAs revealed distinct expression patterns from early embryo to adult stages in An. stephensi. In both An. stephensi and Ae. aegypti, the expression of miR-x2 was restricted to adult females and predominantly in the ovaries. A significant reduction of miR-x2 level was observed 72 hrs after a blood meal. Thus miR-x2 is likely involved in female reproduction and its function may be conserved among divergent mosquitoes. A mosquito homolog of miR-14, a regulator of longevity and apoptosis in D. melanogaster, represented 25% of all sequenced miRNA clones from 17-day old An. stephensi female mosquitoes. An. stephensi miR-14 displayed a relatively strong signal from late embryonic to adult stages. miR-14 expression is consistent during the adult lifespan regardless of age, sex, and blood feeding status. Thus miR-14 is likely important across all mosquito life stages.ConclusionThis study provides experimental evidence for 23 conserved and four new microRNAs in An. stephensi mosquitoes. Comparisons between miRNA gene clusters in Anopheles and Aedes mosquitoes, and in D. melanogaster suggest the loss or significant change of two miRNA genes in Ae. aegypti. Expression profile analysis of eight miRNAs, including the four new miRNAs, revealed distinct patterns from early embryo to adult stages in An. stephensi. Further analysis showed that miR-x2 is likely involved in female reproduction and its function may be conserved among divergent mosquitoes. Consistent expression of miR-14 suggests that it is likely important across all mosquito life stages from embryos to aged adults. Understanding the functions of mosquito miRNAs will undoubtedly contribute to a better understanding of mosquito biology including longevity, reproduction, and mosquito-pathogen interactions, which are important to disease transmission.

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Dataset Information

Cloning, expression and transmission-blocking activity of anti-PvWARP, malaria vaccine candidate, in Anopheles stephensi mysorensis.

Background

Methods

Results

Conclusions

Publications

Cloning, expression and transmission-blocking activity of anti-PvWARP, malaria vaccine candidate, in Anopheles stephensi mysorensis.

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