2014 Fiscal Year Research-status Report
A genomic approach to the identification of transmission blocking antigens of malaria parasites
| Project/Area Number |
25870525
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| Research Institution | Nagasaki University |
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Principal Investigator |
カレトン リチャード 長崎大学, 熱帯医学研究所, 准教授 (10503782)
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| Project Period (FY) |
2013-04-01 – 2016-03-31
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| Keywords | malaria / genomics / bioinformatics / LGS / WGS / Gametocyte / vaccine |
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| Outline of Annual Research Achievements |
We have made major breakthroughs in our project to identify the targets of strain specific immunity in malaria parasites. We have developed new technologies and new analyses methodologies which have allowed us to investigate genes involved in malaria pathogenesis much more deeply. Due to these breakthroughs, we are currently preparing two major manuscripts for submission to high impact factor journals;
i)We have developed a quantitative whole genome resequencing system for measuring the proportion of alleles from two parental malaria parasites in a recombinant progeny population.
ii) We have developed a robust bioinformatics strategy to identify those genes within selection valleys that are statistically likely to be under selection. This involves the use of repeated crosses and selections and the mathematical identifications of apparent non-neutral marker movement following selection
iii) We have shown that there is a sharp decrease in the infectivity of gametocytes throughout the course of an infection in mice, and, using IFN-Gamma and MyD88 KO mice, that this is independent of the innate immune response. We have used sera transfer to demonstrate that sera from mice with long-term infections suppresses the ability of early-infection stage gametocytes to transmit
iv) We are currently investigating the feasibility of performing LGS experiments with P. falciparum. Using the strains 3D7 and HB3, we have cultured pure gametocytes, and purified crude protein, to produce immune sera in rabbits. We have acquired the uncloned recombinant progeny of a cross between these two strains.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
We initially intended to identify strains of the rodent malaria parasite P. yoelii that can induce transmission blocking immunity specific to the strains. We have achieved this through the demonstration that laei infection stage sera from P. yoelii strain CU, can, when transferred to mice with early infections (Day 3 post-infection) significantly reduce the transmission of parasites to mosquitoes (measured as the percentage of infected mosquitoes, and the number of oocysts per infected mosquito), compared to naive sera. Sera transfer from mice infected with late-stage infections of another strain, P. yoelii 17X1.1pp, did not significantly reduce transmission of early stage infections of P. yoelii CU, but did supress infections of the homologous strain. We have produced a genetic cross between these two strains for LGS analysis.
We have developed a robust bioinformatics and statistical analysis protocol for the interrogation of selected progeny
Finally, we have begun investigations into strain specific immunity using the human malaria parasite P. falciparum. We plan to perform a full LGS with this parasite using in vitro culture and mosquito transmission. We have successfully purified gametocytes of 3D7 and HB3 strains, and prepared crude antigen for production of transmission blocking sera. We have also acquired the uncloned recombinant progeny of a cross between the two parasite strains for downstream LGS experiments. We hoped to have completed these studies this year, but are being held up by technical difficulties in transmitting P. falciparum to mosquitoes consistently.
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| Strategy for Future Research Activity |
We haveembarked on studies with Plasmodium falciparum. This was due to the realisation that we can directly investigate genes in the human parasite through transmission blocking assays involving in vitro membrane feeding of cultures of gametes in human blood. This exciting new route at tackling our initial problem requires additional work-up and experimental testing. We have now procured the uncloned recombinant progeny of a cross between two strains of P. falciparum for our LGS purposes, and will perform whole genome sequencing on these both before and after transmission blocking selection. The first priority is to optimise the strain specific immunity blocking protocol. Currently, our experiments involve the transfer of sera from mice with late stage infections of parasites to mice with early stage infections. This results in a significant reduction in both the percentage of mosquito infected and the numbers of oocysts per infected mosquito, but does not completely block transmission. For the LGS experiments, it is essential that transmission be blocked as thoroughly as possible. We will, therefore, attempt to maximise the transmission-blocking efficacy of immune sera by a) immunising mice directly with crude antigen derived from purified gametocytes of each strain, and b) switching to membrane feeding rather than in vivo feeding of mosquitoes. Following this optimisation, we will perform the LGS experiments, involving selection of recombinant progeny, whole genome resequencing, and vaccine candidate identification.
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| Causes of Carryover |
26年度に、新たに開発したヒト血液中で培養したマラリア原虫配偶子の経膜摂取による感染抑制試験を行う事で直接ヒト寄生虫の遺伝子機能解析が実施可能になった。この結果、熱帯熱マラリア原虫を用いた実験系の構築と検証が必要となり、計画を変更し次世代ゲノム解析を行うこととしたため、未使用額が生じた。
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| Expenditure Plan for Carryover Budget |
次世代ゲノム解析と感染抑止血清の制作に費用がかかるため、その経費に未使用額を充当する。
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[Journal Article] Plasmodium knowlesi: Clinical Presentation and Laboratory Diagnosis of the First Human Case in a Scottish Traveller2014
Author(s)
Cordina, C., Culleton, R., Jones, B.L., Smith, C., McConnachie, A., Coyne, M, Alexander, C.L
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Journal Title
Journal of Travel Medicine
Volume: 21
Pages: 357-360
DOI
Peer Reviewed / Open Access
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