Studies of hepatic latency in human ovale malaria

Lead Research Organisation: London Sch of Hygiene and Trop Medicine
Department Name: Infectious and Tropical Diseases

Abstract

Malaria in humans is caused by several species of parasite belonging to the Plasmodium group, which pass from infected mosquitoes into the person's liver, where after a period of incubation the infection emerges into the blood, causing the fevers and other symptoms we associate with malaria. One of the species causing malaria in humans is Plasmodium ovale, which was first discovered in 1922. It was not until 2010, however, that we were able to provide convincing evidence that these were actually two different parasites which could not be distinguished from each other just by looking down a microscope. We developed some simple DNA testing methods which allow health workers to tell which of these two species is present; using these it is now clear that this type of malaria is actually more widespread than had been thought. Although ovale malaria does not commonly cause a life-threatening form of malaria, two features make it hard to control, and a serious challenge for efforts to eliminate malaria altogether in some countries. Firstly, it is often present in people's blood in relatively small numbers at the same time as other malaria parasites which are more abundant, and so is under-recognised. Secondly, it shares with one other species, Plasmodium vivax, the ability to "hibernate" at the liver stage for months or years, being resistant to almost all known malaria drugs when it is in this resting phase. Efforts to study these "sleeping" forms in the liver for P. vivax malaria have not been very successful, and although we have some ideas about them, these "sleeping" stages remain largely a mystery of modern biology. As host institution for the UK Malaria Reference Laboratory, we receive blood samples from malaria cases across the UK, and each year there are more than fifty ovale cases among them. We thus expect approximately one per week, and therefore have sufficient numbers coming through to plan a new study of these parasites. We have developed a way to purify the parasites in patient blood away from human blood cells, and we will seek to feed these to mosquitoes, and then infect liver cells obtained from the ongoing research work of colleagues at UCL, nearby. If this is successful, we will have a new approach for studying liver-stage malaria, and in particular a new window into the mysterious world of the "sleepy" liver-stage parasites. This information should also help us to better understand these stages in P. vivax malaria, also.

Technical Summary

Ovale malaria is caused by two closely related parasite species, Plasmodium ovale curtisi (Poc) and P. o. wallikeri (Pow). These have recently been shown to differ in the duration of the latent period prior to development of a symptomatic blood-stage infection, implying differences in liver stage biology. The literature contains a single study on liver-stage schizonts (1954) and none on hypnozoites of ovale malaria, yet recent data suggest that ovale parasites can evade chemotherapy in some individuals, presumably through the emergence of hypnozoites into the blood after drug levels have fallen. Thus P. ovale spp. liver-stage biology has important public health consequences for malaria control and elimination efforts in sub-Saharan Africa, where the parasite is widespread. We have established a transient 48 - 72 hour culture method with ex vivo ovale parasites from blood samples submitted to the UK Malaria Reference Laboratory that sustains schizont and gametocyte maturation, but not-erythrocyte invasion. This will permit studies of mRNA expression, sex ratio and ex-flagellation in ovale sexual stage parasites for the first time. We will attempt experimental mosquito infections and initiation of in vitro liver stage cultures by inoculation of primary human hepatocytes and cell lines with sporozoites from both Poc and Pow. The ex vivo blood stage material will also be used to for next-generation sequencing on the LSHTM in-house Illumina platform. Bioinformatic approaches will be used to identify genes shared with the relapsing parasite P. vivax, but not with P. knowlesi, as a means to generate a list of candidate genes important in the formation of hypnozoites. Patterns of ovale relapse in Malian children treated with ACT for falciparum malaria will be deduced from screening of follow-up blood samples in large clinical trials currently underway; this will add to our knowledge of the role of hypnozoites in maintaining ovale transmission in Africa.

Planned Impact

This project will have impact on the following stake-holders:
-travel medicine specialists, as we hope to produce better understanding of relapse patterns with ovale malaria in Africa
-drug development groups beyond academia as new pathways to the identification of targets in Plasmodium spp. hypnozoites may be found
-malaria control programmes in endemic countries, as a better understanding of ovale malaria relapse (whether symptomatic or asymptomatic) following ACT treatment of falciparum malaria cases is likely to suggest changes to treatment policy and a need for more specific diagnostics
-immunologists and vaccine developers, as the Poc and Pow sporozoite-infected mosquitoes could be used for irradiation experiments and elicitation of ovale-specific antibodies in mice.

Publications

10 25 50

publication icon
Assefa SA (2014) estMOI: estimating multiplicity of infection using parasite deep sequencing data. in Bioinformatics (Oxford, England)

publication icon
Kristan M (2019) Mosquito and human hepatocyte infections with Plasmodium ovale curtisi and Plasmodium ovale wallikeri. in Transactions of the Royal Society of Tropical Medicine and Hygiene

publication icon
Liu W (2014) African origin of the malaria parasite Plasmodium vivax. in Nature communications

 
Description British Society for Antimicrobial Chemotherapy - Research Grants
Amount £50,000 (GBP)
Funding ID GA2014_015R 
Organisation British Society for Antimicrobial Chemotherapy 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2014 
End 09/2015
 
Title Mosquito rearing and transmission of malaria parasites 
Description Through refurbishment of a disused room, we have established a dedicated mosquito-rearing facility for our parasite transmission work; the MRC project team have now been trained in mosquito husbandry and dissection techniques, and practise experiments to test the system by transmission to Anopheles mosquitoes of cultured P falciparum are being carried out. 
Type Of Material Improvements to research infrastructure 
Provided To Others? No  
Impact Preparedness for suitable Plasmodium ovale sample, should one be available in coming weeks. 
 
Title Transmission of ovale parasites to mosquitoes from patient samples, generating sporozoites infectious to human hepatocytes 
Description We have derived a protocol for taking EDTA blood smaples from patients diagnosed with ovale malaria )of either species) and transmit to Anopheles coluzzi mosquitoes in our laboratory at LSHTM. Sporozoite-positive salivary glands from these mosquitoes have then been used to isolate sporozoites for subsequent infection of cultured human hepatocytes. Antibody to the parasite protein HSP70 were then used to demonstrate the presence of parasite liver-stage forms in intra-cellular locations inside the host cells, successfully achieved in the final year of the project (2016). We have presented preliminary results in our recent research proposal to the MRC for further project funding to commence (if successful) later in 2017. 
Type Of Material Biological samples 
Provided To Others? No  
Impact This methodology is not fully developed yet, but will be disseminated in due course, when this has been achieved. The impact of this may be development of an in vitro system for testing antiomalarial drugs that target liver-stage malaria infections. 
 
Description COmparative genetic epidemiology of ovale parasites in Ethiopia and Bangladesh 
Organisation University of Veterinary Medicine Vienna
Country Austria 
Sector Academic/University 
PI Contribution We are providing sequence data from our parasite sample set, which does not include the two counties in which our collaborator, Dr Hans-Peter Fuehrer, has been working - Ethiopia and Bangladesh.
Collaborator Contribution Dr Hans-Peter Fuehrer has provided sequence data from parasite isoaltes derived from his work in Ethiopia and Bangladesh.
Impact Draft manuscript - still a work in progress. This collaboration encompasses sequence analysis and genetic relatedness only.
Start Year 2015
 
Description Comparison of neoplastic and non-neoplastic immortalised human hepatocyte cell-lines as hosts for Plasmodium ovale spp. sporozoite invasion 
Organisation King's College London
Department Institute of Liver Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We are generating Plasmodium ovale spp.-infected mosquitoes, from which sporozoites are isoalted for infection of hepatocytes of different origins.
Collaborator Contribution Dr Shilpa Chokshi has provided us with SV40-immortlaised but non-neoplastic human hepatocyte cells, and continues to provide advice on maintenance of these cells. Dr Chokshi, together with co-Investigator Dr O-Brien, have assisted in our planning of the hepatic stuides componenet of the project.
Impact Successful generation of ovale-infected hepatocytes.
Start Year 2014
 
Description Genome sequencing partnership - P. ovale curtisi and P. ovale wallikeri 
Organisation King Abdullah University of Science and Technology (KAUST)
Country Saudi Arabia 
Sector Academic/University 
PI Contribution We have provided malaria parasite DNA, analysed data, and gene sequence information. We have also contributed to writing of a majpr manuscript which is currently in submission.
Collaborator Contribution Partners provided data analysis, parasite DNA sequence data, bioinformatic infrastructure and wrote the major part of the joint manuscript.
Impact A major manuscript has been submitted to the International Journal for Parasitology.
Start Year 2015
 
Description Genome sequencing partnership - P. ovale curtisi and P. ovale wallikeri 
Organisation Nagasaki University
Country Japan 
Sector Academic/University 
PI Contribution We have provided malaria parasite DNA, analysed data, and gene sequence information. We have also contributed to writing of a majpr manuscript which is currently in submission.
Collaborator Contribution Partners provided data analysis, parasite DNA sequence data, bioinformatic infrastructure and wrote the major part of the joint manuscript.
Impact A major manuscript has been submitted to the International Journal for Parasitology.
Start Year 2015
 
Description Ovale malaria surveys in Mali 
Organisation University of Bamako
Department Malaria Research and Training Centre (MRTC) Bamako
Country Mali 
Sector Academic/University 
PI Contribution We have provided resources from the MRC award to long-term collaboraotr Prof Abdoulaye Djimde, University of Bamako, to conduct additional field surveys in a rural area of Mali where ovale malaria prevalence (both species) is relatively high. For the first time in our studies of this pair of parasite species, there is a longitudinal component to this field work.
Collaborator Contribution Prof Djimde's team are collecting longitudinal data on PCR-confirmed carriers of either of the ovale species in this area of Mali.
Impact Unpublished parasite prevalence data, so far.
Start Year 2011
 
Description Sequence anlaysis of apioplast and mitochondrion genomes in P. ovale spp. 
Organisation University of Veterinary Medicine Vienna
Country Austria 
Sector Academic/University 
PI Contribution We are providing previously collected genome sequence data, parasite DNA samples, laboratory results, study design, intellectual input and manuscript writing.
Collaborator Contribution Our partners are providing parasite DNA samples, laboratory results, intellectual input and manuscript writing.
Impact None to date.
Start Year 2014