Online workshop on optimisation of the production of sporozoite stabilate for Theileria parva

25 Jan 2022

East Coast Fever (ECF) is an important disease of cattle in a large area of eastern, southern and central sub-Saharan Africa that is caused by the tick-borne apicomplexan parasite Theileria parva. At present the only vaccine commercially available for T. parva is the ‘infection-and-treatment method’ (ITM), in which animals are concurrently inoculated with T. parva sporozoites and treated with long-acting oxytetracycline.

Generation of T. parva sporozoites requires the use of the tick vector Rhipicephalus appendiculatus (brown ear tick of cattle) in a process that enables the tick to ‘pick-up’ T. parva piroplasms from the blood of infected cattle and then, during a subsequent partial feed, mature the parasite to the sporozoite stage. This process is long, complex, resource- and time-intensive and involves use of a significant amount of animals. There remain a number of key ‘bottlenecks’ and challenges in the production of T. parva sporozoites for ITM vaccination.

This workshop aims to bring together groups actively involved in the industrial-scale production of T. parva sporozoites, groups researching methods that could be used to improve sporozoite production and others interested in this area of work to:

  1. Share information on ongoing research projects.
  2. Identify what are the most important obstacles to optimising sporozoite production.
  3. Explore how groups can collaborate to most effectively address these challenges.

Meeting programme

  1. Welcome and introduction – Dr Timothy Connelley (Roslin Institute).
  2. The process of T. parva sporozoites generation – Dr David Kalenzi (Centre for Tick and Tick-Borne Diseases, Malawi).
  3. Challenges in stabilate production – Professor Maxime Madder (Clinglobal).
  4. Short invited presentations:
    • Improvement of Theileria parva sporozoite vaccine against East Coast Fever – Dr Massaro Ueti (US Department of Agriculture).
    • A method for dissection-free purification of sporozoites from mosquitos – Alexander Fyfe (Imperial College London).
    • Use of tick cell culture systems to support research objectives – Dr Lesley Bell-Sakyi (University of Liverpool).
  5. Open discussion – led by panel members.
  6. Concluding remarks.

Meeting attendees

See the table below for a list of attendees.

Name Organisation Country
Timothy Connelley Roslin Institute United Kingdom
Lucas Lefevre Roslin Institute United Kingdom
Alex Fyfe Imperial College London United Kingdom
C Ducker Imperial College London United Kingdom
Maxime Madder Clinglobal Netherlands
Lindsay Fry USDA Agricultural Research Service United States
Massaro Ueti USDA Agricultural Research Service United States
Ard Nijhof Freie Universit├Ąt Berlin Germany
Naftaly Githaka International Livestock Research Institute Kenya
Lesley Bell-Sakyi University of Liverpool United Kingdom
David Kalenzi Centre for Ticks and Tick-Borne Diseases Malawi
Peter Billingsley Sanaria Inc United States
Ndawula Junior Charles National Livestock Resources Research Institute Uganda
Dirk Werling Royal Veterinary College United Kingdom
Ahmed Saheed University of Ibadan Nigeria
David Oluwaseun Adisa College of Veterinary Surgeons Nigeria
Nyasha Chin'ombe University of Zimbabwe Zimbabwe
Gad Milton Oqido International Livestock Research Institute Ethiopia
Ellen Knuepfer Royal Veterinary College United Kingdom
Chimvwele Namantala Choopa Central Veterinary Research Institute Zambia
Qamar-un- Nisa University of Veterinary and Animal Sciences Pakistan
Choolwe Malabwa Central Veterinary Research Institute Zambia
Raheela Akhtar University of Veterinary and Animal Sciences Pakistan
Md Sabri Bin Mohd Yusoff Universiti Putra Malaysia Malaysia
Albert Iribagiza University of Burundi Burundi
Amany Hassan Alexandria University Egypt
Edward Robb BioPharmaPotentials United States
Myounus Dasti University of Veterinary and Animal Sciences Pakistan
Abdalla Latif University of KwaZulu-Natal South Africa
Abiodun Fatoba University of KwaZulu-Natal South Africa
Ambreen Khalid University of Veterinary and Animal Sciences Pakistan
Phil Toye International Livestock Research Institute Kenya
Ssali Enock Makerere University Uganda
Jeannine Kolakowski Royal Veterinary College United Kingdom
Shahin Tajeri Soronne University France
Antoinette Miyunga International Livestock Research Institute Kenya
Kim Lee Sim Sanaria Inc United States
Eric James Sanaria Inc United States

Notably, in addition to delegates from areas where ECF is an endemic disease, there were several attendees from other parts of Africa (e.g. Nigeria) and Asia (e.g. Pakistan) where the topics discussed are of potential relevance to related parasites (e.g. Theileria annulata).

Also attending the meeting were delegates who have a primary interest in malaria. In keeping with some of the work being presented, this reflected the interest in translational knowledge between groups working with human and veterinary pathogens.

Notes from the meeting

A video recording of the workshop is available below. A version with automated transcription, along with the chat discussion from the meeting, is available here.

The meeting started with Dr Tim Connelley (Roslin Institute) providing a brief introduction to T. parva, its importance as the causal agent of East Coast Fever, its lifecycle and the bovine immune response against this pathogen. The talk highlighted that there is as yet no alternative vaccine for ECF and that therefore ITM, and consequently the production of T. parva sporozoites, remains central to vaccine-based control strategies for ECF.

Dr David Kalenzi (Centre for Tick and Tick-Borne Diseases) then provided a comprehensive and clear overview of the production of sporozoites at a commercial scale. The CTTBD has recently generated 3 batches of sporozoites and is currently generating a 4th batch which will hopefully provide sufficient material for ~1x106 doses. David’s talk highlighted the long time required to produce a batch of sporozoites (18 months), the number of different processes involved and the rigorous QC required for the final product (viability, safety, efficacy and external QC validation by PanVac). A key point made by David was how the processes involved in sporozoite production haven’t changed significantly since the ITM vaccine was first produced ~40 years ago.

Professor Maxime Madder (Clinglobal) then provided his perspective on where there remain major challenges in sporozoite production and potential alternative routes to circumvent these. The challenges (and possible solutions) included:

  1. Challenge: The need to balance parasitaemia with welfare on cattle used for tick ‘pick-up’ of T. parva.
    • Potential solution: In vitro systems for generating piroplasm-infected red blood cells and using these to infect ticks - thus removing animals from this stage of the production cycle
  2. Challenge: The need to use large number of rabbits to mature the parasite to the sporozoite stage (‘pre-feed’ of infected ticks)
    • Potential solution: In vitro feeding system for maturation of sporozoites (see presentation by Dr Massaro Ueti) to remove animals from this stage of the production cycle
  3. Challenge: The difficulty in effectively and efficiently harvesting sporozoites and separating them from contaminating tick material
    • Potential solutions:
      1. In vitro feeding system for maturation of sporozoites with collection of sporozoites in medium after emission from the ticks (see presentation by Dr Massaro Ueti) so that there is no need to separate tick material from the sporozoites
      2. High throughput sporozoite purification system (see presentation by Alexander Fyfe) - transfer of technology from malaria research.
  4. Challenge: The need to use large number of cattle (up to 60-70 animals) for the titration of sporozoites to be used in ITM to achieve robust quantification of viable sporozoites
    • Potential solutions:
      1. Purification of sporozoites may assist in permitting better quantification of sporozoites - reducing the range over which in vivo titrations are required (see above)
      2. Reproducible in vitro system for quantification - a standardised WBC infection assay that correlates to in vivo infectivity
      3. Systems to accurately and directly quantify the number of viable sporozoites.
  5. Challenge: the need for cold chain distribution of the sporozoites when used for vaccination.
    • Potential solution: lyophilisation? Has been demonstrated to work in a ‘one-off’ experiment but has not been repeated

Dr Massaro Ueti then gave a presentation on work to develop an in vitro R. appendiculatus feeding system. This work uses devices that can feed approx. 800 ticks per unit. Feeding of adults using silicone membranes in this unit has been successfully used to mature sporozoites. The feeding system uses an alternate blood (21hr)/harvest medium (3hr) cycle, with sporozoites then being recovered directly from the harvested medium. This allows sporozoites to be i) generated without the need for rabbits (Challenge 2) and ii) harvested free from contaminating tick material (Challenge 3/4). The system can easily be scaled up making it of potential utility in industrial-scale production but has not yet been successfully adapted to the feeding of nymphs and this remains an area of research in the hope that it could also be used to achieve tick ‘pick-up’ of infection (Challenge 1 - although this would still require the infection of animals to provide a source of piroplasm-infected RBC).

Alexander Fyfe (Imperial College) presented work that has been ongoing in the laboratory of Professor Jacob Baum to develop a method of dissection-independent purification of Plasmodium sporozoites from mosquitoes (‘MalPure’). This work uses a combination of size-exclusion filtration and free-flow electrophoresis to generate purified Plasmodium sporozoites from homogenised infected mosquitos. The work in malaria has demonstrated the ability to generate purified sporozoites in a (near) aseptic technique with minimal contamination of mosquito proteins. Purified sporozoites are infective in both in vitro models and in vivo mouse experiments and have also been shown to generate immunity in a murine infection and challenge experiment (including by sporozoites administration by intra-muscular as well as intra-venous routes). Working in collaboration with the group at Roslin there is an attempt to transfer the technology to the purification of T. parva sporozoites (Challenge 3).

Dr Lesley Bell-Sakyi gave the final presentation which focused on the use of tick cell and organ culture systems to look at fundamental aspects of tick biology and tick/pathogen interactions. Lesley currently runs the ‘Tick Cell Biobank’ project at the University of Liverpool, which is in the process of establishing satellite units (Outposts) in Kenya, Malaysia and Brazil. Lesley and the Tick Cell Biobank have provided training and materials to many working in the field of tick research and continue to expand the accessibility of specialised resources and knowledge to researchers across the globe. There are a number of R. appendiculatus cell lines in the Tick Cell Biobank collection, although none of these are known to support T. parva growth or development (they do however support the growth of a number of other important veterinary pathogens such as Nairobi Sheep Disease Virus and Ehrlichia ruminantium). Although not able to directly address the challenges identified, resources in the Tick Cell BioBank can be used to answer basic biological questions that in turn will support the development of technologies that do.

After the presentation there was a number of questions from the meeting attendees across a broad range of issues including: the parameters of the in vitro tick-feeding system, techniques for quantifying T. parva sporozoites using qPCR, details of the lyophilisation technology used for T. parva, the use of alternative cryoprotectants (and the approval by regulatory bodies), details of the industrial-scale production of sporozoites (size of production runs, stability and viability assessment) amongst other issues.

Conclusions

The meeting was set up with three objectives:

1. Share information on ongoing research projects

Information from two ongoing projects were shared:

  • The WSU/Clinglobal/CTTBD collaboration looking at adoption of in vitro feeding for Rhipicephalus appendiculatus funded by the Livestock Vaccine Innovation Fund programme of the IDRC (Canada).
  • The Roslin/Imperial collaboration looking at transfer of sporozoites purification technology from malaria to Theileria funded by the BMGF

The opportunity to bring the participants of these projects together and enable them to share information with each other and the wider community was very beneficial in initiating discussion between these collaborative groups. No other ongoing research projects were identified by meeting attendees.

2. Identify what are the most important obstacles to optimising sporozoite production

Maxime Madder gave a summary of the main challenges that are faced in the production of T. parva stabilate and the deployment of the ITM vaccine:

  1. The need to balance parasitaemia with welfare on cattle used for tick ‘pick-up’ of T. parva.
  2. The need to use large number of rabbits to mature the parasite to the sporozoite stage (‘pre-feed’ of infected ticks).
  3. The difficulty in effectively and efficiently harvesting sporozoites and separating them from contaminating tick material.
  4. The need to use large number of cattle (upto 60-70 animals) for the titration of sporozoites to be used in ITM to achieve robust quantification of viable sporozoites.
  5. The need for cold chain distribution of the sporozoites when used for vaccination.

Other challenges are known (e.g. the need for parasite diversity in the vaccines deployed in some geographical areas) but these largely summarise the challenges to the optimised production and delivery of sporozoites. The two projects discussed are largely relevant to challenges 2 and 3 (in vitro feeding for the maturation of sporozoites to reduce rabbit use and the purification of sporozoites using high throughput technology).

3. How groups can collaborate to most effectively address these challenges

It was discussed that some of the other identified challenges could best be achieved by recruiting other groups with complementary expertise (in for example in vitro culture of apicomplexan parasites) into the Theileria research field.

The other issue that became apparent during the meeting was divergence of methodologies being used (for example the media used for suspension of the sporozoites and the technique used for grinding ticks during sporozoites harvesting). Additionally there is ambiguity in details in some of the steps in protocols (e.g. in the rate of addition of glycerol cryopreservant) and in some cases protocol variants have not been directly compared to identify the optimal method (e.g. different cryoprotectant formulations such as trehalose, combination of glycerol with a sugar-cryopreservant and commercially available cryopreservants have not be compared for efficiency against sucrose or glycerol). Furthermore, there were other areas where standardised methods could be beneficial (e.g. in the in vitro titration assay which currently uses PBMC from locally accessible animals – so lacks a global standard). Many of these issues could be addressed by the community coming together to produce a set of standardised protocols and compile a list of steps in the production pipeline that need further research to be optimised.