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

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.