The first research area focuses on the generation and validation of viruses (virus vectors) that contain Chlamydia genes that can be used as vaccines.
Genes were selected, which encoded recombinant proteins from the Chlamydia abortus bacterium that have been shown to be recognised by white blood cells from sheep immune to Chlamydia. The genes were synthetically made and individually inserted into separate virus vectors. Not all of the genes were successfully inserted to produce new virus vectors. Two of these were progressed to the validation stage of the virus vector production process. Only a single virus vector has been validated for use in the final part of this project, to assess it as a vaccine candidate to Chlamydia infection.

The second research area focuses on establishing Neospora parasites that lack some of their genes and evaluation if these parasites have lost the ability to cause persistent infections and could therefore be used as vaccines. Technology called CRISPR/Cas9 was used to delete genes within the parasite by inserting an antibiotic resistance gene (pyrimethamine) in their place to allow us to select the altered parasites. Three genes thought to be essential for the parasite to cause persistent infections in animals were targeted. This new approach has resulted in the generation of parasites that were maintained under continuous antibiotic selection pressure for ~12 weeks, showing that the antibiotic resistance genes have been inserted into the parasites. During this time, numerous passages of each parasite were preserved by freezing. The effect of deleting these genes on the ability of the parasite to cause persistent infection are being studied in cultures in the lab and successful parasite lines will be used in year 5 to see if they cause persistent infections in mice.

The project involved the following Knowledge Exchange activities this year:
Scientists involved in the project attended the Royal Highland Show in June and vaccines to reproductive diseases where highlighted within the Moredun marquee and developments were discussed with farmers and veterinarians. Moredun research was showcased at the Moredun Press Day in December. A scientist attended the International Veterinary Immunology Symposium, Vienna, August 2025 presenting research on vaccine development to academic and industrial audiences.
 

Impacts:

Policy: 
An update of our world leading vaccine approaches was given to members of the Animal Health & Welfare team within the Scottish Government during the Plant and Animal Health Theme meeting, which was held at the Moredun Research Institute in May. As these vaccines are still at an early stage of their development there are no policy relevant outputs yet, except for informing policy advisors about reproductive diseases, and current management options. It also highlighted that reductions in animal losses will result in greater productivity for the same amount of greenhouse gas emissions.

Industry: 
Scientist from this project have had talks with representatives from veterinary pharma companies who have expressed a specific interest in vaccines against reproductive diseases in ruminants. During these talks we discussed the potential of progressing and commercialising our vaccines. Talks are ongoing with two separate companies, where one is interested in the Chlamydia vaccine and the other company is interested in the Neospora vaccine. The availability of reproductive vaccines and management approaches to minimise the impact of reproductive diseases was discussed with farmers and veterinarians at the Highland Show in June, North Sheep in June and Welsh Sheep in May. The farmers who we engaged with said that they will try to adopt the management approaches we suggested to minimise their reproductive losses.

General Public: 
We engaged with members of the general public about the importance of vaccines at Open Farm Sunday at a farm in Pathhead in June, where the audience consisted of children, their parents and other members of the general public; at the Highland Show at Ingleston in June, where the audience also included children and the general public; and at the Scottish Small Holders Festival at Ingleston in October, where the audience was crofting families. During these events we make science and veterinary advances more accessible to the general public. Our activities are perceived by the public as informative, engaging and enjoyable.

Academia: 
Our vaccine progress was presented at the Veterinary Immunology Symposium, Vienna, August 2025, where the need for reproductive vaccines for ruminants was discussed as it not only reduces economic losses to the farming industries but also results in a reduction of greenhouse gas emissions. Moredun scientists are members of the “REPRODIVAC - Next-generation vaccines and diagnostics to prevent livestock reproductive diseases of worldwide impact” consortium, consisting of academics and industry stakeholders, which facilitates interactions between academics and industry, aiding commercialisation of academic research. A presentation at a REPRODIVAC webinar resulted in questions from industry experts and subsequent meetings have led to the production of target product profiles, separate lists of desired characteristics for the two types of Chlamydia abortus vaccines, which are under development. One relates to the Chlamydia vaccine that has existing commercial partner interest and the second relates to a new recombinant protein vaccine. It is anticipated that these documents could describe features of future vaccine products
 

All objectives and Knowledge Exchange activities for Year 2 were completed on time. The project remains on track to deliver improved reproductive vaccines for livestock.

Objective 1 – Chlamydia abortus (sheep)
Researchers optimised the production of 19 synthetic proteins identified as surface-expressed or secreted by C. abortus, targeting them as potential vaccine candidates. Protein yield and solubility varied significantly. Immune sheep were bred and inoculated with the pathogen during pregnancy to generate blood samples for downstream immunological testing following lambing in April 2024.
 

Objective 2 – Neospora caninum (cattle)
A gene-editing system was developed to silence selected Neospora genes. While the initial construct targeted an essential gene and was not viable, four additional targets were pursued.
A tissue culture system was established to test parasite stage transitions and screen for edited lines unable to complete their lifecycle, a potential route to vaccine development.
 

Knowledge Exchange
Project outcomes were shared at the Royal Highland Show, IVIS, and the Moredun Press Day.
 

Vaccine delivery systems for controlling reproductive chlamydial infections in livestock

We have identified the proteins that are on the surface of the bacterium and which are all a target for the immune system. These could therefore be used to provide protection from infection and so represent potential candidate proteins for testing in new vaccine formulations. Thus far we have produced eight synthetic versions of one of the predominant proteins on the bacterial surface and tested its reactivity to sheep antibodies from diseased animals to determine their suitability for vaccine production. The proteins were not found to be optimal however and therefore we are currently producing newer versions for future testing as well as evaluating other surface proteins.

Development of a vaccine for Neosporosis in cattle

We have conducted a literature review and in silico analysis to identify Neospora genes that are essential for the formation of bradyzoites/tissue cysts, which are the lifecycle stages responsible for persistent infections in cattle. We have identified four main genes, which will be targeted for silencing using CRISP-Cas9 gene editing technology. The resulting parasites generated would not be able to cause a persistent infection which would make them good candidates for vaccine strains. We have also started on the methodology to be able to select transfected parasites by incorporating genes essential for CRISPR-Cas9 editing as well as genes used in antibiotic selection and fluorescent markers into a transfection plasmid. This plasmid has successfully been used to transiently transfect Neospora tachyzoites, making these parasites fluorescent and resistant to Bleomycin for a short time.

Knowledge exchange and Impact

We have discussed the development of vaccines for reproductive diseases at the Royal Highland Show, the 6th International Meeting of Apicomplexan Parasites in Farm Animals (ApiCOWplexa 2022), the British Society for Immunology Congress and Moredun Press Day. Five publications focusing on livestock abortion, the development of a new Chlamydia vaccine and effect of adjuvants on vaccine efficacy has been published in open access journals, while we have also presented at the European Chlamydia meeting and at an EU Consortium meeting, following the successful acqusition of Horizon Europe funding. We have set up a new collaboration with the University of Dundee to improve the CRISPR-Cas9 editing of Neospora to assist vaccine development. 

We have had discussions with a company regarding vaccine delivery and proof-of-concept antigen testing in their proprietary vaccine delivery system. We have identified research gaps and priorities for the control of chlamydial infections in animals as part of the EU Discontools project and signed a licensing agreement with an industrial partner to commercialise a new Chlamydia vaccine for use in sheep. New reproductive vaccines for ruminants were also discussed when speaking with farmers and/or veterinarians, at the Royal Highland Show, other farming events, or by phone and also during two webinars given to international audiences of over 100 veterinarians and pharma representatives.