Developing bovine immune organoids for the screening of vaccine candidates

A range of diseases commonly affect cattle in the UK and worldwide including bovine mastitis, brucellosis, bluetongue, bovine viral diarrhoea, foot and mouth disease, Johne's disease and bovine TB. These have major implications for animal health, food security and the economy. Vaccination is widely considered the most effective control strategy, but in most cases vaccines still need to be developed or improved, necessitating screening studies, and all licenced vaccines need to be regularly batch-tested. Current in vitro models fail to capture the complex features of the adaptive immune response required to assess antigen-specific vaccine immunogenicity in a satisfactory way, and rodents are a poor model due to key differences in their immune systems compared with cattle. Thus in vivo cattle experiments are commonly used, which are ethically, logistically and economically costly.

We propose to develop bovine organoids based on secondary lymphoid organs as a new tool for screening bovine vaccines to replace the use of cattle (and rodents) in early vaccine development and vaccine batch-testing. Organoids are self-organised 3D tissue that mimic the key functional, structural and biological complexity of an organ. Bovine organoids have been developed for several other anatomical locations in cattle including the mammary glands and intestine, indicating feasibility. Immune organoids have recently been reported in humans, with demonstrated ability to measure primary and memory responses to antigens, adjuvants and vaccines. Our preliminary data shows that it is possible to grow organoids from cryopreserved bovine lymph node material, with the expected reaggregation of cells and good survival over an extended culture period.

We aim to optimise, validate and implement the bovine immune organoid model using bovine TB as an exemplar for proof-of-concept in collaboration with the Animal and Plant Health Agency. Project Partner Lisa Wagar from the University of Irvine, California, pioneered the human immune organoid model and will provide hands-on training and expertise. For the optimisation phase, we will obtain secondary lymphoid tissue from naïve and BCG vaccinated cattle, and use this to identify the most appropriate tissue type and assay conditions including composition and concentrations of the components as well as the kinetic of response over time. We will also assess reproducibility at multiple levels.

We will then validate the model by demonstrating that it can mirror immunogenicity read-outs from in vivo studies. The approach to this will be three-fold: i) comparing immune responses induced by the Ad85A bovine TB vaccine in vivo in previously-conducted studies in the literature with those induced in our organoid model; ii) comparing immune responses induced by a panel of vaccine adjuvants in a previously-conducted study in the literature with those induced in our organoid model; and iii) directly comparing immune responses induced by four novel bovine TB vaccine candidates using an in vivo cattle study and our immune organoid model in parallel.

To demonstrate that the model is fit for purpose, we will then implement it to screen an expanded panel of bovine TB vaccine candidates. Finally, we will establish a 3Rs legacy locally and globally by actively disseminating methods, protocols and results; by supporting training and tech-transfer to relevant end-user laboratories; and by generating a small-scale bovine lymphoid tissue bank.

The advantages of bovine immune organoids include the clear 3Rs benefits in replacing the use of cattle and rodents in early bovine vaccine screening and batch-testing, the scientific benefits in offering a high-throughput and tractable model to expedite bovine vaccine development, cost-effectiveness, transferability including to resource-limited settings, and adaptability for use across a range of bovine diseases as well as other ungulate species of importance in vaccine development.

Many diseases commonly affecting cattle in the UK and worldwide have major implications for animal health, food security and the economy. The development or improvement of bovine vaccines is critical as an effective control strategy, necessitating immunogenicity screening of novel candidates and regular batch-testing of licenced vaccines. However, the evaluation of vaccines in cattle is ethically, logistically and economically costly, and mice are a poor model due to immunological and pathophysiological differences. No validated in vitro/ex vivo tools exist, and current approaches fail to capture the complexity of adaptive immune features required to assess antigen-specific vaccine responses.

The aim of this project is to establish bovine immune organoids as a high-throughput tool for bovine vaccine development, to replace the use of cattle (and rodents) in immunogenicity screening and down-selection studies and provide an alternative for vaccine batch-testing. We will optimise, validate and implement the bovine immune organoid model using bovine TB as an exemplar for proof-of-concept in collaboration with the Animal and Plant Health Agency.

Bovine secondary lymphoid tissue will be obtained from naïve and BCG vaccinated cattle and used to generate organoids for the assessment of primary and memory antigen-specific immune responses respectively. Organoid conditions will be optimised for cell survival, spatial organisation, sensitivity to observe responses to antigen stimulation and reproducibility. The optimised bovine organoids will then be validated against in vivo immunogenicity outcomes for novel bTB vaccine candidates and a panel of adjuvants. Finally, to demonstrate it is fit for purpose, the model will be implemented to screen an expanded panel of protein and adjuvant vaccine combinations. A 3Rs legacy will be established by disseminating methods and results, supporting tech-transfer, and generating a bovine lymphoid tissue bank for end-users.