A Refined Approach to Producing Polyclonal Antibodies in Chickens – Completely Replacing All Invasive Elements by Combining Immunizations with Routine Aerosol-based Vaccinations (finished project)

Otto Kalliokoski

A large proportion of all laboratory animals worldwide are used for production rather than experimentation: Polyclonal antibodies represent a billion-dollar industry, where the immune systems of mice, rats, rabbits, goats, and other mammals are used to produce serum that can be applied, for example, in disease diagnostics. The animals are repeatedly injected with disease-relevant proteins combined with an adjuvant (a solution that enhances the immune response), after which blood samples are collected and the antibodies are isolated.

These polyclonal antibodies play an essential role in modern medicine and biomedical research and offer significant societal benefits. However, the production process can be improved in terms of animal welfare. If antiserum is produced in chickens, blood sampling can be avoided. A chicken egg is packed with antibodies, as the egg is evolutionarily adapted to protect potential offspring by utilizing the hen’s immune system. By isolating antibodies from eggs, the need for blood sampling in antibody production can be eliminated (and an egg yields significantly more antibodies than a blood sample from, for example, a rabbit). The only remaining invasive procedure is the injection of locally irritating adjuvants. We aim to investigate whether these interventions can also be eliminated—i.e., whether the production of polyclonal antibodies can become a completely non-invasive process when conducted in chickens.

Chickens—those that lay the eggs we eat—are routinely vaccinated against various diseases. Vaccines against respiratory diseases are often administered as aerosols that the chickens inhale. The attenuated virus in the vaccine can still enter the chicken’s body via the mucous membranes of the respiratory tract. There, it encounters immune cells that quickly build immunity against the virus.

We aim to utilize this process by attaching the protein to these viral particles. By allowing the proteins to follow the virus into the chicken’s body, we hope to achieve two goals simultaneously: vaccinating the chicken while also immunizing it against a specific protein. Antibodies against the protein can then be isolated from the chicken’s eggs in a process that causes no more discomfort than the routine vaccinations already administered to our laying hens.

Our project vision was to devise and demonstrate a method for producing antibodies in hens without using invasive methods. By using hens instead of the commonly used mammalian models for antibody production – e.g. rabbits, mice, rats – we could circumvent the need for drawing large-volume blood samples, instead extracting antibodies from unfertilized eggs.

Moreover, hens are routinely vaccinated against respiratory tract viruses – specifically infectious bronchitis virus – using an inhaled aerosol of inactivated virus particles. If we, instead of injecting the hens with our immunogen – the template molecule we want to raise antibodies against – could “piggy-back” it onto the inactivated virus particles, we could immunize the animals while they are undergoing mandatory vaccinations.

This would remove the only remaining invasive procedure. Due to the large amounts of antibodies that can be obtained from an egg, the outlined approach has potential to reduce the use of laboratory animals. Removing the need to inject immunogens and draw blood constitutes a considerable refinement of procedures. Suffering could be entirely removed: There would be no need to break the skin with needles, no need for stressful restraint, no anemia, and no need for inflammation-potentiating adjuvants.

Globally, a considerable number of laboratory animals are used for the production of antibodies, and despite great advances in antibody-based methods, we will not replace the use of animals fully within a foreseeable future. Consequently, refining procedures and reducing the numbers of animals needed in antibody production can have a great impact for a number of years to come.

The support of the Danish 3R Center allowed us to develop all the technical procedures needed for the outlined proof-of-concept. The commercially available chicken vaccines are, however, of so poor a quality as to prevent us from demonstrating the approach in chickens at present. We have thus chosen to hold off on carrying out experiments on chickens. The chances of success are, currently, too low; this would simply constitute a pointless use of laboratory animals – something we are opposed to.

Moving forward, we will look into means for disseminating the methods we have developed and will keep looking for opportunities to obtain a better model vaccine capable of finally demonstrating our idea in full.

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