Exploring the antigenic response to multiplexed immunizations in a chicken model of antibody production.

Hens have a tremendous capacity for producing polyclonal antibodies that can subsequently be isolated in high concentrations from their eggs. An approach for further maximizing their potential is to produce multiple antisera in the same individual through multiplexed (multiple simultaneous) immunizations. An unknown with this approach is how many immunogens a single bird is capable of mounting a sizeable antigenic response toward. At what point does it become counter-productive to add more immunogens to the same immunization regimen? In the present study we were able to demonstrate that the competing effects of co-administering multiple immunogens effectively limit the antibody specificities that can be raised in a single individual to a fairly low number. Two potent model immunogens, KLH and CRM197, were administered together with competing antigens in various concentrations and complexities. With an upper limit of 1 mg protein material recommended for chicken immunizations, we found that the maximum number of immunogens that can be reliably used is most likely in the low double digits. The limiting factor for a response to an immunogen could not be related to the number of splenic plasma cells producing antibodies against it. When administering KLH alone, up to 70% of the IgY-producing splenic plasma cells were occupied with producing anti-KLH antibodies; but when simultaneously being exposed to a plethora of other antigens, a response of a comparable magnitude could be mounted with a splenic plasma cell involvement of less than 5%. Two breeds of egg-layers were compared with respect to antibody production in an initial experiment, but differences in antibody productivity were negligible. Although our findings support the use of multiplexed immunizations in the hen, we find that the number of immunogens cannot be stretched much higher than the handful that has been used in mammalian models to date.