Abstract
The humanization of animal model immune systems by genetic engineering has shown great promise for antibody discovery, tolerance studies and for the evaluation of vaccines. Assessment of the baseline antibody repertoires of unimmunized model animals will be useful as a benchmark for future immunization experiments. We characterized the heavy chain and kappa light chain antibody repertoires of a model animal, the OmniRat, by high throughput antibody sequencing and made use of two novel datasets for comparison to human repertoires. Intra-animal and inter-animal repertoire comparisons reveal a high level of conservation in antibody diversity between the lymph node and spleen and between members of the species. Multiple differences were found in both the heavy and kappa chain repertoires between OmniRats and humans including gene segment usage, CDR3 length distributions, class switch recombination, somatic hypermutation levels and in features of V(D)J recombination. The Inference and Generation of Repertoires (IGoR) software tool was used to model recombination in VH regions which allowed for the quantification of some of these differences. Diversity estimates of the OmniRat heavy chain repertoires almost reached that of humans, around two orders of magnitude less. Despite variation between the species repertoires, a high frequency of OmniRat clonotypes were also found in the human repertoire. These data give insights into the development and selection of humanized animal antibodies and provide actionable information for use in vaccine studies.
Highlights
The humanization of animal model immune systems by genetic engineering has shown great promise for antibody discovery, tolerance studies and for the evaluation of vaccines
Sequence diversity is again enhanced in the germinal center by somatic hypermutation (SHM) and/or class switch recombination (CSR), two processes that depend on activation induced cytidine deaminase (AID)
The resulting amplicons were subjected to high throughput sequencing in conjunction with preprocessing and annotation by the Abstar analysis pipeline[17] (Methods) which resulted in a mean of ~3 × 106 processed heavy chain sequences and ~1.5 × 106 processed kappa chain sequences per transgenic animal (Table S1)
Summary
The humanization of animal model immune systems by genetic engineering has shown great promise for antibody discovery, tolerance studies and for the evaluation of vaccines. We characterized the heavy chain and kappa light chain antibody repertoires of a model animal, the OmniRat, by high throughput antibody sequencing and made use of two novel datasets for comparison to human repertoires. A major challenge in human vaccine science is finding appropriate models for studying antibody responses Animals such as mice, rabbits and monkeys have typically been used in the past and the small animals, in particular, have been favored for ease of immunization, cost reasons and the ability to extensively biopsy post-immunization. Many new transgenic animal models have been developed including rodents, chickens, rabbits and cows[8] These animal models have been used extensively for the discovery of monoclonal antibodies (mAbs)[9], tolerance studies[10] and more recently for modelling human antibody responses to vaccine candidates[3,4]. We provide the most thorough description of humanized transgenic rodent antibody repertoires to date and leverage a novel extremely deep human dataset to make comparisons with implications of immediate use as a reference for OmniRat immunization studies
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