Characterizing adjuvants’ effects at the murine immunoglobulin repertoire level

We developed an improved library prep protocol and standardized the data analysis pipeline for accurate repertoire profiling. In addition, two metrics were implemented to assess repertoire clone properties. We then studied systemically the effects of two adjuvants, CpG and Alum, on the Ig heavy chain repertoire using the ovalbumin (OVA) challenged mouse model. Ig repertoires of different tissues (spleen and bone marrow) and isotypes (IgG and IgM) were examined and compared in terms of sequence mutation frequency, IGHV gene usage, CDR3 length, rescaled Hill numbers for clonal diversity, and clone selection strength. As a result, Ig repertoires of different tissues or isotypes exhibited distinguishable profiles at the non-immunized steady state. Adjuvanted immunizations further resulted in statistically significant alterations in Ig repertoire compared with PBS or OVA alone immunized groups. Lastly, we applied unsupervised machine learning techniques – multiple factor analysis and clustering – to identify Ig repertoire signatures in different compartments and under varying immunizations. Age-matched C57BL/6J mice were immunized with ovalbumin with or without adjuvants (PBS control, Ova alone, Ova+CpG and Ova+Alum). There were three mice in each group. A week after the fourth immunization, BMs and spleens were harvested, total RNA were isolated, and IgSeq library was prepared. Sequencing was done on the Illumina HiSeq 2500 platform with pair-end 2x250 chemistry. Each sample received on average 1M paired reads. The data were demultiplexed and preprocessed with an newly-developed pipeline. Then we studied Ig repertoire profiles in different tissues (BM vs spleen) and under different adjuvant effects (CpG vs Alum) by comparing IGHV gene segment usage, mutation frequency, CDR3 length, clonal diversity, and clone selection strength.