Grouped immunogens are hierarchically recognized by host to generate antibodies

Introduction: Microorganisms with dozens, hundreds, or thousands of proteins invade hosts. Whether the hosts simultaneously recognize all or a part of them is unknown. Methods: recombinant outer membrane (OM) proteins were pooled to immunize mice, and the resulting antisera were analyzed using a protein microarray. Proteins recognized in each cycle were removed, and the remaining proteins were re-pooled for subsequent immunizations. Bioinformatics analysis of B cell epitope scores and GO functional categories was performed across the identified immunogen hierarchies. Results: Among the 68 OM proteins, only 18 were recognized to generate antibodies, designated as the first hierarchy of immunogens. When the other 50 proteins were grouped to immunize mice, 15 were detected to have their corresponding antibodies, designated as the second hierarchy of immunogens. This procedure was repeated four times, leading to the identification of 16 and eight proteins as the third and the fourth hierarchy of immunogens, respectively, and 11 residual proteins as the fifth hierarchy of immunogens. Bioinformatics analysis showed a negative correlation of B cell epitope score with increasing hierarchies, suggesting that the score plays an essential role in hierarchy recognition. In addition, more proteins with transport and fewer proteins with metabolic process were found in the combination of the first, second, and third than the combination of the fourth and fifth hierarchies of immunogens. Discussion: The host's immune system hierarchically recognizes antigens to mount antibody responses. This finding highlights the way in understanding differentially neutralizing antibodies during infections caused by different microorganisms.