Preprint / Version 1

Tuning Hapten Density on Protein Conjugates Programs Vaccine Immunity via Carrier Protein Processing

This article is a preprint and has not been certified by peer review.

Authors

    Rui Liu,  
    Rui Liu
    • China Agricultural University
    • College of Veterinary Medicine
    Xuezhi Yu,  
    Xuezhi Yu
    • China Agricultural University
    Yingjie Zhang,  
    Yingjie Zhang
    • China Agricultural University
    Bochen Lv,  
    Bochen Lv
    • China Agricultural University
    Li Nan,  
    Li Nan
    • China Agricultural University
    Linhui Zhang,  
    Linhui Zhang
    • China Agricultural University
    Yinkun Liu,  
    Yinkun Liu
    • China Agricultural University
    Fanjian Zhang,  
    Fanjian Zhang
    • China Agricultural University
    Jing Zhang,  
    Jing Zhang
    • China Agricultural University
    Kai Wen,  
    Kai Wen
    • China Agricultural University
    Jianzhong Shen,  
    Jianzhong Shen
    • China Agricultural University
    Zhanhui Wang
    Zhanhui Wang
Categories
Immunology & Microbiology
Keywords
Hapten density; Antigen processing; Conformational rigidity; Chemical immunology; Vaccine design

Abstract

Hapten-protein conjugates are widely used in vaccines against tumors and addictive drugs, antidotes to toxins, and diagnostic antibodies for hazard detection. However, how hapten density of conjugates governs the ensuing immune response remains poorly defined, hindering rational hapten-based vaccine design. Here, we systematically investigate the relationship between hapten density and immunogenicity using a model system of AMDH-BSA conjugates with precisely controlled and well-characterized hapten densities spanning 1.8 to 36.3. We demonstrate that low-to-moderate hapten densities (1.8-17.2) elicit robust humoral immunity, characterized by potent germinal center B cell responses, CD4+ T cell activation, and high-affinity antibody production. In contrast, excessive hapten loading (26.7 to 36.3) compromises immune responses through a chemically defined mechanism whereby the carrier protein is stabilized into a rigid, α-helix-rich conformation that sterically hinders proteolytic cleavage, thereby reducing the diversity of processed peptides. This impaired antigen processing leads to defective CD4+ T cell activation, attenuated germinal center reactions, and ultimately compromised humoral immunity. Bulk and single-cell transcriptomics corroborate this mechanism, revealing global downregulation of antigen-processing and B cell receptor signaling pathways, while TCR/BCR repertoire sequencing shows that higher hapten density conjugates altered V(D)J usage and fail to drive effective clonal expansion. Our findings establish hapten density as a tunable chemical parameter for programming vaccine immunity, providing a quantitative strategy for the rational design of next-generation hapten-based immunogens.

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Liu, R., Yu, X., Zhang, Y., Lv, B., Nan, L., Zhang, L., Liu, Y., Zhang, F., Zhang, J., Wen, K., Shen, J., & Wang, Z. (2026). Tuning Hapten Density on Protein Conjugates Programs Vaccine Immunity via Carrier Protein Processing. LangTaoSha Preprint Server. https://doi.org/10.65215/LTSpreprints.2026.09.21.000348

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