Introduction: The immune protection conferred by Bacillus Calmette-Guérin (BCG) is insufficient, underscoring the urgent need for novel strategies to control tuberculosis (TB). Although antibody-mediated responses targeting mycobacterial cell wall antigens have been observed during infection, the precise mechanism by which antibodies resist TB pathogens remains unclear.

Objectives: This study aims to elucidate the molecular mechanism by which protective monoclonal antibodies (mAbs) against Mycobacterium bovis (M. bovis) cell wall antigens resist M. bovis infection.

Methods: In this study, we employed immunization with M. bovis cell wall antigens to screen for protective mAbs and identify their corresponding antigens. The underlying mechanisms were investigated using Western blotting, molecular docking, CRISPR/Cas9, flow cytometry, ELISA, surface plasmon resonance (SPR) and animal experiments.

Results: MAb 2H3 targeted a conserved conformational epitope (161KKAAPAKKAAPAKKAAPA178) in HBHA's heparin-binding domain, inhibiting bacterial attachment to epithelial cells and phagocytosis by macrophages and reducing bacterial loads in mice. HBHA interacted specifically with NRP1-coupled HS chain, whose interaction can be disrupted by mAb 2H3. ATWLPPR peptide TFA, a selective NRP1 inhibitor, reduced attachment and virulence of M. bovis through decreasing the NRP1 expression on cell membrane.

Conclusion: By screening of a protective mAb (2H3) against cell wall antigens and elucidating its mechanism in blocking the interaction between adhesin (HBHA) and host cell co-receptor (NRP1), this work establishes a valuable framework for advancing anti-tuberculosis research and prophylactic development.

Keywords: Entry; HBHA; M. bovis; Novel mycobacterialco-receptor NRP1; Protective mAb.