Single-Cell BCR and Transcriptome Analysis Revealed Potent Neutralizing Antibodies in Response to HAdV-4 Infection

Human adenovirus type 4 (HAdV-4) is a predominant serotype causing acute respiratory infections, for which no antibody drugs have been clinically approved to date, resulting in an urgent unmet medical need. This study aimed to identify highly potent neutralizing antibodies by dissecting the B cell immune response after infection, thereby laying a foundation for vaccine design and antiviral therapy. Peripheral blood mononuclear cells (PBMCs) were isolated from a convalescent patient who exhibited the most robust neutralizing antibody titers among 16 HAdV-4 convalescent volunteers. A total of 2710 B cells were isolated from the peripheral blood of this patient, and the characteristics of B cell subsets and B cell receptor (BCR) repertoires were comprehensively analyzed by integrating scRNA-seq and scVDJ-seq technologies. Candidate antibodies were subsequently selected for a series of functional and mechanistic validations based on the characteristics of BCRs and origination. Transcriptomic profiling classified memory B cells into three functionally heterogeneous subpopulations designated as MB sub1, sub2 and sub3. Combined with the characteristics of BCR, 92 selected antibodies from sub2 and sub3 were synthesized for further functional validation. Three potent neutralizing antibodies were successfully identified, among which 1QH7 demonstrated potentially potent virus neutralization capability in vitro and conferred robust in vivo protection in the Stat1-deficient mouse model. We further employed AlphaFold 3 to predict the complex structure of 1QH7 with the HAdV-4 Hexon protein and validated the key binding residues through site-directed mutagenesis. Additionally, the study elucidated the viral neutralization mechanism mediated by TRIM21. We established an efficient antibody screening strategy based on integration of scRNA-seq and scVDJ-seq and isolated a potent neutralizing antibody, 1QH7. This study lays a solid foundation for the rapid pathogen-bait-free, cell subset-guided screening antibody route for novel emerging infectious disease outbreaks.