Background: Human adenovirus type 4 (HAdV-4), the sole member of species Human mastadenovirus E (HAdV-E), is of zoonotic origin and has established stable human transmission through recombination, conferring distinctive host adaptation and pathogenicity. It causes respiratory and ocular diseases, with a significant risk of severe pneumonia in children. No targeted antivirals are approved for routine use, leaving supportive care as the primary management. China bears a relatively high HAdV-4 disease burden in Asia. Methods: To generate neutralizing nanobodies (Nbs) against HAdV-4, we employed an alpaca immunization strategy using hexon protein from Ad4-RI67 strain, followed by the isolation of hexon-specific nanobodies. The epitope competition and molecular docking was employed to analysis the binding site of the Nbs’. We engineered VHH-Fc fusions by conjugating VHH domains to human IgG1 Fc. The lead candidate, NVA17, showed efficacy in both in vitro and in vivo (Stat1^+/− mouse model). Flow cytometric analysis was employed to assess the downstream immune effects of NVA17 in vivo. Its intracellular neutralization mechanism was further investigated through confocal microscopy by examining co-localization in TRIM21-overexpressing and knockdown cells. Results: The isolated nanobodies revealed epitopes distinct from those targeted by known antibodies. The lead candidate NVA17 demonstrated potent neutralizing activity in vitro (IC₅₀ < 10 ng/mL). In the Stat1^+/− mouse model, NVA17 provided complete protection against lethal challenge, significantly reduced viral load in the lungs, and ameliorated pathological damage. NVA17 treatment dose-dependently reversed the virus-induced reduction in immune cell counts and enhanced cytotoxicity, suggesting a systemic immunomodulatory effect. Mechanistic studies indicated that the antiviral activity of NVA17 partly depends on the TRIM21-mediated antibody-dependent intracellular neutralization (ADIN) pathway, whereby TRIM21 terminates the viral life cycle by promoting viral degradation via K48-linked ubiquitination. Conclusions: We have identified multiple antibody candidates, particularly NVA17, with significant therapeutic potential for developing antibody-based treatments against HAdV-4. This offers a targeted intervention strategy to counter the current lack of specific antiviral therapies.

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.