Chemical cross-linking facilitates antigen uptake and presentation and provides improved protection from Mpox with a dual-antigen subunit vaccine.

Antigen uptake, processing, and presentation are crucial for the immune responses of protein-based vaccines. Herein, we introduced a reversible chemical cross-linking strategy to engineer protein antigens, which can be tracelessly removed upon antigen-presenting cell (APC) uptake and cellular reduction. The chemically cross-linked antigen proteins presented significantly enhanced uptake and epitope presentation by APC. We applied this strategy to monkeypox virus antigens A29L and A35R to construct dual-antigen subunit vaccines. Our results revealed that chemical cross-linking was robust enough to improve both proteins' APC uptake and lymph node accumulation, with each protein being chemically cross-linked and administered separately. In vivo validation revealed that the chemical cross-linking of the two antigen proteins improved immune responses, with increases in antigen-specific antibody and live virus-neutralizing antibody production. Monkeypox virus challenge experiments revealed that dual-antigen vaccines prepared via the chemical cross-linking strategy mitigated tissue damage, reduced the virus load, and extended mouse survival, which proved that the chemical cross-linking strategy is valuable for protein-based subunit vaccine development. In consideration of the current threats from the monkeypox virus and potential future emerging pathogens, the chemical cross-linking strategy provide powerful tools.