Abstract
Orthopoxviruses are zoonotic DNA viruses of continuing public health importance, as highlighted by recent global mpox outbreaks. The high antigenic similarity among orthopoxviruses enables cross-reactive immune responses but complicates the development of serological diagnostic systems for serosurveillance, assessment of previous exposure, evaluation of vaccine-induced immunity, and species differentiation. This review summarizes current knowledge regarding the structure, biological functions, and diagnostic and vaccine potential of major orthopoxvirus recombinant antigens. Particular attention is given to the genomic organization of Orthopoxvirus, differences between intracellular mature virion (IMV) and extracellular enveloped virion (EV) forms, and the characteristics of key antigens, including A29, M1, H3, A35, and B6. Comparative phylogenetic analyses demonstrated a high degree of conservation among structurally important orthopoxvirus proteins while also identifying variable regions potentially relevant for differential diagnostics. Available evidence indicates that IMV-associated proteins are primarily involved in virus neutralization, whereas EV-associated antigens contribute mainly to limiting viral dissemination. Consequently, multicomponent antigen strategies combining IMV- and EV-associated proteins appear to provide the greatest potential for the development of effective serological assays and next-generation subunit vaccines. Overall, rational antigen selection should be based on integrated evaluation of structural, functional, and immunological properties to optimize orthopoxvirus diagnostic and vaccine platforms.