A new comparative review published in the journal Animal Diseases synthesizes what is known about the antigenic proteins of poxviruses, the large double-stranded DNA viruses that include the agents of smallpox, mpox, lumpy skin disease and orf. Written by Siyang Pei, Saqib Ali, Shixin Ma, Aizhen Guo and Pan Tao, the review organizes poxvirus antigens not as a simple catalog of immunogenic molecules but according to virion localization, sequence conservation, antibody function and translational readiness, spanning the genera Orthopoxvirus, Capripoxvirus and Parapoxvirus.
The authors frame their synthesis against a persistent disease burden. Poxviruses complicate this task with a complex virion architecture: two principal infectious forms, the intracellular mature virion and the extracellular enveloped virion, and genomes encoding roughly 130 to 220 proteins depending on the species. Mature virion antigens are typically tied to attachment, entry and neutralization, whereas enveloped virion antigens are linked to cell-to-cell spread, dissemination and antibody effector mechanisms.
Using vaccinia virus as the reference framework, the review details the major mature virion surface antigens. H3, a 324-amino-acid protein of about 37.5 kilodaltons, is an immunodominant component that binds cell-surface heparan sulfate and mediates virion attachment. Its ectodomain adopts a mixed beta/alpha/beta fold with a broad positively charged surface consistent with a glycosaminoglycan-binding interface. Human H3-specific antibodies induced by vaccination have shown cross-neutralization or protection against multiple orthopoxviruses, including mpox and cowpox viruses, in experimental models. Notably, epitope mapping showed that V13I or D15N substitutions did not abolish antibody binding but converted a neutralizing epitope into a non-neutralizing one, indicating that neutralization depends on structural context rather than binding alone.
D8, a roughly 32-kilodalton mature virion membrane protein, promotes adsorption by binding chondroitin sulfate, particularly chondroitin sulfate E, through a carbonic anhydrase homology domain with a positively charged central dome. Antibody mapping revealed that monoclonal antibodies whose footprints overlap this dome differ markedly in their ability to block the D8-chondroitin sulfate interaction, and the neutralizing antibody LA5 prevents engagement of chondroitin sulfate by traversing the binding surface. Unlike H3, D8 appears restricted to Orthopoxvirus, lacking detectable homologs in the representative capripoxvirus and parapoxvirus genomes analyzed, which makes it especially relevant for orthopoxvirus-focused serology.
A27, a small 14-kilodalton surface protein, carries a flexible N-terminal heparin-binding domain and assembles mainly as a homotrimer whose coiled-coil architecture may organize multiple binding domains to increase avidity. Antibodies mainly recognize the N-terminal region spanning amino acids 21 to 40 and a C-terminal region spanning amino acids 81 to 110, but neutralizing activity concentrates among antibodies targeting the heparin-binding region. Even among closely related homologs, limited substitutions matter: the mpox virus homolog A29 differs by only three residues in this domain, yet a mpox-specific monoclonal antibody recognizing the A29 N terminus is non-neutralizing and fails to bind other orthopoxvirus A27 homologs broadly.
L1 stands apart from these attachment proteins. It is a myristoylated membrane protein that functions in a glycosaminoglycan-independent entry pathway as part of the entry-fusion complex, and its N-myristoylation is critical for producing infectious virions. L1-specific neutralizing antibodies target conformational epitopes, with residue D35 identified as a key determinant of a shared vulnerable surface recognized by antibodies 2D5 and 7D11. L1 is highly conserved across the family, and recent mapping of the mpox virus homolog M1 showed that the D35-centered surface is retained there; alanine substitution of D35 markedly reduces antibody binding to the virus.
On the enveloped virion, A33 and B5 dominate. A33 is a glycosylated protein forming a butterfly-shaped disulfide-linked homodimer with a C-type lectin-like domain. Antibodies targeting the variable apical loop show restricted breadth, whereas the antibody A27D7, which recognizes the more conserved dimerization groove, binds multiple orthopoxvirus homologs and protects in a mouse model. Human anti-A35 antibodies isolated from mpox-convalescent individuals target the homologous groove, block viral spread and protect mice and macaques. B5, a type I transmembrane glycoprotein with four short consensus repeat motifs, accounts for much of the enveloped virion-neutralizing capacity of vaccinia immune globulin, and efficient neutralization generally requires complement, meaning binding titers alone do not predict protection.
On the vaccine side, the review concludes that single-antigen strategies are usually insufficient. H3 alone protected roughly 80 percent of mice against a low-dose intranasal vaccinia challenge but failed to protect in a multivalent mpox mRNA vaccine study, whereas the multivalent formulation achieved complete protection. A27-based protection proved highly dependent on antigen format and challenge model, and a variola virus A27 formulation induced high antibody titers but failed to protect mice. L1 performed best when conformationally intact and well-adjuvanted, with a pseudovirus nanoparticle displaying L1 providing complete survival after lethal challenge. The classical four-antigen concept combining A27 and L1 with A33 and B5 illustrates the guiding principle: complementary coverage of both infectious forms and of antibody effector mechanisms.
For diagnostics, the review distinguishes exposure-level screening from differentiation of infected from vaccinated individuals. Conserved, immunodominant antigens such as H3, D8 and A27 homologs maximize sensitivity but cannot reliably distinguish mpox infection from prior vaccinia-based vaccination. The mpox homolog E8 provided the strongest separation between mpox-positive and pre-outbreak negative samples in recent multiplex evaluations. Differential strategies instead exploit antigenic differences: B22-family proteins, absent from vaccinia virus, support mpox-specific and capripoxvirus DIVA assays, while ratio-based comparisons of vaccinia B5 and mpox B6 reactivity improved discrimination between infection- and vaccination-associated responses.
The authors emphasize that cross-reactive binding, direct neutralization, spread inhibition, animal protection and cross-protection must be treated as distinct evidence levels, and that conserved structural motifs should remain testable hypotheses until functional endpoints are demonstrated. The vaccinia-centered framework transfers most reliably within Orthopoxvirus, where direct evidence for neutralization and protection is concentrated; its extension to capripoxviruses and parapoxviruses requires antigen-by-antigen, epitope-level validation in natural hosts. For vaccines and antibody countermeasures, the strongest current candidates are multivalent or multi-epitope targeting of mature and enveloped virion antigens, especially L1, A33 and B5, while future work points toward single-cell B-cell profiling, deep mutational scanning and structural prediction to identify functional conformational epitopes that survive authentic-virus and in vivo testing.
Subject of Research: Comparative review of antigenic proteins of poxviruses and their applications in vaccines, serological diagnostics and antibody countermeasures
Article Title: Poxvirus antigenic proteins: from fundamental biology to translational applications
Article References: Pei, S., Ali, S., Ma, S., Guo, A., & Tao, P. (2026). Poxvirus antigenic proteins: from fundamental biology to translational applications. Animal Diseases, 6(1), Article 51. https://doi.org/10.1186/s44149-026-00269-z
Image Credits: AI Generated
DOI: 10.1186/s44149-026-00269-z
Keywords: poxvirus, Orthopoxvirus, Capripoxvirus, Parapoxvirus, antigenic proteins, vaccinia virus, mpox, subunit vaccines, serological diagnostics, DIVA, monoclonal antibodies, neutralization
News Source: Kristina Jarvis. (October 29, 2026). Mapping Poxvirus Antigens From Virion Structure to Vaccines and Diagnostics. Scienmag.
