IntroductionThe very recent reports (January, March 2025) of foot-and-mouth disease (FMD) episodes in Germany1 (last outbreak, 1988), Hungary and Slovakia2 (last outbreaks, 1970s), are a salutary reminder that even highly-surveilled regions of the world are vulnerable to the reemergence of this worrying disease. A highly contagious viral infection affecting cloven-hoofed animals like cattle, sheep, and pigs, FMD remains a persistent concern for the livestock industry3,4. Infection quickly disseminates among susceptible animals, often culminating in widespread epidemics causing illness, discomfort, and serious productivity losses. Classified by the World Organization for Animal Health (WOAH) as a reportable disease, FMD prompts trade restrictions by FMD-free countries on animals and products from endemic areas, with severe economic and social repercussions5,6. In regions where FMD is prevalent, such as Asia and Africa, vaccination remains the primary control method, mainly by inactivated virus vaccines7,8. Although such classic vaccines have demonstrated efficacy in disease control and prevention9, limitations such as the risk of live virus escape during production10, plus the broad antigenic variability of FMDV, with seven different serotypes11, each subdivided into genetically and geographically distinct topotypes, complicate control efforts12,13,14 and call for alternative approaches.In this regard, subunit vaccines, by definition free of any infectious agent, are currently viewed as smarter, safer alternatives to virus-based formulations. Within the subunit class, vaccines based on the multiple antigenic peptide concept pioneered by Tam (1988)15 are an increasingly attractive choice, with advantages like unquestioned safety, antigenic versatility, easy production and handling, and especially, marker features allowing distinction between infected and vaccinated animals (DIVA concept)16,17. In such peptide-based formulations, selected antigenic motifs can be purposefully integrated into higher-order assemblies eliciting both B- and T-cell immune responses18,19, conferring protection, adaptability, and multivalency20,21.Previously, we developed a vaccine prototype named B₂T, incorporating two copies of a conserved B-cell epitope from VP1 (residues 140–158) and one copy of a T-cell epitope from nonstructural protein 3A (residues 21–35), both derived from type-O FMDV. These epitopes were tethered onto a well-defined dendrimeric scaffold using chemoselective ligation methods22. A single dose of B2T, at either 2 mg or as little as 0.5 mg, sufficed to induce a rapid and robust immune response. By 15 days post-immunization (dpi), animals had developed high levels of FMDV-neutralizing antibodies and IFN-γ-secreting T cells. Moreover, 80% of the B2T-vaccinated animals were fully protected, exhibiting no FMD signs following viral challenge at 25 dpi23,24.Importantly, this dendrimeric platform has also been successfully adapted to harbor epitopes from other FMDV serotypes (e.g., C-S8) tailored for pig and cattle, as well as from other pathogens such as classical swine fever virus, demonstrating its versatility and potential for broader vaccine applications25,26,27. Also, given the relevance of swine major histocompatibility complex (SLA) polymorphism in immune response and vaccine design, SLA class II typing was performed in B2T-immunized animals, revealing associations between specific low-resolution haplotypes (Lr-Hp) and the magnitude of T cell responses, and to a lesser extent, neutralizing antibody levels28.Building upon these results, we have designed a new construct, B₂T-TB₂, generated by fusing two B₂T units via click chemistries (thiol-ene addition and copper(I)-catalyzed azide-alkyne cycloaddition, CuAAC), resulting in a dendrimer containing four B-cell and two T-cell epitope copies29. The aim was to enhance immunogenicity through increased valency and epitope density while retaining the molecular definition and safety of the original platform. Importantly, the increased potency allows for protection at reduced antigen doses, an essential feature for emergency use and scalable field application. Moreover, the modular design of B₂T-TB₂ enables rapid adaptation to emerging FMDV variants, as epitopes can be interchanged or updated without the lengthy production cycles required for conventional inactivated vaccines. This flexibility, combined with its potent and durable immunogenicity persisting for up to four months after immunization, positions B₂T-TB₂ as a promising next-generation FMD vaccine platform30.Moving on from these findings, we now evaluate for the first time the efficacy of B2T-TB2 in a challenge model in pigs, one of the two main FMDV hosts, as a crucial step toward validating the platform’s potential for real-world application. Our in-depth evaluation shows full clinical protection, broad intraserotype cross-neutralization, and early induction of neutralizing antibodies and IFN-γ-secreting T-cells following a single low-dose of B₂T-TB₂, all of them key features supporting a possible role in emergency FMD vaccination strategies. In addition, SLA typing has shown all analyzed pigs to have homogeneous haplotypes, among the most common in European farmed pigs.ResultsDesign and synthesis of the multiepitopic B2T-TB2 dendrimeric vaccine platform using click chemistryIn a quest for chemically well-defined peptide vaccines that display several immunorelevant epitopes in multivalent fashion, we have designed the dendrimeric B2T-TB2 platform. An outline of the synthetic route to this construct, where several peptide moieties are assembled by chemoselective conjugation techniques, is shown in Fig. 1.Fig. 1: Click chemistry synthesis of B2T-TB2.General scheme of the chemoselective click approach used for the synthesis of a B2T-TB2 multiepitopic peptide platform.Full size imageThe main components of B2T-TB2 were peptide building blocks embodying B- and T-cell epitopes [VP1(140–158), 3A(21–35), light green and light blue respectively, Fig. 1] of FMDV isolate O/UK/11/2001. They were readily produced by Fmoc solid phase peptide synthesis (SPPS), with site-specific extra residues bearing functional groups enabling chemoselective Lego-like buildup of B2T-TB2 by two distinct click chemistries. Thus, the T-cell moiety (pale blue) incorporated a Lys residue providing a branching point (dark blue), with both Nα and Nε amino groups derivatized as maleimide (orange). For its part, the B-cell epitope (light green) had an extra C-terminal Cys residue (green) for subsequent thiol−ene addition. Likewise, azide- or alkyne-functionalized residues (purple and red, respectively), enabling click CuAAC, were integrated into the T-cell peptide sequence as required. Finally, Lys-Lys dipeptide motifs were placed at both ends of the T-epitope sequence (wavy lines within light blue segment) to serve as a cleavage site for cathepsin D, a protease involved in in vivo antigen processing for MHC class II presentation31.With the above constituents in hand, Michael type thiol−ene click chemistry was applied to make the first two selectively functionalized units of B2T, itself an effective vaccine prototype22,23,24. Next, the two B2T variants were fused tail-to-tail by click CuAAC to give the B2T-TB2 dimeric end product (Fig. 1), where tetra- and bivalent display of B- and T-cell epitopes, is respectively achieved. Further details, including solutions to some often-ignored pitfalls found on assembling constructs of such complexity and size, can be found in (29).Single-dose B2T-TB2 vaccination confers complete clinical protection against FMDV challenge in swineThe B2T-TB2 vaccine described above has been reported to elicit robust B- and T-cell-specific responses in swine, the natural FMDV host, at reduced doses30. Animals immunized with B2T-TB2 show a consistent trend to antibody titers equal or higher than those elicited with the B2T prototype, which is known to provide full, long-lasting protection against viral challenge23,24. Here we compare the protective responses to FMDV challenge afforded by a low dose (0.5 mg) of either B2T-TB2 or its monomer version B2T, including clinical signs and other immune parameters associated to protection.All immunological and clinical data presented in this study were obtained from the same vaccination and challenge experiment, which included both vaccinated and control animals. The results of protection experiments are summarized in Table 1. Domestic pigs, in two different groups, were immunized once at day 0 with B2T or B2T-TB2, or non-immunized (PBS+adjuvant), and subsequently challenged with FMDV O-UK/11/2001 at 25 dpi. Animals were examined daily for clinical signs (see “Methods”) and judged protected if lesions were observed only at the infection site and/or at a single site32.Table 1 Evidence for protection in animals immunized with B2T-TB2 and B2T vaccine candidatesFull size tableAs expected, non-immunized (PBS-inoculated) pigs #11 and #12 showed typical clinical FMD signs at 4–5 dpc, with vesicular lesions on the snout and all four feet. In contrast, peptide-immunized pigs (#1 to #10) showed no clinical FMD signs after challenge with O-UKG/11/2001 (100% protection). Pig #8 died spontaneously at 25 dpi, prior to challenge, for causes unrelated to the experiment, hence no protection and viremia data are reported for this animal. In general, a correlation between low body temperature and protection was observed. Thus, animals protected by B2T or B2T-TB2 immunization did not develop fever (