African Swine Fever Virus (ASFV) is a large dsDNA virus that encodes at least 150 proteins. The complexity of ASFV and lack of knowledge of effector immune functions and protective antigens have hindered the development of safe and effective ASF vaccines. In this study, we constructed four Orf virus recombinant vectors expressing individual ASFV genes B602L, -CP204L, E184L, and -I73R (ORFV^Δ121-ASFV-B602L, -CP204L, -E184L, and -I73R). All recombinant viruses expressed the heterologous ASFV proteins in vitro. We then evaluated the immunogenicity of the recombinants by immunizing four-week-old piglets. In two independent animal studies, we observed high antibody titers against ASFV p30, encoded by CP204L gene. Using Pepscan ELISA, we identified a linear B-cell epitope of 12 amino acids in length (Peptide 15) located in an exposed loop region of p30 as an immunodominant ASFV epitope. Additionally, antibodies elicited against ASFV p30 presented antibody-dependent cellular cytotoxicity (ADCC) activity. These results underscore the role of p30 on antibody responses elicited against ASFV and highlight an important functional epitope that contributes to p30-specific antibody responses.

Influenza A viruses (IAV-S) belonging to the H1 subtype are endemic in swine worldwide. Antigenic drift and antigenic shift lead to a substantial antigenic diversity in circulating IAV-S strains. As a result, the most commonly used vaccines based on whole inactivated viruses (WIVs) provide low protection against divergent H1 strains due to the mismatch between the vaccine virus strain and the circulating one. Here, a consensus coding sequence of the full-length of HA from H1 subtype was generated in silico after alignment of the sequences from IAV-S isolates obtained from public databases and was delivered to pigs using the Orf virus (ORFV) vector platform. The immunogenicity and protective efficacy of the resulting ORFV^Δ121conH1 recombinant virus were evaluated against divergent IAV-S strains in piglets. Virus shedding after intranasal/intratracheal challenge with two IAV-S strains was assessed by real-time RT-PCR and virus titration. Viral genome copies and infectious virus load were reduced in nasal secretions of immunized animals. Flow cytometry analysis showed that the frequency of T helper/memory cells, as well as cytotoxic T lymphocytes (CTLs), were significantly higher in the peripheral blood mononuclear cells (PBMCs) of the vaccinated groups compared to unvaccinated animals when they were challenged with a pandemic strain of IAV H1N1 (CA/09). Interestingly, the percentage of T cells was higher in the bronchoalveolar lavage of vaccinated animals in relation to unvaccinated animals in the groups challenged with a H1N1 from the gamma clade (OH/07). In summary, delivery of the consensus HA from the H1 IAV-S subtype by the parapoxvirus ORFV vector decreased shedding of infectious virus and viral load of IAV-S in nasal secretions and induced cellular protective immunity against divergent influenza viruses in swine.

Serological assays are essential for infectious disease surveillance and vaccine evaluation because they detect antibody responses that persist beyond the period of active viral replication. The reliability of these assays, however, depends heavily on the structural and functional stability of the antigens used. Recombinant proteins—such as the SARS‑CoV‑2 Spike—are vulnerable to degradation from suboptimal handling, storage, and repeated freeze–thaw cycles, which can compromise assay performance. To address this concern, we systematically assessed the impact of three commonly used aliquoting and freezing approaches on the long‑term stability of a recombinant SARS‑CoV‑2 Spike protein: (1) aliquoting on wet ice followed by storage at −80 °C, (2) aliquoting on dry ice followed by storage at −80 °C, and (3) aliquoting on dry ice followed by snap‑freezing in a methanol–dry ice bath. Aliquots from two manufacturing lots were evaluated over six months using multiple quality metrics, including purity, structural integrity [Analytical Size Exclusion Chromatography (AnSEC) and Thermal Shift Assay (TSA)], and functional performance in a validated IgG ELISA. Across all conditions and time points, antigen purity remained within the ideal A260/A280 range (0.6–0.8), with minimal nucleic acid contamination. ELISA performance demonstrated consistently low background and high sensitivity, specificity, and precision. Structural analyses confirmed stable protein size and conformation (AnSEC elution peaks within ±1.0 mL of baseline) and unchanged thermal stability (Tm within ±2.0 °C). These findings indicate that all tested methodologies effectively preserved antigen integrity under controlled laboratory conditions, providing a foundation for standardized SOPs while acknowledging that real‑world variability may influence outcomes.