Background and Aim: African swine fever (ASF) is a highly virulent and contagious viral disease caused by the ASF virus (ASFV). It has a significant impact on swine production throughout the world, while existing vaccines and specific treatments remain ineffective. ASFV p30 is a potent antigenic protein that induces protective antibodies immediately after infection; however, most recombinant p30 is insoluble. This study aimed to improve the solubility, yield, and purity of recombinant p30 by tagging it with a small ubiquitin-like modifier (SUMO) and modifying the protein purification process. Materials and Methods: SUMO fused with ASFV p30 (SUMO-p30) and p30 alone were cloned and expressed in Escherichia coli. SUMO-p30 and p30 solubility and expression levels were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein purification was modified by combining ammonium sulfate precipitation method with affinity chromatography. In addition, large-scale production of all versions of p30 were compared using SDS-PAGE and western blotting, and the purified p30 was used to develop the indirect enzyme-linked immunosorbent assay (ELISA). Results: The solubility and expression levels of SUMO-p30 were dramatically enhanced compared with that of p30. Modification of the purification process significantly increased purified and soluble SUMO-p30 and p30 yields by 6.59 and 1.02 μg/mL, respectively. Large-scale production confirmed that this procedure increased the quantity of recombinant p30 while maintaining protein purity and immunogenicity. The p30-based indirect ELISA was able to discriminate between positive and negative serum samples with statistically significant differences in mean optical density 450 values (p < 0.001). Conclusion: This study demonstrates the enhancement of solubility, purity, and yield of ASFV p30 expressed in E.coli by SUMO fusion tagging and combining ammonium sulfate precipitation with affinity chromatography for protein purification. These positive effects were sustained in large-scale production. Cleavage and removal of hexahistidine-SUMO tag from the fusion protein by protease may not be suitable when handling a large amount of the protein. However, the SUMO-fused p30 retained strong immunoreactivity to convalescent swine serum, indicating its application in immunization and diagnostic purposes. The expression and purification procedures in this study could be applied to increase solubility, quality, and quantity of other recombinant proteins as well. Keywords: African swine fever, diagnosis, indirect enzyme-linked immunosorbent assay, p30, protein expression, purification.

Background/Objectives: African swine fever (ASF) is a devastating disease affecting the swine industry globally. Development of safe and effective vaccines is an urgent need. This study aimed to evaluate, caASFV001-MA52, a cell-adapted ASFV strain derived from serial passaging in MA-104 cells, as a promising live-attenuated vaccine (LAV) candidate against virulent ASFV infection. Methods: Seven-week-old, crossbred pigs were immunized with caASFV001-MA52 (at a dose of 10⁵ TCID₅₀) and subsequently challenged with a lethal dose of virulent ASFV. Vaccine efficacy was measured through clinical monitoring, immunological and virological analyses, and pathological assessments of tissue protection and viral load reduction. Safety was critically assessed, particularly regarding its profile in animals with concurrent endemic porcine infections, including PCV, PRRSV and S. suis. Results: caASFV001-MA52 exhibits partial protection (70–80%) against the lethal challenge. Vaccinated and challenged survivors exhibited reduced viral loads and significantly alleviated pathological lesions compared to controls. Safety evaluations revealed that the vaccine’s profile is susceptible to concurrent infection. Pigs co-infected with endemic porcine pathogens showed increased virulence and mortality following vaccination. Although vaccination temporarily reactivated latent viral infections (PCV2, PCV3, and PRRSV), most surviving pigs effectively controlled and eliminated these co-infections. Conclusions: The caASFV001-MA52 strain demonstrates promising immunogenicity and protection against lethal challenges, supporting its continued development as an LAV candidate. However, the observed safety concerns regarding concurrent infections emphasize the critical need for veterinary health surveillance during its future practical application.

Background: Middle East Respiratory Syndrome Coronavirus (MERS-CoV) causes severe respiratory illness in humans and currently lacks an approved vaccine. The Newcastle disease virus (NDV) vector is a well-established, safe, and effective platform for vaccine development. With recent advancements in stabilizing coronavirus spike proteins to enhance their antigenicity, this study aimed to determine whether modifications to the MERS-CoV spike protein could improve its presentation on NDV particles, allowing the resulting virus to be used as an inactivated vaccine. Methods: We codon-optimized the gene encoding the ectodomain of the MERS-CoV spike protein and incorporated modifications at the S1/S2 and S2’ cleavage sites, along with a proline substitution at residues V1060-L1061. This modified spike gene was inserted into the NDV genome to create the NDV-S_MERS virus. After purification and inactivation, the vaccine’s immunogenicity was assessed in mice. Results: Mice immunized with the inactivated NDV-S_MERS vaccine developed robust anti-spike IgGs, neutralizing antibodies, and cellular immune responses. The study demonstrated that modifications to the MERS-CoV spike protein were essential for its effective presentation on NDV particles. Additionally, the spike gene insert remained stable through five egg passages, confirming the vector’s stability. Conclusions: Engineering the MERS-CoV spike protein is crucial for its successful display on NDV particles. The strong immune responses elicited by the NDV-S_MERS vaccine in mice highlight that NDV is a promising, safe, and effective platform for MERS-CoV vaccination.