Quil a

All animal experimental protocols were approved by the Estonian Project Authorization Committee for Animal Experiments on November 5, 2020, and April 16, 2021 (approval numbers 177 and 191, respectively); all experiments were performed in accordance with the European Communities Directive of September 2010 (2010/63/EU); and the study was performed in compliance with the ARRIVE guidelines. Cows (Estonian Holstein breed) were intramuscularly immunized twice with SARS CoV-2 S1 RBD protein (0.1 mg of antigen per injection) followed by 1 boost with SARS-CoV-2 trimeric S protein (0.5 mg per injection) in proper adjuvant solutions. For adjuvant, a mixture of Quil-A (0.5 mg/mL, Invivogen, Toulouse, France) and Imject Alum (20 mg/mL, ThermoFisher Scientific) was used for the immunization procedure, except for 5 cows for which Quil-A (0.5 mg/mL, Invivogen) alone was used. The initial immunization was performed 56–72 days before the expected calving date. The second immunization was administered three weeks after the first injection, and the boost was performed two weeks after the second injection. After calving, the cows continued to receive reimmunizations, equal to the initial immunizations, with proteins at 0.1 mg or 0.15 mg of different VoCs (Table 1.; Alpha, Beta, Gamma or Delta) once every 5–6 weeks.

The animal experiments were conducted as previously described (Lee et al., 2022 (link); Kim et al., 2023 (link)). We performed animal experiments to evaluate the potential of isoprinosine to serve as a vaccine adjuvant and validate the protective effects mediated by the FMD vaccine containing isoprinosine in the early stages of viral infection. The vaccine composition used in the experiments for the PC group was as follows: purified antigens (O PA2 and A YC) (0.375 + 0.375 μg/dose/100 μL), ISA 206 (Seppic, Paris, France; 50% w/w), 10% Al(OH)₃, and 15 μg/dose/mouse Quil-A (InvivoGen, San Diego, CA, USA), in a total volume of 100 μL. The Exp group received vaccines with the same formula as the PC group, adding 100 μg isoprinosine/dose/mouse as an adjuvant via the same route. Mice in the NC group were administered an equal volume of PBS (pH 7.0) via the same route. Mice (6–7 weeks old, n = 5/group) were vaccinated by means of an I.M. injection [0 days post-vaccination (dpv)] and challenged with FMDV (100 LD₅₀ of O/VET/2013 or 100 LD₅₀ A/Malay/97) by means of an I.P. injection at 7 dpv. Survival rates and body weight were monitored up to 7 dpc (Lee et al., 2022 (link); Kim et al., 2023 (link)).

To assess the induction of early, mid-, and long-term immune responses of the FMD vaccine with D-galacto-D-mannan in target animals, a study was conducted as previously described (7 (link), 29 (link)). Animals were randomly divided into three groups; NC, PC, and Exp group (n = 5–6/group). The vaccine formula for the PC group was as follows: purified antigens type O (O PA2) and type A (A YC) (15 + 15 μg/dose/mL), 10% Al(OH)₃, ISA 206 (Seppic; 50% w/w), and 150 μg/dose/pig Quil-A (InvivoGen), in a total volume of 1 mL. The Exp group received test vaccines with the same formula as the PC group, with the addition of 1 mg of D-galacto-D-mannan/dose/pig as an adjuvant, via the same route. NC group pigs were administered an equal volume of PBS. After primary vaccination via IM injection, a booster shot was administered via the same route 28 dpv. Sera were collected from the vaccinated pigs 0, 7, 14 (early), 28, 42 (mid-term), 56, and 84 (long-term) dpv for serological assays. The local reaction was confirmed at the vaccination site (neck) by sacrificing pigs at 84 dpv. Under the observation of a veterinarian, both sides of the neck were cut into circular shapes with a diameter of 20 cm and a length of 40 cm and cut into 1.5-cm-thick pieces; the effect of suppressing abnormal meat formation was confirmed through visual inspection.