Inactivated African Swine Fever Virus Vaccine Induces Safe and Efficient Immune Protection Through Mucosal Immunity

1. Our Method for Preparing Inactivated Vaccines Is As Follows:

① Improved sample processing. Sterilely collect liver and spleen tissues from typical African swine fever-infected pigs, remove fat and connective tissue, weigh them, grind thoroughly, mix with 1×PBS buffer solution at a ratio of 1:10, homogenize, freeze-thaw three times, centrifuge at 5000r/min for 15 minutes, take the supernatant, filter through a 0.45μm sterilizing filter, and set aside at 4 °C [6].

② viral load testing. ASFV real-time PCR Kit (Beijing MingRiDa Technology Development Co.,Ltd., Beijing, China) was used according to the manufacturer’s protocol. The test results showed that the CT value of the prepared samples was less than 30.

③ Preparation of inactivated vaccines. Add 0.1% β-propiolactone to the viral suspension with a real-time PCR detection CT value less than 30, incubate at 4°Cwith shaking to inactivate, add another 0.1% β-propiolactone after 24 hours, inactivate for 96 hours, then hydrolyze β-propiolactone at 37 °C for 2 hours, and store at 4 °C [7].

2. Safety Testing:

After streaking the inactivated vaccine on a blood agar medium and incubating it at 37°C for 72 hours, no contaminants were detected. Subsequently, mucosal vaccination was performed on five healthy, non-immunized piglets. Each piglet was inoculated with 1 ml vaccine nasally and 10 ml orally every 7 days for 3 times. During the 30-day observation period, no adverse reactions or abnormal manifestations were noted in these piglets. This outcome demonstrates the outstanding safety of the vaccine [8].

3. Effective in Application:

One hundred and fifty ASFV-antigen-and-antibody-negative pigs, each weighing about 20 kg, were randomly divided into a 140-pig experimental group and a 10-pig control group.The experimental group received mucosal immunization with an inactivated vaccine: 1 ml intranasally and 3 ml orally per pig, administered every 7 days for 3 times. The control group was given 4 ml of PBS buffer per pig, following the same immunization schedule.After the three-round immunization, all 150 pigs were placed in an ASFV-infected pig farm. They were randomly penned in groups of 10. This setup enabled natural ASFV exposure through cohabitation with infected pigs, simulating a precise cross-infection scenario.The 100-day experiment included randomly sampling 5 pigs from each group every 10 days. Blood was centrifuged to obtain serum, which was pooled separately for each group for analysis. Real-time PCR was used to detect the ASFV viral load in the serum, and pen-level mortality was closely monitored.As presented in Table 1, the experimental group's viral load decreased steadily and vanished by the 70th day. By the end of the experiment, 98.6% of the experimental group pigs survived, with no adverse reactions or abnormalities. In contrast, the control group suffered 100% mortality within 25 days.

4. Discussion

The inactivated ASF vaccine, once judged ineffective upon injection, might induce elevated levels of secretory immunoglobulin A (SIgA) following mucosal immunization via means like nasal spraying and oral intake. Mucosal immunization with this vaccine not only achieves outstanding results but is also straightforward and safe. The century-old problem has been effortlessly solved.