Abstract:

The aim of this study was to develop a nanoparticle vaccine targeting the E2 protein of classical swine fever virus (CSFV) and analyze its immunogenicity. Using the E2 gene sequence of Shimen strain of CSFV as template, the human codon was optimized in its extracellular domain. E2, E2-pFc (fusion expression of E2 with Fc fragment of porcine antibody), SpyTag-E2 (fusion expression of E2 with SpyTag) and the nanoskeleton protein LS (Lumazine Synthase-protein A) were prepared by 293F mammal cell eukaryotic expression system. Nanoskeleton protein mi3 (mi3-SpyCatcher) was purified by prokaryotic expression system and then LS and mi3 assembled into nanoparticles LS-E2-pFc NP and mi3-E2 NP, which were identified by transmission electron microscopy and dynamic light scattering test, and each protein was emulsified with ISA201 adjuvant to prepare subunit vaccines. Thirty-five Japanese white rabbits (1.5-2.0 kg) were randomly divided into 7 groups. Blank control group (n=3), PBS negative control group (n=3), E2 group (n=5), E2-pFc group (n=5), LS-E2-pFc NP group (n=5), mi3-E2 NP group (n=5), and CSFV E2 commercial subunit vaccine group (n=3). On 0 d and 21 d, intramuscular immunization was administered, and on 42 d, CSFV C strain was challenged with 100 RID₅₀ via ear vein. The specific antibody level, neutralizing antibody level, body temperature response and spleen viral load of rabbits were evaluated by blocking ELISA, neutralizing test, temperature monitoring and RT-qPCR. The results showed the successful expression of all component proteins, with single-band profiles and high purity. The average particle size of mi3-E2 nanoparticles (68 nm) was larger than mi3 (42.73 nm), and LS-E2-pFc nanoparticles (42.4 nm) was larger than LS (13 nm). Immunization results showed that E2, E2-pFc, LS-E2-pFc NP, mi3-E2 NP vaccine and commercial subunit vaccine all had good immune effect, and 7 days before challenge, the specific antibody blocking rate in serum was above 89%, with no significant difference between immunized groups (P>0.05). After challenge, the PBS negative control group showed typical stereotyped heat reaction, while the other immune groups showed no obvious changes in body temperature. Seven days post-challenge, the titers of neutralizing antibodies of E2, E2-pFc, LS-E2-pFc NP, mi3-E2 NP vaccine and commercial subunit vaccine groups were 1:10 392.4, 1:37 168.4, 1:75 473.6, 1:20 201.8 and 1:2 407, respectively, with the LS-E2-pFc NP group showing the highest neutralizing antibody levels. RT-qPCR results showed that one rabbit in the E2 group had a low viral load of 5.83 copies ·μL^-1, while no viral load was detected in other immunized groups. The above results showed that LS-E2-pFc NP immunized group had the best effect and the highest titer of neutralizing antibody. This "marker" vaccine allows for differential diagnosis of naturally infected and vaccinated animals, helping to eradicate CSF in swine farms.

Key words: classical swine fever virus, E2 subunit vaccine, self-assembled nano vaccines, neutralizing antibody