Synthesis Of Empty Capsid Particles Of Asia 1 Foot-and-Mouth Disease Virus In Insect Cells And Their Immunogenicity In Guinea Pigs

The outbreaks of foot-and-mouth (FMD) serotype Asia 1 in large areas of China had caused great economic losses since it occurred in Jiangsu Province in 2005. The FMD virus (FMDV) strain that cause these outbreaks was a new isolate different from the history strain Asia 1/YNBS/1958 once appeared in Yunnan province. This new isolate was given the name Asia 1/JS/2005. The virus strain mainly affects ruminant animals and cause clinical and subclinical infection in cattle and sheep, however, pig could also be infected and several clinical cases were reported. In this study, we investigated the assembly of Asia 1 foot-and-mouth disease virus empty capsid particles in insect cells using baculovirus expression system based on this new incurred Asia 1 FMDV, and with the hope to achieve a safe, high potency subunit vaccine based on empty capsid particles for Asia 1 FMDV.The P12A and 3C sequences of cell passaged Asia 1/JS/2005 FMDV were amplified and sequenced. The results showed that the whole length of structural protein coding region was 2145 base pairs (bp) and encoded 715 amino acids (aa). The four capsid proteins, VP4, VP2, VP3, and VP1, were 207bp, 648bp, 657bp, 633bp in length, respectively, and the deduced amino acid sequences were 69aa, 216aa, 219aa, and 211aa. Residues at the VP4/VP2, VP2/VP3, and VP3/VP1 cleavage sites were Ala/Asp, Glu/Gly, and Gln/Thr, which could be recognized and processed by 3C proteinase. The RGD motif in VP1 protein of FMDV changed into RDD in the cell passaged Asia 1/JS/2005. Positions 140aa and 143aa in VP3 were histidine residues which were likely involved in acid sensitivity of empty capsid particles. 2A was 48bp in length and the deduced amino acid sequences were about 16aa, and 3C was 639bp in length and the deduced amino acid sequences were about 213aa.Three different strategies were used to investigate the assembly of empty capsid particles of Asia 1 FMDV. (1) Structural protein P12A and proteinase 3C genes were inserted into the baculovirus transfer vector pMelBac B with a melittin secretion signal sequence to test whether the target proteins could be secreted into culture medium, respectively. (2) A recombinant baculovirus that simultaneously expressed the genes for the P12A and 3C proteins from individual promoters was constructed using baculovirus transfer vector pFastBac? Dual to assembly empty capsid particles in insect cells. (3) Study the factors that affect the capsid stability after assembly in insect cell medium with pH value 6.2. It is demonstrated that the icosahedral virus capsid of FMDV dissociates into 12 pentamers at pH below 7. In this study, two histidine residues at positions 140 and 143 in VP3 were changed into leucine by site-directed mutation to improve acid resistance of empty capsid particles.The results showed: (1) Only a small amount of expressed P12A and 3C proteins were secreted into cell supernatant, and lots of them were unable to secrete into the medium and accumulated in the cell, which indicated the existence of rate-limiting steps in the secretion of foreign proteins. Therefore further study was needed to improve the construction of new cDNA cassettes and conditions for secretory expression of foreign proteins in insect cells. (2) The capsid proteins expressed in High Five? insect cells were successfully processed by viral 3C protease, as shown by Western blotting. Both empty capsid particles with a diameter of about 25~30nm and pentamers with a diameter of approximately 10 nm were observed using immunoelectron microscopy. Furthermore, the empty capsid particles or intermediates induced high levels of FMDV-specific antibodies in guinea pigs following immunization, and neutralizing antibodies were induced in the second week after vaccination. These recombinant, non-infectious, FMDV empty capsids are potentially useful for the development of new diagnostic techniques and vaccines. (3) The modified P12A and 3C were successfully expressed in insect cells by Western blotting, and that expressed capsid proteins were also processed by expressed viral 3C proteinase. Furthermore, only empty capsid particles were observed in lystate of High Five? cells infected with recombinant baculoviruses with mutated P12A under electron microscope, and no pentamers were detected, suggesting that replacing the histidines with leucines could, indeed, improve the stability of empty capsid particles under acidic conditions, but somewhat reduced the efficiency of assembly.