Development Of The Multiple-epitopes Vaccines To Foot-and-mouth Disease Virus Type Asia 1 And O

Foot-and-mouth (FMD) is a highly contagious and economically devastating viral disease of cloven-hoofed live-stock including pig, cattle, sheep and goats that causing by foot-and-mouth disease virus (FMDV). The inactivated FMDV vaccines as one of the systematic implements of prevention and control disease play a crucial role for control and eradication of FMD. However, the inactivated vaccines have several disadvantages including difficulty to discriminate between infected and vaccinated animals, requiring of the safe building biologically. Seriously, there is a potential risk of live virus escaping from vaccine plants. Development of the molecular biology and validation of immunogenicity of the synthetic peptides offer the theories and techniques for development of antigenic epitope vaccines to FMDV. To date, there were a lots of reports about antigenic epitope vaccines to FMDV and their immune potential is promising in the small model. However, they confer the limited immune responses in the natural hosts. In recent years, outbreaks of FMD frequently occur in China, particularly, several FMDV serotypes including types Asia 1, O and A alternately occurred, which the severely impact to the live-stock and animal's products in China. With a aim to control and eradicate FMDV occurred in China, the multiple-epitope vaccines to FMDV type Asia 1 in sheep and FMDV type O in swine have been developed with the molecular biology, proteins engineering and relative techniques, based on the current prevalent FMDV isolates type O and Asia 1. These vaccines as a safe and efficacy alternative will redound to prevention and control of FMD in China in the future.We designed and synthesized a tandem-repeated multiple-epitopes gene (RE) based on residues 135-160 and 197-211 of FMDV VP1 type Asia 1 Jiangsu isolate occurred in China. The ovine immunoglubine G heavy constant region gene (OIgGC) and 3D of FMDV type Asia 1 were amplified by RT-PCR. A series of the recombinant expression plasmids, pRE-OIgGC, p3D and pOIgGC, were constructed by directly inserting RE-OIgGC, 3D and OIgGC into an expression vector, pET-30a, respectively. The RE was purified and subcloned into an expression vector, pGEX-4T-1, which resulted in a recombinant plasmid, pGST-RE. These recombinant proteins were large-scale expressed in E.coli and the high-throughput purification proteins were obtained. All of the recombinant proteins were expressed as a formation of inclusion bodies in E.coli. Western blotting results showed that the recombinant RE-OIgGC, 3D and GST-RE are able to react with anti-FMDV positive serum, and the OIgGC is able to recognized by rabit anti-goat IgG. A series of vaccines, OIgGC(100μg/ml),RE-OIgGC(100μg/ml),RE-OIgGC(100μg/ml)+3D(50μg/ml) and GST-RE(100μg/ml)were performed by emulsification of appropriate proteins with a equal volume of ISA206. Potency of immunity of vaccine candidates in guinea pigs and sheep was evaluated by detection of neutralizing antibodies in serum, lymphocytes proliferation assay and virus challenge test. The results of the vaccinated guinea pigs showed that the recombinant proteins, RE-OIgGC, RE-OIgGC+3D and GST-RE, except groups PBS and OIgGC are able to elicit protective levels of neutralizing antibodies in the vaccinated guinea pigs and to offer compelete protection of guinea pigs against 103 ID50 of virus challenge. The multiple-epitope vaccine containing 3D is able to induce the highest titers of neutralizing antibodies than that of other proteins, which is the same high as that of the inactivated vaccine. Regretingly, the GST-RE elicit the same high levels of neutralizing antibodies as that of the RE-OIgGC. The possible explanation for this phenomenon is that the carrier proteins, GST and OIgGC, are the foreign proteins to the guinea pigs. In addition, the vaccine containing 3D is also able to the highest levels of lymphocytes proliferation than that of other vaccine candidates. These results demonstrated that 3D protein is not only able to improve the humoral immune responses, but to elicit T cell immune responses. The results of the vaccinated sheep showed that two groups, RE-OIgGC and RE-OIgGC+3D, elicit the significant higher level of neutralizing antibodies than that of GST-RE. The neutralizing antibodies is signifcantly rapid raise after booster vaccination compared with the initiation vaccination, which demonstrated that OIgGC play an important function biologically and significantly enhance the immunogenicity of the multiple-epitope vaccine. However, the immune adjuvant function of 3D protein was not especially distinctness in the vaccinated sheep. A tandem-repeated multiple-epitope gene (RE) containing residues 140-160 and 197-211 of VP1 of FMDV type O was designed and synthesized. A swine immunoglubine G heavy constant region (SIgGC) and 3D of FMDV type O were amplified by RT-PCR. The gene 3D, RE and SIgGC were subcloned into a expression vector, pET-22b, which resulted in the recombinant expression plasmids, pRE-SIgGC and p3D. The recombinant proteins, RE-SIgGC and 3D, were large-scale expressed in E.coli and highly throughput purified. The recombinant proteins were expressed as a formation of inclusion bodies in E.coli, and their immunoreactivity was verified with Western blotting. The RE-SIgGC and 3D was purified with Ni-NTA resin and a mixture of RE-SIgGC and 3D was developed according to an appropriate concentration ratio of two proteins. Subsequently, ten different multiple-epitopes vaccines to FMDV type O in swine were developed by emulsification of the mixture of proteins with a equal volume of several adjuvants including Al(OH)3, compelete Freund's adjuvant and ISA206 etc. Potency of immunity of ten vaccines were evaluated by vaccinated swine, and then a vaccine candidate has been screened. The results showed that the potency of immunity in swine vaccinated with single-dose vaccination is the same or higher than that of the traditionally inactivated vaccine. Three sets of vaccine candidates have been developed in our laboratory and their potency of immunity were systematically evaluated including detection of the minimum immune dosage, potency of immunity (PD50), duration of storage, duration of immunity and variation of neutralizing antobides after vaccination. The results showed that a vaccine candidate containing 83μg mixture of antigen is able to elicite the higher titers of neutralizing antibodies and to offer compelete protection of swine agaisnt 103 ID50 of virus challenge, protective ratio is 5/5. Potency of immunity from three sets of vaccine candidates are about 5.20-10.05 PD50 that is higher than the country standard (3.0 PD50). The titers of neutralizing antibodies reach the peak at days 30 post-vaccination, and the titers of neutralizing antibodies are gradually decline over times. Inspiringly, at months 7 post-vaccination, the higher titers of neutralizing antibodies were remained and majority of the vaccinated swine (70%) were compelete protection against virus challenge. Potency of immunity is not affected when vaccine candidate stored at 2-8℃for 12 months. These results indicated that we have developed multiple-epitope vaccine to FMDV type O in swine is a promising vaccine candidate, which may be to replace the traditionally inactivated and to be use for the control and prevention of FMD in China in the future.Conclusions, we have successfully designed and constructed the systems of assembly and expression for the multiple-epitopes vaccines to FMDV type O and Asia 1. The recombinant proteins were large-scale expressed in E.coli and highly throughput purified proteins were obtained. An immunological important molecular, host self immunoglubine G, and 3D of FMDV as a adjuvant were considerated in the designing of multiple-epitopes vaccine, which significantly improve the potency of immunity of vaccine and elicite cell immune responses. The higher potency vaccine candidates were selected by vaccination of guinea pigs, sheep and pigs. Particularly, the selected multiple-epitope vaccine candidate to FMDV type O in swine has several advantages including a higher potency of immunity, lower amount of antigen, longer duration of immunity and good stability stored at 2-8℃. Therefore, we have developed vaccine candidate is a promising vaccine, which will be used for the reference to develep multiple-epitope vaccines to other animal's viruses.