FMD-VP1-Hetero-6: A Recombinant Epitope Vaccine For Foot And Mouth Disease

Foot and mouth disease is a highly contagious disease, which always inflict cloven-hoofed animals such as cattle, sheep and pigs. Foot and mouth disease virus is the pathogen that causes this disease. Because of its dangerous infectivity and its impact upon agriculture, it was enlisted as number on the A list of contagious by OIE. Among varies of farm animals, cattle, sheep and pigs are all sensitive to the infection of FMDV, among which cattle is the most sensitive one. Cattle and pigs display most serious symptoms while sheep only showed very mild sub-clinical infection. The incidence of the disease is almost 100% while the course of the disease is always mild with the mortality of 2-3%. The death rate may reach 50-70% for FMD of calf and piglets. Besides the economical loss caused by the death of farm animals, the suspension of production of milk and meat during the epidemic will lead to large amount of loss. So the breakout of FMD always leads to the ceasing of export and import of farm products of the country and great loss caused. Last FMD breakout in England occurred in 2001, during which half a million animals were sacrificed and direct loss of 1.5 billion pound was caused. And it was showed in an investigation that the income of most of England farmers dropped 70% in recent 6 years. The major route of transmission of FMDV includes pathways through alimentary system, respiratory system, skin or mucosa. FMD will break out anytime of the year, especially at the end of autumn. The epidemic will aggravates during winter and abates in the spring. According to the past record, FMD always break out periodically, which means it will occur in the same region every few years. Foot and mouth disease is caused by the infection of Foot and mouth disease virus (FMDV). FMDV is a member of Picornaviridae family and there are a lot of virus in the family very important for animals and human being such as polio virus, hepatitis A virus, rhinovirus and so on. There are 7 sero-types of FMDV, namely, O, A, C, SAT1, SAT2, SAT3 and Asia. And there was no cross protection between those types. The genome of FMDV is a positive RNA molecule of 8.5 KB. The genome is composed of 5'UTR, 3'UTR and a large ORF between the UTRs. The large ORF contains the following genes, L, P1, P2 and P3. P1 gene encodes 4 major structure proteins of FMDV, namely, VP1, VP2, VP3 and VP4. VP1, VP2 and VP3 are the subunits of the capsid while VP4 locates in the interior of the viral particle. VP1 is the major antigen of FMDV and it contains the major B cell epitope (141-160aa), which is the major immuno-dominant epitope eliciting the protective humoral immunity. VP1 also contains some T cell epitopes such as T cell epitope (20-40aa) and (200-213aa), both of which are very important for the production of neutralizing antibody. Vaccination is the safest and most effective way for the prevention of FMD and good vaccine is the precondition for the eradication of FMD. Early in the 1950s, Waldman developed the earliest FMD vaccine, killed FMDV vaccine. Whole pathogen FMDV vaccines such as attenuated and killed viralvaccine maintained very good immunogenicity and are important for the prevention of the disease. But because of following risky factors such as the recovery of virulence and the incomplete inactivation of the virus and escape of live virus, new type of vaccine which should be safer and more effective are under intensive study worldwide. With the development of molecular biology, more and more genetic engineered vaccines are being developed. In this study, VP1 was chosen as the target for the designed recombinant vaccine. B cell epitope (141-160aa) and 2 T cell epitopes (20-40aa) and (200-213aa) were selected because of their function in the generation of protective humoral immunity. Their genes were connected and repeated several times to form the gene of FMD-VP1-Hetero6. The FMD-VP1-Hetero6 gene was synthesized using PCR and was subsequently cloned into prokaryotic expression system pET28a. After being expressed in the system, the recombinant protein FMD-VP1-Hetero6 was purified through nickel affinity column. Guinea pigs were immunized with this protein and after the guinea pigs were sacrificed the serum collected. ELISA and suckling mice protection experiment were carried out upon the serum respectively. This study was composed of following parts: 1 The design of the FMD-VP1-Hetero6 structure VP1 is the major antigen of FMDV and it contains the major B cell epitope (141-160aa), which is the major immuno-dominant epitope eliciting the protective humoral immunity. VP1 also contains some T cell epitopes such as T cell epitope (20-40aa) and (200-213aa), both of which are very important for the production of neutralizing antibody. The two T cell epitopes were named T1 and T2. BT1 and BT2 genes were constructed separately and the terminate form of the gene is BT1-BT2-BT1-BT2-BT1-BT2. 2 the construction of BT1 and BT2 genes and their TA cloningBT1 and BT2 genes were synthesized separately using specific primers and the products of PCR were confirmed by agarose gel electrophorosis. They were synthesized after 2 round of PCR. BT1 and BT2 genes were subsequently subcloned into pMD18T vector and the product of ligation reaction was transformed into JM109 host strains. After antibiotic screening plasmid was extracted. The recombinant plasmid was digested by EcoRâ… ,Hindâ…¢and a gene fragment of 170 was released. After being sequenced, the gene was confirmed to be exactly the gene designed. pMD18-T-BT2 was digested by XhoI,BamHâ… , and BT2 fragment was released and recovered from the agarose gel. This gene was subcloned into pMD18-T-BT1 at the site of XhoI and BamHI. Using similar method, pMD18-T-BT1-BT2-BT1-BT2 and pMD18-T-FMD-VP1-Hetero6 recombinant plasmid was constructed. 3 the construction of pET28a-FMD-VP1-Hetero-6 The recombinant plasmid pMD18-T-FMD-VP1-Hetero-6 was digested by EcoRâ… and HindIIIand gene of FMD-VP1-Hetero-6 was released and recovered from agarose gel. The gene was introduced into expression vector pET28a at the site of EcoRâ… and HindIII . Recombinant clones were identified after agarose gel electrophoresis. pET28a-FMD-VP1-Hetero-6 was sequenced and the result showed that the sequence was exactly what we have designed. 4 the expression and purification of FMD-VP1-Hetero-6 pET28a-FMD-VP1-Hetero-6 expression plasmid was transformed into expression host BL21 and positive clone was selected and cultured. After being induce in the presence of IPTG, the bacteria was collected. Total protein of the expression host was characterized by SDS-PAGE. The expression of FMD-VP1-Hetero-6 was confirmed by the presence of an additional band ofMW of 43 KD in the induced host bacteria. The size of the protein was the same as predicted. The expression of FMD-VP1-Hetero-6 was further confirmed by Western blot analysis. The recombinant protein was purified through nickel affinity column and the purity of the purified protein was nearly 98%. 5 the immune function of FMD-VP1-Hetero-6 Specifically designed schedule was used to study the ability of the FMD-VP1-Hetero-6 protein to elicit the production of neutralizing antibody of FMDV. In the formulation, alum and killed pertussis bacteria was used as adjuvants. Guinea pigs were immunized intraperitonially for the first time together with the adjuvant. Two booster immunizations were carried out after every 3 weeks intravenously without adjuvants. The mechanism of the method of immunization can be explained as followed: in the primary immunization the antigen adhered to the alum particle was released into the body slowly while killed pertussis bacteria as a kind of complete pathogen elicit strong inflammation we called bystander effect during which cellular and humoral immunity of the host was strongly activated. The titer of antibody may reach a level that common way of immunization never obtained after 2 booster immunizations. 5 days after the last immunization of the FMD-VP1-Hetero-6, the animals were sacrificed and serum collected. Elisa was carried out to test the titers of antibody produced. The result showed that: the immunization of FMD-VP1-Hetero-6 can elicit antibody the titer of which is much higher than FMD-VP1 and nearly to the level of traditional killed vaccine. The titer of the antibody was nearly 1:12800. Suckling mice protection test was also performed. After being serial diluted, the serum of different concentrations was mixed with 100 TCID50 of...