Reverse-genetic engineering of foot and mouth disease virus (FMDV) can improve the productivity, antigen matching, antigen stability, immune response ability, and biological safety of vaccines, so vaccine candidates with anticipated biological characteristics can be promptly achieved. Negative influence in taming of virulent strains can also be decreased or avoided. Reverse genetics not only make up for deficiencies like limitation of viral nature, low success rate, and time and energy consuming, but also realize more active designing of vaccines. Therefore, reverse genetics is significant in improving integral quality and efficiency of vaccines. In this review, we use FMDV vaccines as an example to summarize improvement in biological characteristics of virulent strains and provide a reference for related researches.
Animals ; Antibodies, Viral ; immunology ; Foot-and-Mouth Disease ; immunology ; prevention & control ; virology ; Foot-and-Mouth Disease Virus ; genetics ; immunology ; Reverse Genetics ; Viral Vaccines ; genetics ; immunology

Using mutation PCR, we cloned the target gene containing 421-480nt (141-160aa) and 598-639nt (200-213aa) of VP1 gene of foot and mouth disease virus (FMDV) into the deleted region (508-532aa) of Nsp2 gene of a highly pathogenic porcine reproductive and respiratory syndrome virus derived vaccine strain (HuN4-F112) that was used as vector. The recombinant cDNA was in vitro transcribed followed by transfection of BHK-21 cells for 36 h. Then, the supernatant of the cell culture was continuously seeded to monolayer of MARC-145 cells for recovery of the recombinant virus. CPE was obviously visible after a couple of passages in the seeded MARC-145, and the rescued virus (designated as rPRRSV-F112-O/VP1ep) was identified by Mlu I digestion, sequencing and immunofluorescence assay. Meanwhile, expression of inserted FMDV epitopes was also detected by indirect immunofluorescence assay with polyclonal antibodies against VP1 protein of FMDV. The analysis of biological characteristics shows that the titer of the rescued recombinant PRRSV (TCID50 = -log10(-6.75)/0.1 mL) was similar to its direct parental virus rHuN4-F112-delta508-532, but higher than rHuN4-F112.
Animals ; Antigens, Viral ; immunology ; Base Sequence ; Capsid Proteins ; immunology ; Cell Line ; Cysteine Endopeptidases ; genetics ; Epitopes ; genetics ; Foot-and-Mouth Disease ; immunology ; prevention & control ; Foot-and-Mouth Disease Virus ; genetics ; immunology ; Molecular Sequence Data ; Mutation ; Porcine respiratory and reproductive syndrome virus ; genetics ; immunology ; Recombination, Genetic ; Swine ; Transfection ; Vaccines, Attenuated ; genetics ; immunology ; Viral Envelope Proteins ; genetics ; immunology ; Viral Vaccines ; genetics ; immunology

We investigated the enhanced immune response of a recombinant T cell immunogen as an effective cellular immune adjuvant. The T cell immunogen named TI contained several T cell epitopes from the VP1, VP4, 3A and 3D proteins of foot-and-mouth disease virus (FMDV) and two pan-T helper (T(H)) cell sites to broaden the immunogenicity of the protein. Meanwhile, another fusion protein named OA-VP1 was expressed in bacteria, which contained two VP1 proteins of O and Asia1 type FMDV. Mice were vaccinated with commercially inactivated vaccine or OA-VP1 protein with or without the TI immunogen. The results show that mice inoculated with inactivated vaccine or OA-VP1 protein supplemented with TI immunogen produced significantly higher level of neutralizing antibodies (P < 0.01 or P < 0.05) than the mice only inoculated with inactivated vaccine or OA-VP1 protein by microneutralization assay. An obvious increase in T cell number by flow cytometric analysis and significantly higher concentration of IFN-gamma secreted in culture media of spleen lymphocytes were observed in groups supplemented with TI immunogen (P < 0.01). TI immunogen was an effective stimulator for humoral and cellular immunity and could help improve the immunogenicity of inactivated vaccine or protein subunit vaccine.
Adjuvants, Immunologic ; pharmacology ; Animals ; Capsid Proteins ; genetics ; immunology ; Epitopes, T-Lymphocyte ; genetics ; immunology ; Foot-and-Mouth Disease ; immunology ; prevention & control ; virology ; Foot-and-Mouth Disease Virus ; immunology ; Immunization ; Mice ; Viral Vaccines ; genetics ; immunology ; pharmacology

To develop a sensitive and specific ELISA for detection of antibodies to the nonstructural protein of FMDV. We cloned and expressed FMDV nonstructural protein 3AB in Escherichia coli expression system. The recombinant protein 3AB was purified with Ni-NTA HisBind Resins and characterized by Western blotting. An indirect ELISA based on purified protein 3AB as a coating antigen was established. The specificity and sensitivity of this assay were evaluated by comparison with a commercial 3ABC-ELISA kit in detecion of serum samples. The results showed that the recombinant protein 3AB was expressed as a formation of inclusion bodies in Escherichia coli. The purified protein could specificially react with FMDV infection antibodies in Western blotting assay, but no reaction with the immune antibodies induced with vaccine. Two assays were no significant differences in specificity and sensitivity for detection of field samples (P>0.05). Therefore, we speculated that the recombinant protein 3AB is a promising molecular marker, which may effectively differentiate FMD-infected from vaccinated animals in a herd.
Animals ; Antibodies, Viral ; analysis ; Antigens, Viral ; biosynthesis ; genetics ; immunology ; Cattle ; Cattle Diseases ; diagnosis ; immunology ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; metabolism ; Foot-and-Mouth Disease ; diagnosis ; immunology ; Foot-and-Mouth Disease Virus ; chemistry ; genetics ; isolation & purification ; Genetic Vectors ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Viral Nonstructural Proteins ; biosynthesis ; genetics ; immunology

VP1 is a major antigenic protein of foot-and-mouth disease virus(FMDV), which induces the immune response against FMDV infection, and contains several epitopes of the virus. We designed and chemically synthesized a DNA fragment which encoding a tandem repeat protein of 136-160aa and 198-211aa of a strain of type Asia I FMDV, and cloned the gene of heavy chain constant region of sheep IgG. By using the BamH I, EcoR I and Xho I sites, both genes were cloned into pPROExHTb vector in turn to form a recombinant plasmid pPRO-FshIgG A chimeric protein, named FshIgG, was obtained after transforming the pPRO-FshIgG into Escherichia coli BL21 (DE3) host cell and induced by IPTG. Inoculation with 100 microg FsIgG induced strong neutralizing antibody response in guinea pigs, and FshIgG inoculated guinea pigs were also protected against 200 ID50 FMDV challenge. Our study indicated that the heavy chain constant region of sheep IgG can act as the carrier protein for FMDV peptide epitopes, and FshIgG is a potential multiepitope peptide vaccine candidate to prevent FMDV infection.
Animals ; Antibodies, Viral ; blood ; Capsid Proteins ; biosynthesis ; genetics ; Epitopes ; immunology ; Escherichia coli ; genetics ; metabolism ; Foot-and-Mouth Disease Virus ; genetics ; immunology ; Genetic Vectors ; genetics ; Guinea Pigs ; Immunization ; Immunoglobulin G ; genetics ; Immunoglobulin Heavy Chains ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Sheep ; Viral Vaccines ; genetics ; immunology

In recent years, the potential value of nonstructural protein (NSP) 2C was well documented for distinguishing foot-and-mouth disease virus (FMDV) in infected animals and vaccinated animals. In order to develop a more sensitive approach to detect natural infected FMDV while there is no interact with vaccinated FMDV, we incorporated a major epitope region of 2C with whole 3AB coding region within NSP and expressed in Escherichia coli. We got a 47.6 kD fusion protein named 2C'3AB. The product showed a specific reactivity with FMDV from serum of infected animal by using Western blotting analysis. This suggests that this protein could be applied to distinguish infected FMDV and vaccinated FMDV. We further compared 2C'3AB protein with 3ABC fusion protein, another available protein used for detecting infected FMDV, using indirect ELISA assay. The results showed that 2C'3AB-ELISA had higher sensitivity than that of 3ABC-ELISA for distinguishing infected FMDV and vaccinated FMDV of sera from epidemic region. Therefore, this recombinant protein 2C'3AB is a good candidate protein to develop more sensitive method to differentiate infected FMDV and vaccinated FMDV from vaccinated animals. This finding will increase our capability to check the infectious virus carrier and finally improve FMDV infection control.
Animals ; Antibody Specificity ; Carrier Proteins ; genetics ; immunology ; metabolism ; Epitopes ; immunology ; Escherichia coli ; genetics ; metabolism ; Foot-and-Mouth Disease Virus ; genetics ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Viral Nonstructural Proteins ; genetics ; immunology ; metabolism

We designed and constructed a fuse expression gene OAAT and staphylococcal enterotoxin A (SEA) on the basis of the OAAT designed and constructed which consists of the structural protein VP1 genes from serotypes A and O FMDV, 5 major VP1 immunodominant epitopes from two genotypes of Asia1 serotype, and 3 Th2 epitopes originating from the non-structural protein, 3ABC gene and structural protein VP4 gene. The recombinant plasmids pEA was constructed using SEA as a genetic adjuvant. Expressions of target gene from the pEA in Hela cell were verified by IFA and Western blotting. The experiment of BALB/c mice immunized with the DNA vaccines showed that pA and pEA could induce simultaneously specific antibodies against serotypes A, Asia1, and O FMDV, and the highest antibody titres were found in the pEA and inactivated vaccine groups compared to pA vaccinating mice. Compared with the control, the levels of IL-2, IFN-gamma, IL-4, and IL-10 expression by splenic lymphocytes from mice immunized with pA and pEA were significantly increased. In addition, we found that the levels of IL-2, IFN-gamma and IL-4 from the mice immunized with pEA was higher than mice immunized with pA did. The results of viral challenge in guinea pigs showed the pA, pEA and inactivated vaccine provided full protection in 2/4, 2/4, 3/4, 3/4 and 4/4, 4/4 guinea pigs from challenge with FMDV O/NY00 and Asial/YNBS/58, respectively. The results demonstrated fuse protein OAAT and SEA may be potential immunoge against FMDV, furthermore, SEA may be an effective genetic adjuvant for DNA vaccine.
Adjuvants, Immunologic ; genetics ; Animals ; Antigens, Viral ; immunology ; Capsid Proteins ; genetics ; immunology ; Enterotoxins ; genetics ; immunology ; Epitopes ; immunology ; Foot-and-Mouth Disease ; immunology ; prevention & control ; Foot-and-Mouth Disease Virus ; immunology ; Guinea Pigs ; HeLa Cells ; Humans ; Mice ; Mice, Inbred BALB C ; Peptide Fragments ; genetics ; immunology ; Vaccines, DNA ; immunology ; Viral Structural Proteins ; genetics ; immunology ; Viral Vaccines ; immunology

We constructed a recombinant plasmid encoding VP1 gene of O type foot-and-mouth disease virus fused to a molecular adjuvant, goat complement C3d gene. The goat C3d gene was cloned and three copies were tandem-linked with the linker (G4S)2 sequence. VP1 gene of O type foot-and-mouth disease virus was linked to three tandem repeats of C3d through the linker sequence and cloned into pUC19 to obtain the recombinant plasmid pUC19-VP1-C3d3. The VP1-C3d3 fusion gene was then subcloned into the eukaryotic vector pcDNA3.1(+) that had been modified to contain the tissue plasminogen activator (tPA) leader sequence to obtain pcDNA3.1-tPA-VP1-C3d3. HeLa cells were transfected with pcDNA3.1-tPA-VP1-C3d3 by Lipofectamine 2000. Indirect immunofluorescent assay and Western blot assay showed that VP1-C3d3 fusion gene was successfully expressed in HeLa cells. The fusion protein with the expected size 133 kD could be secreted outside the cells. This study laid a good foundation to further research on the novel vaccine against foot-and-mouth disease virus by using goat C3d as a molecular adjuvant to enhance the immunogenicity of VP1.
Animals ; Capsid Proteins ; biosynthesis ; genetics ; Cloning, Molecular ; Complement C3d ; biosynthesis ; genetics ; immunology ; Female ; Foot-and-Mouth Disease Virus ; genetics ; Goats ; HeLa Cells ; Humans ; Immunologic Factors ; biosynthesis ; genetics ; immunology ; Plasmids ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Transfection

We produced beta1 gene which is about 2400 bp by reverse transcription polymerase chain reaction (RT-PCR) from bovine trachea, reclaimed and purified, then cloned the amplified fragment to pGEM-T easy vector, confirmed by sequencing. The immune-dominant epitope of beta1 gene was chosen by computer analysis and then syncretized ligand-binding domain from 346 bp to 843 bp of ecytoplasm with six histidine, expressed LBD protein massly in E. coli BL21 (DE3), and identified by SDS-PAGE. The fusion protein was purified with Ni-NTA affinity chromatography and immunized New Zealand rabbits preparing of its polyclonal antibody, the specific antibody titer was above 1:12,800 detected by indirect ELISA, the result of Western blot showed that this antibody could be recognized by LBD fusion protein.
Animals ; Antibodies, Monoclonal ; biosynthesis ; Cattle ; Escherichia coli ; genetics ; metabolism ; Foot-and-Mouth Disease Virus ; physiology ; Integrin alpha1beta1 ; biosynthesis ; genetics ; immunology ; Ligands ; Protein Binding ; Protein Interaction Domains and Motifs ; Rabbits ; Receptors, Virus ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

Entire 3ABC sequence of FMDV containing a 6 x his tag coding sequence at the N-terminal was obtained through PCR amplification using a pair of specific primers, subcloned into shuttle plasmid of pMelBac-B with a melittin secretion signal sequence and finally constructed recombinant plasmid of pMel-3ABC. After co-transfected the recombinant plasmid and linearized Bac-N-Blue DNA into Sf9 insect cell under intermediary agent of the Cellfectin, the result showed that we have already acquired recombinant baculovirus by screen of plaque assay and identification of PCR. Though the recombinant baculovirus infecting the Sf9 cells again, experiments indicated that 3ABC gene could express in insect cells and the expressed protein was secreted in the supernatant of Sf9 cell culture possessing favourable biological activities detected by adopting two methods of SDS-PAGE and Western blot. The result verified that the protein could respond with sera derived from FMDV infected animals, but have no responsibility with sera derived from health animals and vaccinated animals detected by indirect ELISA using antigen of expressed protein after purification with Ni-NTA his bind resin. Therefore, this study has established a solid foundation for establishing an effective diagnosis method to discriminating the FMDV infected animals from vaccinated animals.
Animals ; Antigens, Viral ; genetics ; immunology ; metabolism ; Blotting, Western ; Cattle ; Cell Line ; Cloning, Molecular ; Culture Media, Conditioned ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Foot-and-Mouth Disease ; immunology ; virology ; Foot-and-Mouth Disease Virus ; genetics ; immunology ; metabolism ; Gene Expression Regulation, Viral ; Immune Sera ; immunology ; Plasmids ; genetics ; Recombinant Proteins ; immunology ; secretion ; Sheep ; Spodoptera ; Swine ; Transfection ; methods ; Viral Nonstructural Proteins ; genetics ; immunology ; secretion