B cell epitopes within VP1 of type O foot-and-mouth disease virus for detection of viral antibodies

In this study, the coding region of type O FMDV capsid protein VP1 and a series of codon optimized DNA sequences coding for VP1 amino acid residues 141–160 (epitope1), tandem repeat 200–213 (epitope2 (+2)) and the combination of two epitopes (epitope1–2) was genetically cloned into the prokaryotic expression vector pP_ROExHTb and pGEX4T-1, respectively. VP1 and the fused epitopes GST-E1, GST-E2 (+2) and GST-E1-2 were successfully solubly expressed in the cytoplasm of Escherichia coli and Western blot analysis demonstrated they retained antigenicity. Indirect VP1-ELISA and epitope ELISAs were subsequently developed to screen a panel of 80 field pig sera using LPB-ELISA as a standard test. For VP1-ELISA and all the epitope ELISAs, there were clear distinctions between the FMDV-positive and the FMDV-negative samples. Cross-reactions with pig sera positive to the viruses of swine vesicular disease virus that produce clinically indistinguishable syndromes in pigs or guinea pig antisera to FMDV strains of type A, C and Asia1 did not occur. The relative sensitivity and specificity for the GST-E1 ELISA, GST-E2 (+2), GST-E1-2 ELISA and VP1-ELISA in comparison with LPB-ELISA were 93.3% and 85.0%, 95.0% and 90%, 100% and 81.8%, 96.6% and 80.9% respectively. This study shows the potential use of the aforementioned epitopes as alternatives to the complex antigens used in current detection for antibody to FMDV structural proteins.     

Virus-like particles of recombinant PCV2b carrying FMDV-VP1 epitopes induce both anti-PCV and anti-FMDV antibody responses

Mixed infection of porcine circovirus type 2 (PCV2) and foot-and-mouth disease virus (FMDV) is devastating to swine populations. To develop an effective vaccine that can protect the pigs from the infection of PCV2 and FMDV, we used the neutralizing B cell epitope region (aa 135–160) of FMDV to replace the regions aa 123–151 and aa 169–194 of the PCV2b Cap protein to generate a recombinant protein designated as Capfb. The Capfb protein was expressed in Escherichia coli system and the purified Capfb protein assembled into virus-like particles (VLPs) through dialysis. The ability of the Capfb protein to induce effective immune response against FMDV and PCV2b was tested in mice and guinea pigs. The results showed that the Capfb-VLPs could elicit anti-PCV2b and anti-FMDV antibody response in mice and guinea pigs without inducing antibodies against decoy epitope. Moreover, the Capfb-VLPs could enhance the percentage and activation of B cells in lymph nodes when the mice were stimulated with inactivated FMDV or PCV2b. These data suggested that the Capfb-VLPs could be an efficacious candidate antigen for developing a novel PCV2b-FMDV bivalent vaccine.

     

High-yield production of the VP1 structural protein epitope from serotype O foot-and-mouth disease virus in Escherichia coli

For effective control of foot-and-mouth disease (FMD), the development of rapid diagnostic systems and vaccines are required against its etiological agent, FMD virus (FMDV). To accomplish this, efficient large-scale expression of the FMDV VP1 protein, with high solubility, needs to be optimized. We attempted to produce high levels of a serotype O FMDV VP1 epitope in Escherichia coli. We identified the subtype-independent serotype O FMDV VP1 epitope sequence and used it to construct a glutathione S-transferase (GST) fusion protein. For efficient production of the FMDV VP1 epitope fused to GST (VP1e–GST), four E. coli strains and three temperatures were examined. The conditions yielding the greatest level of VP1e–GST with highest solubility were achieved with E. coli BL21(DE3) at 25 °C. For high-level production, fed-batch cultures were conducted in 5-l bioreactors. When cells were induced at a high density and complex feeding solutions were supplied, approximately 11 g of VP1e–GST was obtained from a 2.9-l culture. Following purification, the VP1 epitope was used to immunize rabbits, and we confirmed that it induced an immune response.     

Structures of Foot-and-mouth Disease Virus with neutralizing antibodies derived from recovered natural host reveal a mechanism for cross-serotype neutralization

The development of a universal vaccine against foot-and-mouth disease virus (FMDV) is hindered by cross-serotype antigenic diversity and by a lack of knowledge regarding neutralization of the virus in natural hosts. In this study, we isolated serotype O-specific neutralizing antibodies (NAbs) (F145 and B77) from recovered natural bovine hosts by using the single B cell antibody isolation technique. We also identified a serotype O/A cross-reacting NAb (R50) and determined virus-NAb complex structures by cryo-electron microscopy at near-atomic resolution. F145 and B77 were shown to engage the capsid of FMDV-O near the icosahedral threefold axis, binding to the BC/HI-loop of VP2. In contrast, R50 engages the capsids of both FMDV-O and FMDV-A between the 2- and 5-fold axes and binds to the BC/EF/GH-loop of VP1 and to the GH-loop of VP3 from two adjacent protomers, revealing a previously unknown antigenic site. The cross-serotype neutralizing epitope recognized by R50 is highly conserved among serotype O/A. These findings help to elucidate FMDV neutralization by natural hosts and provide epitope information for the development of a universal vaccine for cross-serotype protection against FMDV.     

A recombinant, arginine-glycine-aspartic acid (RGD) motif from foot-and-mouth disease virus binds mammalian cells through vitronectin and, to a lower extent, fibronectin receptors

The cell-binding abilities of a recombinant, RGD-containing peptide from foot-and-mouth disease virus (FMDV) have been characterized in HeLa and BHK cells. This peptide represents the aa sequence of the solvent-exposed G-H loop of protein VP1 which is involved in cell recognition and infection. The efficiency of the viral motif in promoting cell attachment and spreading is comparable to that shown by fibronectin or vitronectin. Cell binding is inhibited by a monoclonal antibody directed against a viral, RGD-involving B-cell epitope and also by sera against vitronectin (_Vβ₃/β₅) and fibronectin (₅β₁) receptors. In addition, a synthetic RGD peptide, which is a ligand for both integrins, prevents the cell binding mediated by the FMDV domain. These data demonstrate that the FMDV RGD motif is a potent ligand for cell-receptor integrins and sufficient to promote cell attachment to susceptible cells mainly through the vitronectin receptor.     

Fine mapping of a foot-and-mouth disease virus epitope recognized by serotype-independent monoclonal antibody 4B2

VP2 is a structural protein of the foot-and-mouth disease virus (FMDV). In this study, a FMDV serotype-in-dependent monoclonal antibody (MAb), 4B2, was generated. By screening a phage-displayed random 12-peptide library, we found positive phages displaying the consensus motif ETTXLE (X is any amino acid (aa)), which is highly homologous to ⁶ETTLLE¹¹ at the N-terminus of the VP2 protein. Subsequently, a series of GST-fusion proteins expressing a truncated N-terminus of VP2 were examined by western blot analysis using the MAb 4B2. The results indicated that the motif ⁶ETTLLE¹¹ of VP2 may be the minimal requirement of the epitope recognized by 4B2. Moreover, a 12-aa peptide ²KKTEETTLLEDR¹³ was shown to be the minimal unit of the epitope with maximal binding activity to 4B2. Alanine-scanning analysis demonstrated thatThr⁷, Thr⁸, and Leu¹⁰ are the functional residues of the 4B2 epitope Glu⁶ and Leu⁹ are required residues, and Glu¹¹ plays a crucial role in the binding of MAb 4B2. The fine mapping of the epitope indicated that MAb 4B2 has the potential to be used in FMDV diagnosis.     

Synthesis, cloning, and expression of artificial genes, coding antigenic determinants of the foot-and-mouth virus substrain A22]

Chemical-enzymatic synthesis and cloning in Escherichia coli of double-stranded DNAs, coding for simple and complex antigenic determinants of foot-and-mouth disease virus (FMDV) strain A22, have been carried out. The simple antigenic determinants are a part of the viral coat protein VP1 (amino acid sequence 131-152 or 131-160) whereas the complex antigenic determinants comprise additionally the amino acid sequence 200-213 of VP1 linked to N-terminus of simple antigenic determinants through a tetrapeptide spacer Pro-Pro-Ser-Pro. Recombinant DNAs containing genes for antigenic determinants of FMDV fused with C-terminus of gene for human tumor necrosis factor (hrTNF) have been constructed. Expression of the hybrid genes and properties of the proteins coded were studied. All recombinant proteins were shown to interact specifically with polyclonal antibodies both against hrTNF and FMDV strain A22. The recombinant proteins produced by bacteria are perspective for study as a vaccine against FMDV.     

Foot and mouth disease virus polyepitope protein produced in bacteria and plants induces protective immunity in guinea pigs

The goal of this project was to develop an alternative foot and mouth disease (FMD) vaccine candidate based on a recombinant protein consisting of efficient viral epitopes. A recombinant gene was designed that encodes B-cell epitopes of proteins VP1 and VP4 and T-cell epitopes of proteins 2C and 3D. The polyepitope protein (H-PE) was produced in E. coli bacteria or in N. benthamiana plants using a phytovirus expression system. The methods of extraction and purification of H-PE proteins from bacteria and plants were developed. Immunization of guinea pigs with the purified H-PE proteins induced an efficient immune response against foot and mouth disease virus (FMDV) serotype O/Taiwan/99 and protection against the disease. The polyepitope protein H-PE can be used as a basis for developing a new recombinant vaccine against FMD.     

一种新型T细胞免疫原的原核表达及对口蹄疫疫苗的免疫增强作用

主要探讨了T细胞免疫原TI对口蹄疫疫苗的免疫增强作用。设计并原核表达产生了一种包含口蹄疫病毒VP1,VP4,3A和3D蛋白上多个T细胞表位与两个通用T细胞表位的T细胞免疫原,命名为TI;同时表达了O和Asia 1两个型口蹄疫病毒 VP1 蛋白的串联编码基因,表达产物命名为OA-VP1。将上述T细胞免疫原分别与OA-VP1和口蹄疫灭活疫苗按不同剂量组合免疫小鼠,于免疫后不同时间测定各组小鼠的体液与细胞免疫应答情况。采用微量中和试验检测小鼠O型和Asia1型中和抗体,采用流式细胞检测技术和测定γ-干扰素的水平来分析不同免疫组小鼠细胞免疫的水平。结果显示,与灭活疫苗或OA-VP1单独免疫组相比,添加TI抗原后灭活疫苗 (P＜0.01) 和OA-VP1免疫组(P＜0.05)小鼠均能产生高水平的特异性中和抗体;且CD4+ T细胞数量显著增多,IFN-γ产生水平显著升高 (P＜0.01)。说明TI抗原具有很好的诱导特异性体液与细胞免疫应答的作用,是一种很好的免疫增效剂,可作为口蹄疫蛋白亚单位疫苗和灭活疫苗中的一种有效成分,以提高疫苗的免疫效果。     

Identification of short hairpin RNA targeting foot-and-mouth disease virus with transgenic bovine fetal epithelium cells

Background

Although it is known that RNA interference (RNAi) targeting viral genes protects experimental animals, such as mice, from the challenge of Foot-and-mouth disease virus (FMDV), it has not been previously investigated whether shRNAs targeting FMDV in transgenic dairy cattle or primary transgenic bovine epithelium cells will confer resistance against FMDV challenge.

Principal Finding

Here we constructed three recombinant lentiviral vectors containing shRNA against VP2 (RNAi-VP2), VP3 (RNAi-VP3), or VP4 (RNAi-VP4) of FMDV, and found that all of them strongly suppressed the transient expression of a FLAG-tagged viral gene fusion protein in 293T cells. In BHK-21 cells, RNAi-VP4 was found to be more potent in inhibition of viral replication than the others with over 98% inhibition of viral replication. Therefore, recombinant lentiviral vector RNAi-VP4 was transfected into bovine fetal fibroblast cells to generate transgenic nuclear donor cells. With subsequent somatic cell cloning, we generated forty transgenic blastocysts, and then transferred them to 20 synchronized recipient cows. Three transgenic bovine fetuses were obtained after pregnant period of 4 months, and integration into chromosome in cloned fetuses was confirmed by Southern hybridization. The primary tongue epithelium cells of transgenic fetuses were isolated and inoculated with 100 TCID₅₀ of FMDV, and it was observed that shRNA significantly suppressed viral RNA synthesis and inhibited over 91% of viral replication after inoculation of FMDV for 48 h.

Conclusion

RNAi-VP4 targeting viral VP4 gene appears to prevent primary epithelium cells of transgenic bovine fetus from FMDV infection, and it could be a candidate shRNA used for cultivation of transgenic cattle against FMDV.     

Multiserotype protection elicited by a combinatorial prime-boost vaccination strategy against bluetongue virus

Bluetongue virus (BTV) belongs to the genus Orbivirus within the family Reoviridae. The development of vector-based vaccines expressing conserved protective antigens results in increased immune activation and could reduce the number of multiserotype vaccinations required, therefore providing a cost-effective product. Recent recombinant DNA technology has allowed the development of novel strategies to develop marker and safe vaccines against BTV. We have now engineered naked DNAs and recombinant modified vaccinia virus Ankara (rMVA) expressing VP2, VP7 and NS1 proteins from BTV-4. IFNAR((-/-)) mice inoculated with DNA/rMVA-VP2,-VP7-NS1 in an heterologous prime boost vaccination strategy generated significant levels of antibodies specific of VP2, VP7, and NS1, including those with neutralizing activity against BTV-4. In addition, vaccination stimulated specific CD8(+) T cell responses against these three BTV proteins. Importantly, the vaccine combination expressing NS1, VP2 and VP7 proteins of BTV-4, elicited sterile protection against a lethal dose of homologous BTV-4 infection. Remarkably, the vaccine induced cross-protection against lethal doses of heterologous BTV-8 and BTV-1 suggesting that the DNA/rMVA-VP2,-VP7,-NS1 marker vaccine is a promising multiserotype vaccine against BTV.     

Development of a vaccine marker technology: display of B cell epitopes on the surface of recombinant polyomavirus-like pentamers and capsoids induces peptide-specific antibodies in piglets after vaccination

Highly immunogenic capsomers (pentamers) and virus-like particles (VLPs) were generated through insertion of foreign B cell epitopes into the surface-exposed loops of the VP1 protein of murine polyomavirus and via heterologous expression of the recombinant fusion proteins in E. coli. Usually, complex proteins like the keyhole limpet hemocyanin (KLH) act as standard carrier devices for the display of such immunogenic peptides after chemical linkage. Here, a comparative analysis revealed that antibody responses raised against the carrier entities, KLH or VP1 pentamers, did not significantly differ up to 18 weeks, demonstrating the highly immunogenic nature of VP1-based particulate structures. The carrier-specific antibody response was reproducibly detected in the meat juice after processing. More importantly, chimeric VP1 pentamers and VLPs carrying peptides of 12 and 14 amino acids in length, inserted into the BC2 loop, induced a strong and long-lasting humoral immune response against VP1 and the inserted foreign epitope. Remarkably, the epitope-specific antibody response was only moderately decreased when VP1 pentamers were used instead of VLPs. In conclusion, we identified polyomavirus VP1-based structures displaying surface-exposed immunodominant B cell epitopes as being an efficient carrier system for the induction of potent peptide-specific antibodies. The application of this approach in vaccine marker technology in livestock holding and the meat production chain is discussed.     

Sequences derived from the highly antigenic VP1 region 140 to 160 of foot-and-mouth disease virus do not prime for a bovine T-cell response against intact virus.

Although VP1 region 140 to 160 of foot-and-mouth disease virus (FMDV) is able to elicit neutralizing antibody in cattle, the protection against virus challenge that is conferred by peptide immunization is often poor. Here, we show that bovine T cells primed with peptides derived from this region generally show no reactivity to intact FMDV. In contrast, T-cell epitope VP4[20-34] is able to prime for a virus-specific response.     

Foot-and-mouth disease virus capsid protein VP2 activates the cellular EIF2S1-ATF4 pathway and induces autophagy via HSPB1

Foot-and-mouth disease virus (FMDV) can result in economical destruction of cloven-hoofed animals. FMDV infection has been reported to induce macroautophagy/autophagy; however, the precise molecular mechanisms of autophagy induction and effect of FMDV capsid protein on autophagy remain unknown. In the present study, we report that FMDV infection induced a complete autophagy process in the natural host cells of FMDV, and inhibition of autophagy significantly decreased FMDV production, suggesting that FMDV-induced autophagy facilitates viral replication. We found that the EIF2S1-ATF4 pathway was activated and the AKT-MTOR signaling pathway was inhibited by FMDV infection. We also observed that ultraviolet (UV)-inactivated FMDV can induce autophagy. Importantly, our work provides the first piece of evidence that expression of FMDV capsid protein VP2 can induce autophagy through the EIF2S1-ATF4-AKT-MTOR cascade, and we found that VP2 interacted with HSPB1 (heat shock protein family B [small] member 1) and activated the EIF2S1-ATF4 pathway, resulting in autophagy and enhanced FMDV replication. In addition, we show that VP2 induced autophagy in a variety of mammalian cell lines and decreased aggregates of a model mutant HTT (huntingtin) polyglutamine expansion protein (HTT103Q). Overall, our results demonstrate that FMDV capsid protein VP2 induces autophagy through interaction with HSPB1 and activation of the EIF2S1-ATF4 pathway.     

以猪IgG重链恒定区为抗原载体的抗口蹄疫病毒DNA疫苗的研制

口蹄疫(Foot-and-Mouth Disease, FMD)是当今世界上最为严重的家畜传染病之一,主要危害猪、牛、羊等偶蹄动物.FMD的致病原为FMD病毒(FMDV),属小RNA病毒科口蹄疫病毒属,有A、O、C、SATⅠ、SATⅡ、SATⅢ及AsiaⅠ共7个血清型.FMDV结构较简单,完整的病毒颗粒由4种结构蛋白VP1、VP2、VP3及VP4各60个拷贝构成的衣壳包裹一条单股正链RNA组成,其中VP1是主要的抗原蛋白[1].     

DNA vaccination against foot-and-mouth disease via electroporation: study of molecular approaches for enhancing VP1 antigenicity

BACKGROUND: Foot-and-mouth disease virus (FMDV) affects susceptible livestock animals and causes disastrous economic impact. Immunization with plasmid expressing VP1 that contains the major antigenic epitope(s) of FMDV as cytoplasmic protein (cVP1) failed to elicit full protection against FMDV challenge. MATERIALS AND METHODS: In this study, mice were immunized via electroporation with four cDNA expression vectors that were constructed to express VP1 of FMDV, as cytoplasmic (cVP1), secreted (sVP1), membrane-anchored (mVP1) or capsid precursor protein (P1), respectively, to evaluate whether expression of VP1 in specific subcellular compartment(s) would result in better immune responses. RESULTS: Electroporation enhanced immune responses to vectors expressing cVP1 or P1 and expedited the immune responses to vectors expressing sVP1 or mVP1. Immunization of mice via electroporation with mVP1 cDNA was better than sVP1 or cVP1 cDNA in eliciting neutralizing antibodies and viral clearance protection. Vaccination with P1 cDNA, nonetheless, yielded the best immune responses and protection among all four cDNAs that we tested. CONCLUSIONS: These results suggest that the antigenicity of a VP1 DNA vaccine can be significantly enhanced by altering the cellular localization of the VP1 antigen. Electroporation is a useful tool for enhancing the immune responses of vectors expressing VP1 or P1. By mimicking FMDV more closely than that of transgenic VP1 and eliciting immune responses favorably toward Th2, transgenic P1 may induce more neutralizing antibodies and better protection against FMDV challenge.     

Identification of T-cell epitopes in nonstructural proteins of foot-and-mouth disease virus

Porcine T-cell recognition of foot-and-mouth disease virus (FMDV) nonstructural proteins (NSP) was tested using in vitro lymphoproliferative responses. Lymphocytes were obtained from outbred pigs experimentally infected with FMDV. Of the different NSP, polypeptides 3A, 3B, and 3C gave the highest stimulations in the in vitro assays. The use of overlapping synthetic peptides allowed the identification of amino acid regions within these proteins that were efficiently recognized by the lymphocytes. The sequences of some of these antigenic peptides were highly conserved among different FMDV serotypes. They elicited major histocompatibility complex-restricted responses with lymphocytes from pigs infected with either a type C virus or reinfected with a heterologous FMDV. A tandem peptide containing the T-cell peptide 3A[21-35] and the B-cell antigenic site VP1[137-156] also efficiently stimulated lymphocytes from infected animals in vitro. Furthermore, this tandem peptide elicited significant levels of serotype-specific antiviral activity, a result consistent with the induction of anti-FMDV antibodies. Thus, inclusion in the peptide formulation of a T-cell epitope derived from the NSP 3A possessing the capacity to induce T helper activity can allow cooperative induction of anti-FMDV antibodies by B cells.     

High expression level of a foot and mouth disease virus epitope in tobacco transplastomic plants

Chloroplast transformation has an extraordinary potential for antigen production in plants because of the capacity to accumulate high levels of recombinant proteins and increased biosafety due to maternal plastid inheritance in most crops. In this article, we evaluate tobacco chloroplasts transformation for the production of a highly immunogenic epitope containing amino acid residues 135–160 of the structural protein VP1 of the foot and mouth disease virus (FMDV). To increase the accumulation levels, the peptide was expressed as a fusion protein with the β-glucuronidase reporter gene (uidA). The recombinant protein represented the 51% of the total soluble proteins in mature leaves, a level higher than those of the Rubisco large subunit, the most abundant protein in the leaf of a wild-type plant. Despite this high accumulation of heterologous protein, the transplastomic plants and wild-type tobacco were phenotypically indistinguishable. The FMDV epitope expressed in transplastomic plants was immunogenic in mice. These results show that transplastomic tobacco express efficiently the recombinant protein, and we conclude that this technology allows the production of large quantities of immunogenic proteins.     

Formation of virus-like particles from O-type foot-and-mouth disease virus in insect cells using codon-optimized synthetic genes

A recombinant baculovirus was constructed to simultaneously express codon-optimized virus-like particles (VLP), A VP1-2A-VP3 and VP0 of serotype O foot-and-mouth disease virus (FMDV), from individual promoters. The target proteins were expressed in insect cells at high level, as shown by indirect sandwich ELISA; and the expressed VP1-2A-VP3 could autocatalytically be cleaved into the individual proteins, VP1-2A and VP3, as shown by Western-blot analyses. In addition, in the insect cells, the structural proteins, VP0, VP3 and VP1-2A, self-assembled into virus-like particles resembling the authentic FMDV particles. This information should prove useful for the development of more efficient VLP assembly using shorter genes.