Synthetic peptide designs based on immunoactive fragments of the VP1 protein of the foot-and-mouth disease virus strain A22

Peptide constructs consisting of 44-53 aa were synthesized on the basis of sequences 135-159, 170-190 and 197-213 of VP1 from the foot-and-mouth disease A22 strain. Immunogenic and protective properties of the peptide constructs were studied in guinea pigs and mice of three lines. The constructs were shown to induce higher levels of antibodies and exhibit higher protective effects than the separate peptides. The most active among the peptides studied was the construct involving the VP1 fragments 135-160 and 170-190: it protected pigs from the experimental infection by the foot-and-mouth disease virus.     

Antigenic structure of the foot-and-mouth disease virus. II. Synthesis of protective peptides from the major immunogenic region of VP1 protein of foot-and-mouth disease virus type A22

Earlier we found that the immune response and antiviral protection from FMDV can be achieved by immunization with uncoupled FMDV peptides. In a search of approaches to animal protection from FMDV A22 strain we prepared a series of peptides corresponding to the putative antigenic determinants. Synthetic 131-149 and 140-149 sequences afforded 50 to 80% protection, both in the free state and conjugated with keyhole limpet hemocyanin. We believe that the 140-149 segment is so far the smallest peptide capable of eliciting specific antiviral protection without conjugation with a high molecular carrier.     

Antigenic structure of foot-and-mouth virus. IV. Synthesis and immunogenic properties of new fragments of the VP1 protein of food-and-mouth virus strain A22

A synthesis of new fragments of VP1 protein with the specificity of A22 strain of foot-and-mouth disease virus is described. Immunization with the free 136-152 peptide and KLH-conjugates of the peptides 136-152 and 197-213 induced 60-80% protection of guinea pigs against challenge with the A22 virus. Synthetic peptides corresponding to the 10-24, 50-69 and 175-189 sequences of VP1 did not show any protective activity. We have found that uncoupled peptides 175-189 and 197-213 are able to induce antipeptide antibodies. However, these antibodies did not possess any neutralizing activity. Immunization of animals with the mixture of (136-152)O1K and (175-189)A22 has led to inhibition of the immune response to the (136-152)O1K fragment.     

High expression of foot-and-mouth disease virus structural protein VP1 in tobacco chloroplasts

A tobacco chloroplast expression vector, pTRVP1, containing the foot-and-mouth disease virus (FMDV) VP1 gene and the selective marker aadA gene, was constructed and transferred to tobacco by biolistic method. Three resistant lines were obtained through spectinomycin selection, and each transgenic line was subjected to a second round of spectinomycin selection. PCR and PCR southern blot analysis revealed that the VP1 gene had integrated into the chloroplast genome. Western blot and quantification ELISA assays indicated that the VP1 gene was expressed in tobacco chloroplasts and accounted for 2–3% of total soluble protein. This suggested that plant chloroplasts were an efficient expression system for the potential production of recombinant antigens in plants.     

Serotype and VP1 gene sequence of a foot-and-mouth disease virus from Hong Kong (2002)

The nucleotide sequence of the VP1 coding region of foot-and-mouth disease virus (FMDV) strain HKN/2002, isolated from a disease outbreak occurring in Hong Kong in February 2002, was determined and compared with the sequences of other FMDVs. The VP1 coding region was 639 nucleotides in length and encoded a protein of 213 amino acid residues. Comparison of the VP1 nucleotide sequence with those of other isolates indicated that HKN/2002 belonged to serotype O. A VP1-based sequence similarity tree of several South-east Asian FMDV-O isolates showed that HKN/2002 was most closely related to FMDV isolates found in Hong Kong from 1991 to 1999 and Taiwan in 1997. Comparison of the amino acid sequence of the major immunogenic region of HKN/2002 with that of the serotype O vaccine strain, O1/Manisa/Turkey/69, reveals significant similarity, indicating that current serotype O vaccines may offer some degree of protection against HKN/2002.     

Application of VP1 protein to develop monoclonal antibody against foot-and-mouth disease virus Asia1 type

In order to develop an anti-FMDV Asia1 type monoclonal antibody (mAb), BABL/c mice were immunized with recombinant FMDV VP1 protein. Three mAbs, 1B8, 5E1 and 5E2, were then further optimized. The result indicated that prepared anti-FMDV Asia1 mAbs had no cross-reactivity with Swine vesicular disease (SVD) and FMDV O, A and C type antigen. Their titers in abdomen liquor were 1:5×10⁶, 1:2×10⁶ and 1:5×10⁶, respectively. 1B8 was found to be of IgG₁ subtype, 5E1 and 5E2 belonged to IgG_2b subtype. In this study, the prepared mAbs are specific for detecting FMDV type Asia1, and is potentially useful for pen-side diagnosis. Foudation items: The National high Technology Research and Development Program of China (No.2006AA10A204); The National science & Technology Pillar Program (No. 2006BAD06A17)     

Epitopes on foot-and-mouth disease virus outer capsid protein VP1 involved in neutralization and cell attachment.

Foot-and-mouth disease virus structural protein VP1 elicits neutralizing and protective antibody and is probably the viral attachment protein which interacts with cellular receptor sites on cultured cells. To study the relationships between epitopes on the molecule related to neutralization and cell attachment, we tested monoclonal antibodies prepared against type A12 virus, isolated A12 VP1, and a CNBr-generated A12 VP1 fragment for neutralization and effect on viral absorption. The antibodies selected for analysis neutralized viral infectivity with varying efficiencies. One group of antibodies caused a high degree of viral aggregation and inhibited the adsorption of virus to cells by 50 to 70%. A second group of antibodies caused little or no viral aggregation but inhibited the adsorption of virus to cells by 80 to 90%. One antibody, which is specific for the intact virion, caused little viral aggregation and had no effect on the binding of virus to specific cellular receptor sites. Thus, at least three antigenic areas on the surface of foot-and-mouth disease virus which were involved in neutralization were demonstrated. One of the antigenic sites appears to have been responsible for interaction with the cellular receptor sites on the surface of susceptible cells.     

Synthetic peptides simulating the protective epitopes of VP1 protein of foot-and-mouth disease virus type O and A

In a search of novel approaches to cattle protection from foot-and-mouth disease we have prepared a series of peptides from the major antigenic region 130-160 of the VP1 protein. The 144-159 peptide as well as 141-152, 141-148, 148-159 segments (strain O1K) were inactive in all in vitro and in vivo experiments on virus inhibiting. On the other band, synthetic 136-152, 136-148 O1K sequences as well as 131-149, 140-149 A22 sequences afforded 50 to 100% protection, both in the free state and conjugated with keyhole limpet hemocyanin. Therefore the 136-145 region should be considered as an essential part of the major sequential epitope, necessary for full-scale antiviral immune response. We also believe that the 136-152 segment is so far the smallest peptide capable of eliciting virus neutralizing antibodies and antiviral protection without conjugation with a high-molecular carrier.     

Primary structure of the DNA copy of the protein VP1 gene of the foot-and-mouth disease virus A22

The DNA-copy of the major antigen (VP1) coding region of the FMDV A22 sero-type has been cloned and sequenced. A comparison of the respective amino acid sequence with those of other VP1 of A-serotype revealed considerable differences in the structure of antigenic determinants.     

Primary structure of the gene for VP1 protein of the foot-and-mouth disease virus of Asia 1 serotype

The nucleotide sequence of the cDNA for the viral RNA region coding for the main antigenic protein of the epidemic stomatitis virus of Asia 1 serotype has been identified. The amino acid sequences in the regions of VP1 protein antigenic determinants of the serotype Asia 1 virus and other serotypes viruses have been compared.     

A T cell epitope in VP1 of foot-and-mouth disease virus is immunodominant for vaccinated cattle.

Synthetic peptides representing regions of the VP1 protein of foot-and-mouth disease virus strain 01 Kaufbeuren were screened for their ability to stimulate proliferation of PBMC from virus vaccinated cattle. Sites were identified at residue 21-40 (peptide FMDV32) and in the region C-terminal to residue 161. Cells responding to FMDV32 were MHC class II-restricted, CD4+ and secreted IL-2. Thus, this region is defined as a Th site. Of 19 virus vaccinated Friesian cattle, 89% (17/19) responded to purified virus while 37% (7/19; 41% of virus responders) also responded to FMDV32 suggesting that this site is immunodominant for the cattle used. Furthermore, immunisation of FMDV32 responder and non-responder cattle with a related peptide, FMDV5 (FMDV32 co-linearly synthesized with the 141-160 VP1 B cell site), induced neutralizing antibody and a virus-specific T cell population in the FMDV32-responder but not the non-responder animals.     

Nucleotide sequence of the VP1 gene of the foot-and-mouth disease virus strain A Venceslau

The VP1 coat protein of FMDV strain A Venceslau (Aven) consists of 213 amino acid residues. Serum neutralization tests demonstrated that strain Aven is closely related to strain A Argentina/79 (A₇₉) but significantly different from strain A₂₄ Cruzeiro (A₂₄). There is a strong correlation between the amino acid sequences and the serological data. Nucleotide and amino acid sequence analyses of VP1 showed that serologically related viruses (Aven and A₇₉) differ less in this region of the genome than those of serologically distinct viruses (Aven vs. A₂₄). The most significant variation between Aven and A₂₄ occurs at amino acid positions 43 to 46, in which all four residues are different.     

Does the VP1 gene of foot-and-mouth disease virus behave as a molecular clock?

Summary We have carried out a phylogenetic study of the evolution of the VP1 gene sequence from different serological types and subtypes of foot-and-mouth disease virus (FMDV). The maximum-likelihood method developed by Hasegawa and co-workers (Hasegawa et al. 1985) for the estimation of evolutionary parameters and branching dates has been used to decide between alternative models of evolution: constant versus variable rates. The results obtained indicate that a constant rate model, i.e., a molecular clock, seems to be the most plausible one. However, additional information suggests the possibility that the appearance of serotype CS has been accompanied by an episode of rapid evolution (Villaverde et al. 1991). We discuss the possibility that this evolution of RNA viruses was due to episodic positive Darwinian selection, which would have helped the new variant to escape the immunogenic pressure from the hosts. Offprint requests to: A. Moya     

口蹄疫病毒结构蛋白VP1容忍外源标签的能力

【目的】利用口蹄疫病毒的反向遗传操作技术,构建含不同外源标签口蹄疫病毒的全长克隆,鉴定口蹄疫病毒结构蛋白VP1容忍不同外源标签的能力。【方法】通过融合PCR技术,在FMDV O/HN/93全长感染性克隆的VP1 G-H环分别引入V5、TC12、KT3、3FLAG外源标签,构建全长质粒。全长质粒经Not I线化后转染表达T7 RNA聚合酶的稳定细胞,拯救重组病毒。RT-PCR、序列测定、间接免疫荧光鉴定病毒,噬斑和一步生长曲线分析重组病毒的生物学特性。【结果】成功拯救到表达V5或KT3表位标签的重组病毒,未能拯救到表达TC12或3×FLAG的重组病毒。V5和KT3表位标签的插入均影响了口蹄疫病毒的复制能力。【结论】重组口蹄疫病毒的成功拯救为未来标记疫苗以及口蹄疫病毒作为表达载体等的研究奠定了基础。     

Single amino acid substitution of VP1 N17D or VP2 H145Y confers acid-resistant phenotype of type Asia1 foot-and-mouth disease virus

Infection by foot-and-mouth disease virus (FMDV) is triggered by the acidic pH in endosomes after virus uptake by receptor-mediated endocytosis. However, dissociation of the FMDV 146S particle in mildly acidic conditions renders inactivated foot-and-mouth disease (FMD) vaccines much less effective. Type Asia1 FMDV mutants with increased resistance to acid inactivation were selected to study the molecular basis of viral resistance to acid-induced disassembly and improve the acid stability of FMDV. Sequencing of capsid-coding regions revealed four amino acid replacements (VP1 N17D, VP2 H145Y, VP2 G192D, and VP3 K153E) in the viral population of the acid-selected 10th passage. We performed single or combined mutagenesis using a reverse genetic system, and our results provide direct experimental evidence that VP2 H145Y or VP1 N17D substitution confers an acid-resistant phenotype to type Asia1 FMDV.     

口蹄疫病毒结构蛋白VP1在谷氨酸棒状杆菌中的表达及优化

【背景】口蹄疫(foot-and-mouth disease, FMD)是由口蹄疫病毒(foot-and-mouth disease virus, FMDV)引起的感染牛、羊和猪等偶蹄动物的主要疫病之一。口蹄疫病毒的结构蛋白VP1包含多个能够引起机体免疫反应的主要位点,因此VP1是研究亚单位疫苗的方向靶标。谷氨酸棒状杆菌 (Corynebacterium glutamicum)作为安全生产菌株,是医药用蛋白生产的优势细胞工厂。【目的】利用C. glutamicum作为受体菌株表达外源蛋白的优势实现VP1的外源表达。【方法】根据VP1结构蛋白的基因序列、相应功能和C. glutamicum的密码子偏好性设计并合成VP1基因,与pXMJ19载体连接构成重组质粒pXMJ19-VP1。C. glutamicum CGMCC 1.15647菌株用于表达VP1-6×his蛋白,并对蛋白表达元件启动子、5′非翻译端(5′UTR)、目的蛋白自身N端等进行优化,同时对培养条件等进一步优化,采用SDS-PAGE和Western blotting技术检测VP1蛋白的表达情况。最后应用间接酶联免疫吸附试验(enzyme-linked immunosorbent assay, ELISA)测定本研究中生产的VP1的免疫活性。【结果】SDS-PAGE和Western blotting分析结果表明VP1蛋白能在C. glutamicum CGMCC 1.15647菌株成功表达,将P_tac启动子替换为合成型启动子P_H36能提高蛋白产量。在此基础上,通过插入不同的5′UTR序列和VP1蛋白N端氨基酸的改变能进一步提高蛋白产量并可利用CspB信号肽实现分泌表达。发酵试验表明,VP1摇瓶发酵培养最优条件为30 ℃、24 h。ELISA试验表明,本研究中的VP1可与阳性血清特异性结合。【结论】本研究在谷氨酸棒状杆菌中成功表达FMDV的VP1蛋白,并通过优化蛋白表达元件的方法进一步提高了产量,为开发新型FMD免疫诊断试剂和安全高效的亚单位疫苗奠定了良好的基础。     

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.     

Preparation and characterization of monoclonal antibodies against VP1 protein of foot-and-mouth disease virus O/China99

Monoclonal antibodies (McAbs) 1A9 and 9F12 against Foot-and-mouth disease virus (FMDV) serotype O were produced by fusing SP2/0 myeloma cells with splenocyte from the mouse immunized with O/China99. Both McAbs reacted with O/China99 but not with Asia 1, as determined by immunohistochemistry assay. The microneutralization titer of the McAbs 1A9 and 9F12 were 640 and 1 280, respectively. Both McAbs contain kappa light chains, but the McAbs 1A9 and 9F12 were IgG1 and IgM, respectively. In order to define the McAbs binding epitopes, the reactivity of these McAbs against VP1, P20 and P14 were examined using indirect ELISA, the result showed that both McAbs reacted with VP1 and P20. McAbs may be used for further studies of vaccine, diagnostic methods, prophylaxis, etiological and immunological researches on FMDV.     

Further characterization of a protein kinase from foot-and-mouth disease virus.

Acid disruption of foot-and-mouth disease virus released a protein kinase activity that sedimented at less than 7S. This enzyme was separated into three peaks of activity by ion-exchange and hydroxylapatite chromatography. Analysis of the various enzyme fractions by polyacrylamide gel electrophoresis and silver staining revealed that one of the fractions lacked the major virion structural proteins, but still contained two or three other polypeptides. This enzyme phosphorylated mainly one protein (P17) in an in vitro assay.