Immune responses of recombinant adenovirus co-expressing VP1 of foot-and-mouth disease virus and porcine interferon alpha in mice and guinea pigs

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating vesicular disease of cloven-hoofed animals. In this study, we constructed and characterized the immune responses and vaccine efficacy conferred by the recombinant adenovirus co-expressing VP1 of FMDV and porcine interferon alpha as fusion protein (rAd-pIFNalpha-VP1). Six groups of female BALB/c mice each with 18 were inoculated subcutaneously twice 2-week intervals with the recombinant adenoviruses. The results showed that the levels of humoral and cell-mediated immune responses in the group inoculated with rAd-pIFNalpha-VP1 were significantly higher than those in the group inoculated with rAd-VP1+rAd-pIFNalpha (P<0.05). Then four groups of guinea pigs each with six were inoculated two times at 2-week intervals intramuscularly with rAd-pIFNalpha-VP1, commercial inactivated FMD vaccine, wild-type adenovirus (wtAd) or PBS, and the protective efficacy of rAd-pIFNalpha-VP1 was determined. The results indicated that all the guinea pigs vaccinated with rAd-pIFNalpha-VP1 as well as inactivated FMD vaccine were protected from FMDV challenge, even though the levels of neutralizing antibodies (1:32-1:40) of the animals vaccinated with rAd-pIFNalpha-VP1 was lower than that in the group inoculated with inactivated FMD vaccine (1:64-1:128). It demonstrated that the newly recombinant adenovirus rAd-pIFNalpha-VP1 might further be an attractive candidate vaccine for preventing FMDV infection in swine.     

Maturation of functional antibody affinity in animals immunised with synthetic foot-and-mouth disease virus.

A good correlation exists between specific neutralising antibody titre and protection against challenge with foot-and-mouth disease virus (FMDV) in infected or virus-vaccinated cattle, but not in the case of animals immunised with synthetic FMDV peptides. Therefore, mechanisms other than simple neutralisation are likely to be important in vivo. Antibody affinity may influence the protective capacity of sera from immunised animals and experiments were carried out to measure the functional affinity for synthetic FMDV peptide of sera from guinea pigs and cattle given various synthetic vaccines. In guinea pigs given a single dose of synthetic vaccine, antibody affinity increased with time after immunisation. In cattle, however, administration of a second dose of peptide 21 days after the first markedly retarded the process of affinity maturation. For guinea pig sera of equivalent neutralising activity, those of higher functional affinity had higher protective indices than those of lower functional affinity. Knowledge of the importance of antibody affinity in protection against FMD is important for an improved understanding of the mechanisms of protection and for the design of novel vaccines.     

豚鼠动物模型评价牛口蹄疫Asia-1型灭活疫苗效力的研究

以豚鼠为试验动物模型,探索一种应用豚鼠替代牛进行牛口蹄疫Asia-1型灭活疫苗效力检验的方法.豚鼠和牛同步对6批牛口蹄疫Asia-1型灭活疫苗进行PD50效力检验,其中2批进行重复性试验.豚鼠分别在免疫后7、14、21和28天采血检测Asia-1型的中和抗体水平.统计学分析显示,测定的豚鼠PD50和牛PD50之间具有极...     

Protection of cattle and swine against foot-and-mouth disease, using biosynthetic peptide vaccines

A single dose of foot-and-mouth disease (FMD) virus protein 1 (VP1) peptide, expressed in Escherichia coli as a fusion protein with 190 amino acids (AA) of the LE' protein of the tryptophan operon of E coli, elicited an immune response in steers sufficient to withstand the challenge of exposure to animals with acute FMD. The 58-micrograms dose of viral peptide, composed of a segment of the VP1 from the A12 strain (A12) of FMD virus (FMDV; A12-32dimer) in a tandem repeat configuration of AA137 through 168 and emulsified with oil adjuvant, elicited a serologic response in cattle equivalent to that obtained using conventional whole virus vaccines. Two groups of swine were vaccinated, 1 with the A12-32dimer as used in cattle and 1 with AA131 through 157 from VP1 of the A24 strain (A24) of FMDV (A24-peptide), expressed in the same system as A12-32dimer, but as a single copy per molecule. In swine, the 58-micrograms dose of the A12-32dimer repeated at 28 days was an effective immunogen; all swine were protected against A12 and, in addition, the vaccine protected 50% of the swine against A24. The 29-micrograms dose of A24-peptide, administered according to the same schedule, elicited protection against A24 in 50% of the vaccinates and, in addition, protected 25% of those vaccinates against A12. The serologic response elicited by A12-32dimer against A24 virus was considerably greater than the response elicited by A24-peptide against A12 virus. The evidence of multiple immunogenic epitopes between AA131 and AA168 was evaluated.     

猪O型口蹄疫病毒重组结构蛋白的纯化及间接ELISA方法的建立

将猪O型口蹄疫病毒（foot and mouth disease virus,FMDV）的VP0、VP1、VP3基因,通过SUMO融合表达载体在大肠杆菌BL21（DE3）中成功表达,获得大小为55、48和40 ku的融合蛋白,Western blotting检测结果表明,表达的蛋白质具有良好的生物学活性。采用亲和层析法纯化表达产物,分别以纯化的SUMO-VP0、SUMO-VP1、SUMO-VP3蛋白为抗原建立了FMDV的间接ELISA检测方法。200份田间血清样品的检测结果表明建立的SUMO-VP0-ELISA、SUMO-VP1-ELISA、SUMO-VP3-ELISA检测方法均具有良好的特异性和敏感性。     

O型口蹄疫抗体金标试纸盒检测免疫动物抗体效价与攻毒保护关系

本研究目的在于用O型口蹄疫金标试纸条研究免疫动物抗体效价与攻毒保护之间的对应关系以及免疫牛抗体消长规律.用金标试纸条检测了口蹄疫O型疫苗质量标准规定的牛、羊和猪免疫和攻毒血清抗体效价及不同免疫程序的牛血清效价.结果显示,当牛、猪和羊免疫血清稀释≥1∶8时,试纸条是阳性结果,表示有99％的被免疫动物属于抗体水平保护范围;被检测血清稀释≤1∶2时,是阴性结果,表示被免疫动物抗体水平属于不保护范围;血清稀释1∶2～1∶8时,是阳性结果,表示有50％的被免疫动物属于抗体水平保护范围.牛在首次免疫8周后加强免疫可以获得良好的保护率和保护时间.通过本试验确定了金标试纸条检测免疫动物抗体效价与攻毒保护之间的关系及牛加强免疫最佳的时间.     

Evaluation of cross-protection against three topotypes of serotype O foot-and-mouth disease virus in pigs vaccinated with multi-epitope protein vaccine incorporated with poly(I:C)

Epitope-based vaccines are always questioned for their cross-protection against the antigenically variable foot-and-mouth disease virus (FMDV). In this study, we proved the cross-protection effect of a multi-epitope vaccine incorporated with poly(I:C) against three topotypes of O type FMDV. A total of 45 naïve pigs were vaccinated with different doses of multi-epitope protein vaccine incorporated with poly(I:C). At 28 days post-vaccination, 45 vaccinated and 6 unvaccinated control pigs (two pigs for each group) were challenged with three topotypes of virulent O type FMDV, namely, O/Mya/98 (Southeast Asia topotype), O/HN/CHA/93 (Cathay topotype) and O/Tibet/CHA/99 (PanAsia topotype) strains. All unvaccinated pigs developed generalised FMD clinical signs. Results showed that all pigs (n = 15) conferred complete protection against the O/Mya/98 and O/HN/CHA/93 FMDV strains, 11 of which were protected against the O/Tibet/CHA/99 FMDV strain. The 50% protective dose values of the vaccine against the O/Mya/98, O/HN/CHA/93 and O/Tibet/CHA/99 FMDV strains were 15.59, 15.59 and 7.05, respectively. Contact challenge experiment showed that transmission occurred from the donors to the unvaccinated but not to vaccinated pigs. These results showed that vaccination with multi-epitope protein vaccine incorporated with poly(I:C) can efficiently prevent FMD in pigs.     

A serological survey for antibodies against foot-and-mouth disease virus (FMDV) in domestic pigs during outbreaks in Kenya

Foot-and-mouth disease (FMD) is endemic in Kenya and has been well studied in cattle, but not in pigs, yet the role of pigs is recognised in FMD-free areas. This study investigated the presence of antibodies against FMD virus (FMDV) in pigs sampled during a countrywide random survey for FMD in cattle coinciding with SAT 1 FMDV outbreaks in cattle. A total of 191 serum samples were collected from clinically healthy pigs in 17 districts. Forty-two of the 191 sera were from pigs vaccinated against serotypes O/A/SAT 2 FMDV. Antibodies against FMDV non-structural proteins were found in sera from 30 vaccinated and 71 non-vaccinated pigs, altogether 101/191 sera (53 %), and 91 % of these (92/101) also had antibodies measurable by serotype-specific ELISAs, predominantly directed against SAT 1 with titres of 10–320. However, only five high titres against SAT 1 in vaccinated pigs were confirmed by virus neutralisation test (VNT). Due to high degree of agreement between the two ELISAs, it was concluded that positive pigs had been infected with FMDV. Implications of these results for the role of pigs in the epidemiology of FMD in Kenya are discussed, and in-depth studies are recommended.     

Attenuated Salmonella choleraesuis-mediated RNAi targeted to conserved regions against foot-and-mouth disease virus in guinea pigs and swine

In this study, specific sequences within three genes (3D, VP4 and 2B) of the foot-and-mouth disease virus (FMDV) genome were determined to be effective RNAi targets. These sequences are highly conserved among different serotype viruses based on sequence analysis. Small interfering RNA (siRNA)-expressing plasmids (p3D-NT19, p3D-NT56, pVP4-NT19, pVP4-NT65 and p2B-NT25) were constructed to express siRNA targeting 3D, VP4 and 2B, respectively. The antiviral potential of these siRNA for various FMDV isolates was investigated in baby hamster kidney (BHK-21) cells and suckling mice. The results show that these siRNA inhibited virus yield 10- to 300-fold for different FMDV isolates of serotype O and serotype Asia I at 48 h post infection in BHK-21 cells compared to control cells. In suckling mice, p3D-NT56 and p2B-NT25 delayed the death of mice. Twenty percent to 40% of the animals that received a single siRNA dose survived 5 days post infection with serotype O or serotype Asia I. We used an attenuated Salmonella choleraesuis (C500) vaccine strain, to carry the plasmid that expresses siRNA directed against the polymerase gene 3D (p3D-NT56) of FMDV. We used guinea pigs to evaluate the inhibitory effects of recombinant S. cho (p3D-NT56/S. cho) on FMDV infection. The results show that 80% of guinea pigs inoculated with 10⁹ CFU of p3D-NT56/S. cho and challenged 36 h later with 50 ID₅₀ of homologous FMDV were protected. We also measured the antiviral activity of p3D-NT56/S. cho in swine. The results indicate that 100% of the animals treated with 5 × 10⁹ CFU of p3D-NT56/S. cho were protected in 9 days.     

Current status of foot-and-mouth disease vaccines including the use of genetic engineering

SUMMARY Foot-and-mouth disease virus (FMDV) vaccines are used to protect animals against infection by the 7 FMDV serotypes composed of greater than 60 FMDV subtypes. Because of problems of both live attenuated and inactivated FMDV vaccines and also because of the very large market for an effective safe vaccine, research into other types of vaccines has been undertaken. One of the 4 virus structural proteins, VP1, is believed to be the main protein that stimulates virus neutralising antibodies and studies have concentrated on its potential as a subunit vaccine. Genetic engineering has been used to clone the VF1 gene of FMDV and VP1 synthesised from the cloned gene has been used in experimental vaccine studies. The studies in small numbers of cattle and pigs demonstrated that 2 vaccinations with genetically engineered VP1 could confer protection against FMDV challenge. However, there are a number of areas that need further research before such a genetically engineered vaccine could be used commercially. The use of chemically synthesised antigenic fragments of VP1 has recently been reported, and these synthetic fragments appear to be potentially better at producing immunity to FMDV than the whole genetically engineered VP1 protein, perhaps because of conformational problems in the presentation of whole VP1. Other possible future directions in the research and in the development of safe, effective FMDV vaccines are discussed. In conclusion, although very significant progress has been made in cloning FMDV-VP1 genes, we are still far from a genetically engineered VP1-FMDV subunit vaccine. In the meantime, properly inactivated and safety-tested FMDV vaccines will continue to be used and to be of benefit to the livestock industry in countries where foot-and-mouth is endemic or in combating introductions of the disease.     

猪口蹄疫特点、流行情况及疫苗使用现状

口蹄疫是危害我国畜牧业的主要传染病之一,主要感染猪、牛、羊等偶蹄动物,其中猪口蹄疫不同于牛、羊口蹄疫,有其独特特点。本文聚焦猪口蹄疫,从其特点、全球流行情况以及我国疫苗使用现状等方面进行了阐述和分析,以期为我国猪口蹄疫防控提供参考。     

Improved immune response by ID-pVAC: a secretory DNA vaccine construct delivered by PLG micro particles against foot and mouth disease in guinea pigs

Foot and mouth disease (FMD) outbreaks usually have devastating effects on the economy of countries were disease is endemic due to direct and indirect cost; most of them related to international trade embargoes of animals and animal products. Although currently used inactivated vaccine provides protection, it has several drawbacks like short duration of immunity, and the requirement for containment facilities. A DNA vaccine construct which expresses the secretary antigens, delivered through micro particles could be one of the alternate approaches to overcome these limitations. Present study is envisaged to prepare a DNA vaccine construct containing the VP1 sequence of FMDV serotype O in pVAC vector. DNA vaccine was formulated by adsorbing plasmid DNA construct on cationic micro particles and administered in guinea pigs @25 μg DNA vaccine construct per animal intramuscularly. Sera samples collected were analyzed by sandwich ELISA and SNT, shown enhanced immune response in PLG adjuvanted DNA vaccine. MTT and 3H Thymidine incorporation have shown good CMI responses to PLG adjuvanted DNA. When challenged with 100 gpid50 of homologous virus 5 of the six animals were protected.     

The plasmid constructs producing shRNA corresponding to the conserved 3D polymerase of Foot and Mouth Disease virus protects guinea pigs against challenge virus

RNA interference (RNAi) has been used as an effective antiviral strategy for its specific silencing of viral gene expression in mammalian cells. In this study, shRNA targeting two regions of Foot and Mouth Disease Virus (FMDV) i.e. 3D and 5'UTR which are very essential in virus replication were evaluated. The constructs were made using h7K RNA polymerase III promoter. We investigated in vivo inhibitory effect of shRNA on FMDV replication in BHK-21 cells and guinea pigs. The results showed that transfection of 3D shRNA could reduce virus growth by three folds when cells were challenged with 10(2) TCID(50) of FMDV. Pretreated guinea pigs with 3DshRNA were protected 80% with 10(3) GPID(50) of FMDV. As a first report in guinea pigs which are recognized animal model for FMD vaccine potency testing, the study suggests that shRNA could be a viable therapeutic approach to control severity of FMD infection and spread.     

利用SUMO表达系统高效表达猪O型口蹄疫病毒VP0、VP1、VP3基因

根据GenBank中公布的猪O型口蹄疫病毒(foot and mouth disease virus,FMDV)全基因合成了FMDV结构蛋白前体蛋白P1基因,同时设计了扩增FMDV结构蛋白VP0、VP1和VP3基因的引物。以P1基因为模板,分别经PCR扩增获得FMDV VP0、VP1和VP3基因。扩增产物克隆于Blunt载体中,酶切后将目的片段连接到原核表达载体SUMO中,构建重组表达质粒SUMO-VP0、SUMO-VP1和SUMO-VP3,将重组质粒转化大肠杆菌BL21(DE3)plysS进行诱导表达。经SDS-PAGE电泳可见融合蛋白均获得高效表达,融合蛋白表观分子质量分别约为55、48和40 ku。在IPTG浓度为1.0 mmol/L,温度为37 ℃,诱导5 h时融合蛋白表达量最大。Western blotting结果表明,融合蛋白均可被FMDV阳性血清识别,反应原性良好。     

Screening and identification of B cell epitopes of structural proteins of foot-and-mouth disease virus serotype Asia1

The objective of this study was to screen and identify the B cell epitopes of structural proteins of foot-and-mouth disease virus (FMDV) serotype Asia1. The complete amino acid sequence of all the four structural proteins (P1 region) was analyzed using the DNAStar Protean system. Seventeen peptides were predicted and selected as potential B cell epitopes. The potential B cell epitope genes were cloned into the pGEX-6P-1 plasmid, then expressed and purified. The resulting 17 glutathione S-transferase (GST) fusion peptides were detected by Western blot and ELISA for evaluation of their antigenicity. Six of the 17 fusion peptides were identified successfully by sera from rabbits immunized with the purified P1 polyprotein of FMDV type Asia1. The six fusion proteins were epi1-1 (VP1:₁TTTTGESADPVT₁₂), epi1-2 (VP1:₁₇NYGGETQTARRLH₂₉), epi1-6 (VP1:₁₉₄TTQDRRKQEIIAPEKQTL₂₁₁), epi2-2 (VP2:₄₀EDAVSGPNTSG₅₀), epi3-1 (VP3:₂₆YGKVSNPPRTSFPG₃₉), and epi4-2 (VP4:₃₀YQNSMDTQLGDN₄₁). The results of this study lay a foundation for further study of the structure and function of the structural proteins and may aid in the design of an epitope vaccine against foot-and-mouth disease (FMD) type Asia1. This study has also shown that the bioinformatics method, in combination with molecular biology methods can be used to map the B cell epitopes on viral proteins.     

O型口蹄疫病毒结构蛋白VP1的原核表达与抗原性分析

研究分析了O型口蹄疫病毒(FMDV)结构蛋白VP1与当前猪FMDV疫苗血清的免疫反应性.将VP1基因克隆至原核表达载体pET32c,并在大肠埃希菌BL21中得到了表达,Western blot分析表明该重组蛋白与豚鼠O型FMDV标准阳性血清具有良好免疫反应性.目的蛋白经纯化后用ELISA分析其与猪疫苗血清的免疫反应性,结果显示该重组VP1蛋白(rVP1)只能与部分O型FMDV疫苗血清反应.推测当前使用的不同O型FMDV疫苗毒株在VP1重要中和抗原位点G-H环(134 aa～158 aa)与C末端(200 aa～213 aa)存在较大差异.     

The efficacy of FMD vaccine reduced non-structural proteins with a mAb against 3B protein

A monoclonal antibody, 3BIgG, against the prokaryotically expressed foot-and-mouth disease virus (FMDV) non-structural protein (NSP) 3B was obtained. The 3BIgG-sepharose conjugant (3BmAb-6BFF) was prepared by adding the purified 3BIgG into epoxy-activated sepharose 6BFF, incubating with the inactivated FMDV, and then removing the sepharose by centrifugation. The vaccine was made from the supernatant emulsified with oil-adjuvant ISA206. Ten guinea pigs, 26 pigs and six cattle were vaccinated, and a vaccination control group was included without treatment with 3BmAb-6BFF. After 28 days, 9/10 pigs challenged with FMDV were protected, this result was the same as the control group, indicating that the vaccine potency was not reduced after treatment with 3BmAb-6BFF. The other animals were vaccinated weekly for nine weeks, and serum samples were collected to detect 3ABC-antibody titers. The results showed that 3ABC-antibody production was delayed and the positive antibody rates were lower when vaccination was carried out using vaccines treated with 3BmAb-6BFF compared with untreated vaccines. The findings of this study suggest that it is possible to reduce NSPs using a mAb-sepharose conjugant in FMD vaccines without reducing their efficacy.     

腺病毒载体及其在口蹄疫疫苗研究中的应用

腺病毒载体具有高效传递和表达外源基因的能力,常被用作体内和体外基因转移或表达的工具.体内同源重组和体外直接连接是目前构建腺病毒重组体的两种主要方式.口蹄疫病毒基因重组腺病毒活载体疫苗多是以缺失E1和E3基因编码区的人5型腺病毒AdHu5为载体进行构建,有些载体还缺失了E4基因编码区.研究证实,已构建的口蹄疫重组腺病毒疫苗对猪、牛都有一定的免疫保护效果,在口蹄疫预防研究领域具有很好的应用前景.     

口蹄疫灭活疫苗抗原稳定性研究进展

口蹄疫是一种感染牛、羊和猪等偶蹄动物、具有高度传染性的动物疫病。疫苗免疫是控制该病的关键措施之一,口蹄疫灭活疫苗在口蹄疫流行地区广泛使用。灭活疫苗中,完整口蹄疫病毒粒子(146S粒子)是至关重要的免疫抗原,它的数量和稳定性决定了疫苗的免疫效果。不同血清型,甚至同型不同毒株的口蹄疫病毒粒子稳定性不同,146S粒子在一定温度、酸、碱条件下容易分解为五聚体(12S粒子),导致疫苗免疫效力大幅下降。近年来口蹄疫病毒结构及其稳定性的分子基础研究取得了一定进展,为研究口蹄疫病毒稳定性提高疫苗质量,开发新型口蹄疫空衣壳疫苗提供了重要的理论基础。本文简要介绍了口蹄疫病毒结构基础、稳定性研究方法和研究进展。为同行了解口蹄疫病毒结构与免疫的关系,评价新型疫苗提供一些参考信息。