Immunogenicity and protective efficacy of a novel foot-and-mouth disease virus empty-capsid-like particle with improved acid stability

Foot-and-mouth disease virus (FMDV) is the etiological agent of a highly contagious disease that affects cloven-hoofed animal species. The FMDV capsid is highly acid labile and viral particles lose their immunogenicity when they disassemble at mildly acidic pHs. The viral capsid of FMDV serotype O is more sensitive than those of other serotypes, making it more difficult to acquire enough empty-capsid-like particles in the acidic insect cell environment for research. In this study, novel FMDV mutants with increased acid resistance were isolated using BHK-21 cell cultured under low-pH conditions. Amino acid substitutions Q25R, K41E, and N85A in the VP1 capsid protein and K154Q in the VP3 capsid protein were detected in all six mutants. Based on these amino acid replacements, empty-capsid-like particles of FMDV serotype O, which were resistant to the acid-induced dissociation of the capsid into pentameric subunits, were produced in insect cells. We characterized the protective immunity induced by these acid-resistant empty capsid particles. Significant humoral and cellular immune responses were elicited in mice after immunization with the acid-resistant empty capsid particles. The acid-resistant empty-capsid-like particles also induced strong neutralizing antibodies in guinea pigs and protected all the guinea pigs from FMDV challenge. Our results suggest that these acid-resistant empty-capsid-like particles have potential utility as a vaccine against serotype O FMDV infection.     

A recombinant truncated FMDV 3AB protein used to better distinguish between infected and vaccinated cattle

To distinguish the antibodies induced by Foot-and-mouth disease virus (FMDV) infection from those induced by vaccination, a recombinant N-terminal truncated FMDV non-structural protein (NSP) of 3AB, designated as r3aB, was constructed by deleting 80 amino acids displayed about 30% homology to transposase IS4 family protein of Escherichia coli, expressed in E. coli BL21 (DE3) and then purified. The r3aB was majorly expressed in soluble fraction and presented as homogeneous monomers after purification. Using r3aB as coating antigen, an indirect ELISA was established to specifically identify antibodies induced by FMDV infection but not those induced by vaccination. Compared with 3AB, r3aB was more specific to catch antibodies against NSP. The performance of this assay was validated by two commercial FMDV NSP ELISA kits. The result suggested that the r3aB coated ELISA could be developed into a kit to better distinguish between infected and vaccinated cattle.     

Immune response in guinea pigs vaccinated with DNA vaccine of foot-and-mouth disease virus O/China99

In order to obtain the gene P12X3C of foot-and-mouth disease virus (FMDV O/China99) that includes full length P1, 2A, 3C and part of 2B and 3B, the site mutation strategy was used. The recombinant plasmid pcDNA3.1/P12X3C was transfected into BHK-21 cells. The capsid proteins of FMDV expressed in BHK-21 cells were confirmed by sandwich-ELISA and indirect immunofluorescence test. Then the plasmid pcDNA3.1/P12X3C was administered to guinea pigs intramuscularly, and purified FMDV O/China993D protein expressed in yeast cells was injected together with pcDNA3.1/P12X3C. Anti-FMDV antibodies were detected by indirect ELISA, the T-lymphocyte proliferation response was tested by MTT assay, and neutralizating antibodies titers were analyzed by micro-neutralization assay. The result showed that the plasmid pcDNA3.1/P12X3C was able to express immunocompetent proteins of FMDV in BHK-21 cells. Furthermore, anti-FMDV antibodies were elicited and increased by plasmid pcDNA3.1/P12X3C in the second week after vaccination. Neutralizating antibodies were induced and the T-lymphocyte proliferation response was enhanced after vaccination. In the challenge test, all of guinea pigs vaccinated with pcDNA3.1/P12X3C were fully protected from FMDV challenge. However, the result obtained from animals that were injected with protein 3D together with plasmid pcDNA3.1/P12X3C was not satisfied. In conclusion, the results encouraged further work towards the development of a DNA vaccine against FMDV and provided the basis of research for DNA vaccine.     

Characterisation of the antigenic and immunogenic properties of bacterially expressed, sexual stage antigens of the coccidian parasite, Eimeria maxima

Coccidiosis in poultry is caused by the intestinal parasite Eimeria; it causes significant financial losses to the commercial poultry industry worldwide. CoxAbic is the first commercially available subunit vaccine against coccidiosis. The vaccine consists of affinity purified sexual stage (gametocyte) antigens (APGA) isolated from Eimeria maxima. Production of this vaccine is time-consuming and laborious and, therefore, a recombinant subunit vaccine substitute for CoxAbic is desirable. The genes encoding the two immunodominant components of CoxAbic, gam56 and gam82, were cloned into the bacterial expression vector, pTRCHisB, and the proteins expressed and purified. Both recombinant proteins were recognised by protective chicken antibodies that were raised to APGA, by immunoblotting. In a competitive ELISA, a combination of the recombinant proteins inhibited the binding of anti-APGA antibodies to APGA by 76%, which was comparable to the inhibition of 98% observed when APGA was used as the competing protein in the assay. In two breeds of chicken (Australorp and Cobb500), the recombinant proteins alone, or in combination, elicited a dose-dependent, antibody response that recognised APGA by ELISA, and gametocytes by immunoblotting. Together, the results suggested that the development of a recombinant subunit vaccine that maintains the antigenic and immunogenic properties of the native protein vaccine, CoxAbic, is feasible.     

Protective immunity against foot-and-mouth disease virus induced by a recombinant vaccinia virus

We report the construction of a recombinant vaccinia virus expressing the precursor for the four structural proteins of FMD virus (FMDV) (P1) strain C3Arg85 using a procedure for isolation of recombinant vaccinia viruses based solely on plaque formation. Adult mice vaccinated with this recombinant vaccinia virus elicited high titers of neutralizing antibodies against both the homologous FMDV and vaccinia virus, measured by neutralization assays. Liquid phase blocking sandwich enzyme-linked immunosorbent assays (ELISAs) using whole virus as antigen showed high total antibody titers against homologous FMDV, similar to those induced by the conventional inactivated vaccine. When ELISAs were carried out with heterologous strains A79 or O1Caseros as antigens, sera from animals vaccinated with the recombinant virus cross-reacted. Mice boosted once with the recombinant vaccinia virus were protected against challenge with infectious homologous virus. These results indicate that recombinant vaccinia viruses are efficient immunogens against FMDV when used as a live vaccine in a mouse model.     

Protection of cotton rats by immunization with the human parainfluenza virus type 3 fusion (F) glycoprotein expressed on the surface of insect cells infected with a recombinant baculovirus.

The antigenicity, immunogenicity and efficacy of the human PIV3 fusion (F) glycoprotein expressed in insect cells by a baculovirus vector were studied. The results indicate that the PIV3 F glycoprotein expressed by a recombinant baculovirus is antigenically authentic as determined using a panel of PIV3 F specific monoclonal antibodies. Only a low level of antibody was stimulated by immunization of animals with infected cells, but the antibody appeared to be of high quality. Immunized animals were also moderately protected against PIV3 challenge. These results indicate that the baculovirus expression system is a reasonable source of authentic PIV3 F protein for use in a subunit vaccine.     

Analysis of the immune response to FMDV structural and non-structural proteins in cattle in Argentina by the combined use of liquid phase and 3ABC-ELISA tests

The successful sanitary campaign implemented to control the 2000-2002 outbreaks of foot-and-mouth disease virus (FMDV) in Argentina was greatly assisted by the combination of an ELISA test (3ABC-ELISA) that detects antibodies directed against FMDV viral non-structural proteins (NSPs) and a liquid phase blocking competitive ELISA (lpELISA) for the detection of antibodies against the viral structural proteins (SPs). The combined use of these two assays in large-scale analysis of field samples allowed for a clear differentiation between infected and uninfected animals, with high specificity and sensitivity, regardless of the animal's vaccination status. In order to validate the application in indirect vaccine potency assays and assessment of vaccination efficiency, a preliminary correlation between serological response and protection from challenge with O1/Campos and A/Arg/01 FMD virus strains was established with data derived from commercial vaccine series challenge trials. Determination of antibodies to NSPs in vaccinated and revaccinated animals proved helpful in the analysis of vaccine purity. A review and discussion of the epidemiological status of cattle herds and real time monitoring of FMD in Argentina using these assays before, during and after the outbreaks is presented.     

Differentiation of convalescent animals from those vaccinated against foot-and-mouth disease by a peptide ELISA

We have identified continuous antigenic determinants within the amino acid sequences of the conserved nonstructural region containing proteins 2C and 3ABC of foot-and-mouth disease virus which can distinguish between the sera from vaccinated and infected animals. An ELISA based on a 3B peptide gave a positive reaction with sera from cattle, pigs, sheep and guinea pigs infected with all seven serotypes of the virus, but not with sera from vaccinated animals. In experiments with cattle and pigs to determine the duration of the antibody response, positive reactions were obtained as late as one year after infection. The advantages of using peptides from the nonstructural viral proteins instead of recombinant proteins for differentiating vaccinees from infected animals include their exquisite specificity, nonreactivity with antibodies against host cell-derived proteins (e.g. E. coli and insect cell proteins), and their ease of preparation.     

Expression of foot-and-mouth disease virus epitopes in tobacco by a tobacco mosaic virus-based vector

We expressed two immunogenic dominant epitopes of foot-and-mouth disease virus (FMDV) serotype O in tobacco plant using a vector based on a recombinant tobacco mosaic virus (TMV). The recombinant viruses TMVF11 and TMVF14 contained peptides of 11 and 14 amino acid residues, respectively, from FMDV VP 1 fused to the open reading frame of TMV coat protein (CP) gene between amino acid residues 154 and 155. TMVF11 and TMVF14 systemically infected tobacco plant and produced large quantities of stable progeny viral particles assembled with the modified CP subunits. Guinea pigs, mice and swine were used to test the protective effects of the recombinant viruses against FMDV infection. Most guinea pigs were protected against FMDV challenge after parenteral injection with TMVF11, TMVF14, or the mixture TMVF11/TMVF14, but not wtTMV. The TMVF11/TMVF14 mixture protected all animals when challenged with 150 guinea pig 50% infection dosage (GPID(50)) FMDV. Oral administration of the TMVF11/TMVF14 mixture (3mg total) protected 3/8 guinea pigs against the same FMDV challenge. Most of the suckling mice parenterally injected with antiserum from guinea pigs immunized with the TMVF11/TMVF14 mixture, but not with wtTMV, were also protected against FMDV challenge with 10 suckling mouse 50% lethal dosage (SMLD(50)), indicating that antibodies produced in guinea pigs immunized with the TMVF11/TMVF14 mixture specifically neutralized FMDV. Western blot analysis indicated that antiserum from those guinea pigs reacted with the FMDV VP1 protein. The protective effect of TMVF11 was also demonstrated in swine, where preliminary tests showed that nine pigs immunized with TMVF11 in three experiments were protected against FMDV challenge with 20 minimal infecting dose (MID).     

Influenza virus-like particles comprised of the HA, NA, and M1 proteins of H9N2 influenza virus induce protective immune responses in BALB/c mice

Avian influenza viruses represent a growing threat for an influenza pandemic. To develop recombinant vaccine for avian influenza of the H9N2 subtype, we expressed in insect cells virus-like particles (VLPs) consisting of three structural proteins of influenza A/Hong Kong/1073/99 (H9N2) virus. Upon infection of Sf9 cells with recombinant baculoviruses, the hemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins were co-expressed in the infected cells, self-assembled, and released into the culture medium as VLPs of 80–120 nm in diameter. VLPs exhibited functional characteristics of influenza virus including hemagglutination and neuraminidase activities. In BALB/c mice, VLPs elicited serum antibodies specific for influenza A/Hong Kong/1073/99 (H9N2) virus and inhibited replication of the influenza virus after challenge. Thus, VLPs represent a potential strategy for the development of human vaccines against avian influenza H9N2 viruses.     

An experimental marker vaccine and accompanying serological diagnostic test both based on envelope glycoprotein E2 of classical swine fever virus (CSFV)

Envelope glycoprotein E2 is the most immunogenic protein of classical swine fever virus (CSFV). In a proposed model of the antigenic structure of E2, the N-terminal half of E2 forms two independent structural antigenic units, A and BC. E2 without transmembrane region (E2-TMR) is expressed and secreted into the medium of insect cells by use of the baculovirus expression system. The immune response induced by E2 protects pigs against CSFV. Recently, we showed that the protective immune response to a homologous CSFV challenge can be induced by a single unit, A or BC, of E2. An indirect blocking ELISA, or complex trapping blocking assay (CTB) based on both units is routinely used worldwide for serological diagnosis of CSFV infections. Here we show that E2-TMR is secreted into the medium as a homodimer. This E2 homodimer was used to develop a CTB detecting antibodies directed against one immunogenic unit of E2. Thus, the protective immune response induced by E2 containing one unit was not detected with a modified CTB based on the other unit, whereas immune responses induced by a variety of low virulent CSFV strains were detected with such a modified CTB. These results indicate that a deletion E2 protein in combination with a modified CTB are feasible as CSF marker vaccine and accompanying differentiating diagnostic test.     

A recombinant baculovirus-expressed S glycoprotein vaccine elicits high titers of SARS-associated coronavirus (SARS-CoV) neutralizing antibodies in mice

A recombinant SARS-CoV spike (S) glycoprotein vaccine produced in insect cells in a pre-clinical development stage is described. A truncated version of S glycoprotein, containing only the ecto-domain, as well as a His-tagged full-length version were cloned and expressed in a serum-free insect cell line, ExpresSF+. The proteins, purified to apparent homogeneity by liquid column chromatography, were formulated without adjuvant at 3, 9, 27, and 50 microg per dose in phosphate saline and used to immunize mice. Both antigens in each formulation elicited a strong immune response after two or three vaccinations with the antigen. Neutralizing antibody titers correlated closely with standard ELISA reactivity against the S glycoprotein. The truncated S protein was also formulated with an adjuvant, aluminum hydroxide, at 1 microg per dose (+/-adjuvant), and 5 microg per dose (+/-adjuvant). Significantly enhanced immune responses, manifested by higher titers of serum ELISA and viral neutralizing antibodies, were achieved in adjuvanted groups with fewer doses and lower concentration of S glycoprotein. These findings indicate that the ecto-domain of SARS-CoV S glycoprotein vaccine, with or without adjuvant, is immunogenic and induces high titers of virus neutralizing antibodies to levels similar to those achieved with the full S glycoprotein vaccine.     

An approach to a FMD vaccine based on genetic engineered attenuated pseudorabies virus: one experiment using VP1 gene alone generates an antibody responds on FMD and pseudorabies in swine

Foot-and-mouth disease (FMD) and pseudorabies (PR) are two important infectious diseases in swine. An attenuated pseudorabies virus (PRV) has been successfully used as a gene delivery vector for the development of live-viral vaccines. In this study, a recombinant PRV-VP1 virus was constructed by fusioning the VP1 gene of FMD virus in frame to the N-terminal sequence of the gG gene of PRV. To test the protective immunity, 15 FMDV sero-negative white swine were divided into three groups and immunized with the recombinant PRV-VP1 virus, commercial FMD vaccine and vector virus (TK(-)/gG(-)/LacZ(+)), respectively, and challenged intramuscularly with 20 minimal infecting doses (MID) of virulent type O FMDV 4 weeks after booster immunization. Swine vaccinated with PRV-VP1 acquired antibodies against both FMDV and PRV, however, anti-FMDV antibodies were much lower than those vaccinated with the commercial FMD vaccine. Our results suggested that the recombinant PRV-VP1 virus, which only expressed FMDV VP1 gene controlled by PRV gG promoter, could not protect swine from the challenge of 20 MID type O FMDV, but could delay and reduce the clinical symptoms of FMD.     

结核分枝杆菌16KD重组蛋白的纯化及血清学诊断应用价值的研究

目的：纯化获得结核分枝杆菌重组16KD蛋白，并评价其在血清学诊断方面的应用价值。方法：应用亲和层析方法纯化结核分枝杆菌重组16KD蛋白，应用ELISA方法检测220例血清中的抗结核抗体。结果：结核分枝杆菌重组16KD蛋白以包涵体形式表达，以48名正常人血清0D492＋2S为正常限值，57例PPD阳性血清，47例菌阳结核患者血清和68例菌阴结核患者血清阳性检出率分别为19．30％、93．62％和82．35％。结论：结核分枝杆菌16KD重组蛋白以包涵体形式高效表达，具有较强的抗原特异性和免疫反应性。     

水痘-带状疱疹病毒糖蛋白I基因在昆虫细胞中的表达及纯化

目的 将北京水痘-带状疱疹病毒(VZV)84-7株克隆糖蛋白I(gpI)基因在杆状病毒-昆虫细胞表达系统中表达,并对其表达产物进行纯化。方法 采用PCR方法从VZVDNA中扩增gpI全基因序列,并将其插入杆状病毒转移质粒pBacPAK9中,获得重组转移质粒pBacVZVgpI,对pBacVZVgpI中的插入基因进行测序。重组转移质粒与线性杆状病毒BacPAK6DNA(Bsu36Idigested)共转染Sf9昆虫细胞,获得重组病毒BacPAK-gpI。通过亲和层析纯化重组蛋白,并检测其抗原性。结果 PCR扩增得到gpI基因,测序结果表明克隆的外源基因正确。经SDS-聚丙烯酰胺凝胶电泳(SDS—PAGE)、免疫印迹(weotem-blot)方法证明gpI基因在昆虫细胞中获得表达,表达产物在培养72h达到高峰,重组蛋白的相对分子质量约为58000和70000,与理论值相符,蛋白质加工与天然蛋白类似。动物实验结果表明,重组蛋白具有较好的免疫原性,可刺激小鼠产生中和抗体。SDS—PAGE检测纯化的重组蛋白,纯度达80％。纯化蛋白经western-blot和ELISA检测后显示,具有特异的抗体结合活性。结论 应用昆虫细胞表达水痘-带状疱疹病毒gpI基因,可为水痘-带状疱疹病毒抗原定量分析、糖蛋白ELISA的研制和制备亚单位疫苗提供基础。     

Synthesis of fusion proteins containing antigenic determinants of foot-and-mouth disease virus

Part of the genome of foot-and-mouth disease virus (FMDV) type 01,BFS, including the sequence encoding the capsid polypeptide VP1, was cloned in Escherichia coli following a new cloning strategy. The clone containing the VP1 sequence was used for the construction of two expression plasmids encoding VP1 fusion proteins. Subsequently, substantial amounts of the two VP1-beta-galactosidase fusion proteins, containing either one (amino acid region 140-160) or two (amino acid regions 140-160 and 200-213) antigenic determinants of the virus, were synthesized by E. coli bacteria. The protein containing the amino acid region 140-160 of VP1 fused to beta-galactosidase efficiently induced antibodies in rabbits specifically reacting with FMDV type 01,BFS. The same protein was also capable of eliciting neutralizing antibodies. The fusion protein containing both antigenic determinants did not efficiently induce antibodies reacting with FMDV.     

Immunogenic profiling and designing of a novel vaccine from capsid proteins of FMDV serotype Asia-1 through reverse vaccinology

Foot-and-mouth disease is one of the devastating transboundary animal diseases causing heavy losses to the livestock industry. Different vaccines based on the inactivated FMD virus are used against this disease, but lack of immunological memory and the need for high biocontainment are the major drawbacks of these vaccines. A novel vaccine comprising recombinant antigenic regions is effective, as they lack viruses for production. Considering the fact, capsid proteins vp4, vp2, vp3, and vp1 with 3C protease of FMDV serotype Asia-1 were analyzed through reverse vaccinology approaches in this study. The sequence and structural analysis of the proteins is carried out through various bioinformatic tools and the sequence analysis has figured out the acidic nature and thermal stability of the proteins, likewise, the phylogenetic analysis helped us to trace the FMDV isolates, elucidating that selected proteins belong to the strain (Group VII), which is currently circulating in Pakistan. Next, the B-cell and MHC Class-I epitopes are identified from the antigenic proteins by immunoinformatic tools. The highly conserved, antigenic, and non-allergenic epitopes are used to design the vaccine. Accordingly, the codon adaptation and in silico cloning of the corresponding genes is performed. Thus, the bacterial expression vector could be used for efficient expression and large-scale production of the vaccine.     

Immunological properties of FMDV-gP64 fusion proteins expressed on SF9 cell and baculovirus surfaces

In the present report, we characterized the immune response and the protection conferred by recombinant baculoviruses or infected insect cells expressing the fusions gp64-P1 and gp64-site A FMDV antigens. Mice, vaccinated intraperitoneally with gp64-P1 immunogens, showed a low-antibody response and a variable degree of protection. However, when mice received recombinant baculoviruses or infected insect cells expressing the fusion protein gp64-site A, high-ELISA and seroneutralizing titers (SNT) against FMDV were elicited. All mice immunized with Sf9 cells expressing FMDV site A developed a protective immune response against challenge with virulent FMDV, indicating that the baculovirus display of foreign epitopes is a promising approach to biosynthetic vaccines.     

Secretory IgA as an indicator of oro-pharyngeal foot-and-mouth disease virus replication and as a tool for post vaccination surveillance

A serotype-specific ELISA was developed to detect foot-and-mouth disease virus (FMDV) specific IgA antibody in the saliva of cattle, and the method was evaluated for its feasibility in detecting serotype O FMDV carrier animals, particularly amongst vaccinated cattle that had subsequently become sub-clinically infected. For this purpose, saliva samples were collected from na?ve cattle (n = 173), FMDV challenged cattle (n = 10), FMDV vaccinated cattle (n = 40) and FMDV vaccinated-and-challenged cattle (n = 40). A subset of 29 cattle was sampled for 105-168 days after challenge. The FMDV infection status of each of the cattle was determined by virus isolation and RT-PCR tests on oesophago-pharyngeal fluids and the ability of the IgA test to detect viral infection and persistence was compared to an ELISA for the detection of serum antibodies against the 3ABC non-structural proteins of FMDV. Eleven out of twelve vaccinated cattle that were shown to be persistently infected with FMDV up to or beyond 28 days post challenge, were also detected by the IgA test on saliva. With some modification and further validation, this test could be useful in post-vaccination surveillance to help confirm the absence of sub-clinical infection in order to regain the FMD-free status of a region or country.     

Foot-and-mouth disease polyvalent oil vaccines inoculated repeteadly in cattle do not induce detectable antibodies to non-structural proteins when evaluated by various assays

The use of foot-and-mouth disease (FMD) vaccines that do not induce antibodies against non-structural proteins (NSP) is extremely relevant for the demonstration of regions "free of FMDV infection" and control strategies. In this study cattle were primed and boosted with five doses of oil vaccines containing high antigenic payloads on days 0, 90, 130, 160 and 200. The serological response against NSP was measured using four commercially available assays: two 3ABC-ELISAs; one 3B-ELISA (and complementary 3A-ELISA) and an enzyme-immunotransfer blot assay (EITB). Additionally, locally produced NSP antibodies detection reagents and VIAA antibodies were evaluated. A high level of specific immune response against vaccine strains was shown. After four doses of vaccine, non-reactive animals were detected by any of the NSP assays. After the fifth immunization, 2 of 17 animals were reactive in one ELISA kit, but these samples proved negative by confirmatory tests. Antibodies against NSP were not detected in single dose immunized cattle. The principle of the NSP-ELISA used as a screening test for large sero-surveys in South America is established and this paper emphasizes the importance of using vaccines that have demonstrated no interference with NSP antibodies detection assays.