FMDV Vaccine Research Articles

Large-scale production of foot-and-mouth disease virus (serotype Asia1) VLP vaccine in Escherichia coli and protection potency evaluation in cattle.

BackgroundFoot-and-mouth disease (FMD) is an acute, highly contagious disease that infects cloven-hoofed animals. Vaccination is an effective means of preventing and controlling FMD. Compared to conventional inactivated FMDV vaccines, the format of FMDV virus-like particles (VLPs) as a non-replicating particulate vaccine candidate is a promising alternative.ResultsIn this study, we have developed a co-expression system in E. coli, which drove the expression of FMDV capsid proteins (VP0, VP1, and VP3) in tandem by a single plasmid. The co-expressed FMDV capsid proteins (VP0, VP1, and VP3) were produced in large scale by fermentation at 10 L scale and the chromatographic purified capsid proteins were auto-assembled as VLPs in vitro. Cattle vaccinated with a single dose of the subunit vaccine, comprising in vitro assembled FMDV VLP and adjuvant, developed FMDV-specific antibody response (ELISA antibodies and neutralizing antibodies) with the persistent period of 6 months. Moreover, cattle vaccinated with the subunit vaccine showed the high protection potency with the 50 % bovine protective dose (PD50) reaching 11.75 PD50 per dose.ConclusionsOur data strongly suggest that in vitro assembled recombinant FMDV VLPs produced from E. coli could function as a potent FMDV vaccine candidate against FMDV Asia1 infection. Furthermore, the robust protein expression and purification approaches described here could lead to the development of industrial level large-scale production of E. coli-based VLPs against FMDV infections with different serotypes.

A Recombinant Adenovirus Expressing P12A and 3C Protein of the Type O Foot-and-Mouth Disease Virus Stimulates Systemic and Mucosal Immune Responses in Mice.

Foot-and-mouth disease (FMD) is a highly contagious livestock disease of cloven-hoofed animals which causes severe economic losses. The replication-deficient, human adenovirus-vectored FMD vaccine has been proven effective against FMD. However, the role of T-cell-mediated antiviral responses and the mucosae-mediated antiviral responses induced by the adenovirus-vectored FMD vaccine was rarely examined. Here, the capsid protein precursor P1-2A and viral protease 3C of the type O FMDV were expressed in replicative-deficient human adenovirus type 5 vector. BALB/c mice immunized intramuscularly and intraperitoneally with recombinant adenovirus rAdv-P12A3C elicited higher FMDV-specific IgG antibodies, IFN-γ, and IL-4 cytokines than those in mice immunized with inactivated FMDV vaccine. Moreover, BALB/c mice immunized with recombinant adenovirus rAdv-P12A3C by oral and intraocular-nasal immunization induced high FMDV-specific IgA antibodies. These results show that the recombinant adenovirus rAdv-P12A3C could resist FMDV comprehensively. This study highlights the potential of rAdv-P12A3C to serve as a type O FMDV vaccine.

Genetic heterogeneity in the leader and P1-coding regions of foot-and-mouth disease virus serotypes A and O in Africa

Genetic information regarding the leader (L) and complete capsid-coding (P1) region of FMD serotype A and O viruses prevalent on the African continent is lacking. Here, we present the complete L-P1 sequences for eight serotype A and nine serotype O viruses recovered from FMDV outbreaks in East and West Africa over the last 33 years. Phylogenetic analysis of the P1 and capsid-coding regions revealed that the African isolates grouped according to serotype, and certain clusters were indicative of transboundary as well as intra-regional spread of the virus. However, similar analysis of the L region revealed random groupings of isolates from serotypes O and A. Comparisons between the phylogenetic trees derived from the structural coding regions and the L region pointed to a possibility of genetic recombination. The intertypic nucleotide and amino acid variation of all the isolates in this study supported results from previous studies where the externally located 1D was the most variable whilst the internally located 1A was the most conserved, which likely reflects the selective pressures on these proteins. Amino acids identified previously as important for FMDV structure and functioning were found to be highly conserved. The information gained from this study will contribute to the construction of structurally designed FMDV vaccines in Africa.Electronic supplementary materialThe online version of this article (doi:10.1007/s00705-013-1838-9) contains supplementary material, which is available to authorized users.

Foot-and-mouth disease virus-like particles produced by a SUMO fusion protein system in Escherichia coli induce potent protective immune responses in guinea pigs, swine and cattle

Foot-and-mouth disease virus (FMDV) causes a highly contagious infection in cloven-hoofed animals. The format of FMD virus-like particles (VLP) as a non-replicating particulate vaccine candidate is a promising alternative to conventional inactivated FMDV vaccines. In this study, we explored a prokaryotic system to express and assemble the FMD VLP and validated the potential of VLP as an FMDV vaccine candidate. VLP composed entirely of FMDV (Asia1/Jiangsu/China/2005) capsid proteins (VP0, VP1 and VP3) were simultaneously produced as SUMO fusion proteins by an improved SUMO fusion protein system in E. coli. Proteolytic removal of the SUMO moiety from the fusion proteins resulted in the assembly of VLP with size and shape resembling the authentic FMDV. Immunization of guinea pigs, swine and cattle with FMD VLP by intramuscular inoculation stimulated the FMDV-specific antibody response, neutralizing antibody response, T-cell proliferation response and secretion of cytokine IFN-γ. In addition, immunization with one dose of the VLP resulted in complete protection of these animals from homologous FMDV challenge. The 50% protection dose (PD50) of FMD VLP in cattle is up to 6.34. These results suggest that FMD VLP expressed in E. coli are an effective vaccine in guinea pigs, swine and cattle and support further development of these VLP as a vaccine candidate for protection against FMDV.

Co-inoculation of baculovirus and FMDV vaccine in mice, elicits very early protection against foot and mouth disease virus without interfering with long lasting immunity

Baculoviruses (Bvs) potentiate the immune response against soluble antigens. We investigated whether Bv could be used as immunoactivator in foot-and-mouth disease (FMD) vaccines using the BALB/c mouse model.Mice were vaccinated with a single dose of inactivated FMDV (iFMDV), iFMDV+Bv, Bv, or culture medium. Humoral and cellular immune responses were higher in animals immunized with iFMDV+Bv than in mice vaccinated with iFMDV alone. Animals receiving iFMDV+Bv had significantly lower viremia at 2, 4 and 7dpv, than those immunized with iFMDV alone.In order to prolong the immune response, iFMDV oil vaccine was co-inoculated with Bv. Animals receiving iFMDV oil vaccine+Bv were protected two days earlier than those receiving the iFMDV oil vaccine alone. Both formulations protected until 14dpv, the last day of the experiment.This is the first report in which Bv is used as an adjuvant in a FMDV vaccine.

Sequence analysis of capsid coding region of foot-and-mouth disease virus type A vaccine strain during serial passages in BHK-21 adherent and suspension cells

Sequence variability within the capsid coding region of the foot-and-mouth disease virus type A vaccine strain during serial in vitro passage was investigated. Specifically, two methods of virus propagation were utilized, a monolayer and suspension culture of BHK-21 cells. At three positions (VP2131 E-K in both monolayer and suspension passages, VP385 H-R in late monolayer passages and VP3139 K-E in only suspension passages), all mapped to surface exposed loops, amino acid substitutions were apparently fixed without reversion till the end of the passage regime. Interestingly, VP2131, 121 and VP385 which form part of the heparan sulphate binding pocket, showed a tendency to acquire positively charged amino acids in either monolayer or suspension environment probably to better interact with the negatively charged cell surface glycosaminoglycans. At three identified antigenically critical positions (VP279, VP3139 and VP1154), amino acids substitutions even in the absence of immune pressure were noticed. Hence both random drift and adaptive mutations attributable to the strong selective pressure exerted by the proposed cell surface alternate receptors could play a role in modifying the capsid sequence of cell culture propagated FMDV vaccine virus, which in turn may alter the desired potency of the vaccine formulations.

Effects of amino acid substitutions in the VP2 B-C loop on antigenicity and pathogenicity of serotype Asia1 foot-and-mouth disease virus

BackgroundFoot-and-mouth disease virus (FMDV) exhibits a high degree of antigenic variability. Studies of the antigenic diversity and determination of amino acid changes involved in this diversity are important to the design of broadly protective new vaccines. Although extensive studies have been carried out to explore the molecular basis of the antigenic variation of serotype O and serotype A FMDV, there are few reports on Asia1 serotype FMDV.MethodsTwo serotype Asia1 viruses, Asia1/YS/CHA/05 and Asia1/1/YZ/CHA/06, which show differential reactivity to the neutralizing monoclonal antibody (nMAb) 1B4, were subjected to sequence comparison. Then a reverse genetics system was used to generate mutant versions of Asia1/YS/CHA/05 followed by comparative analysis of the antigenicity, growth property and pathogenicity in the suckling mice.ResultsThree amino acid differences were observed when the structural protein coding sequences of Asia1/1/YZ/CHA/06 were compared to that of Asia1/YS/CHA/05. Site-directed mutagenesis and Immunofluorescence analysis showed that the amino acid substitution in the B-C loop of the VP2 protein at position 72 is responsible for the antigenic difference between the two Asia1 FMDV strains. Furthermore, alignment of the amino acid sequences of VP2 proteins from serotype Asia1 FMDV strains deposited in GenBank revealed that most of the serotype Asia1 FMDV strains contain an Asn residue at position 72 of VP2. Therefore, we constructed a mutant virus carrying an Asp-to-Asn substitution at position 72 and named it rD72N. Our analysis shows that the Asp-to-Asn substitution inhibited the ability of the rD72N virus to react with the MAb 1B4 in immunofluorescence and neutralization assays. In addition, this substitution decreased the growth rate of the virus in BHK-21 cells and decreased the virulence of the virus in suckling mice compared with the Asia1/YS/CHA/05 parental strain.ConclusionsThese results suggest that variations in domains other than the hyper variable VP1 G-H loop (amino acid 140 to 160) are relevant to the antigenic diversity of FMDV. In addition, amino acid substitutions in the VP2 influenced replicative ability and virulence of the virus. Thus, special consideration should be given to the VP2 protein in research on structure-function relationships and in the development of an FMDV vaccine.

Evaluation of a genetically modified foot-and-mouth disease virus vaccine candidate generated by reverse genetics

BackgroundFoot-and-mouth disease (FMD) is the most economically important and highly contagious disease of cloven-hoofed animals worldwide. Control of the disease has been mainly based on large-scale vaccinations with whole-virus inactivated vaccines. In recent years, a series of outbreaks of type O FMD occurred in China (including Chinese Taipei, Chinese Hong Kong) posed a tremendous threat to Chinese animal husbandry. Its causative agent, type O FMDV, has evolved into three topotypes (East–South Asia (ME-SA), Southeast Asia (SEA), Cathay (CHY)) in these regions, which represents an important obstacle to disease control. The available FMD vaccine in China shows generally good protection against ME-SA and SEA topotype viruses infection, but affords insufficient protection against some variants of the CHY topotype. Therefore, the choice of a new vaccine strain is of fundamental importance.ResultsThe present study describes the generation of a full-length infectious cDNA clone of FMDV vaccine strain and a genetically modified virus with some amino acid substitutions in antigenic sites 1, 3, and 4, based on the established infectious clone. The recombinant viruses had similar growth properties to the wild O/HN/CHA/93 virus. All swine immunized with inactivated vaccine prepared from the O/HN/CHA/93 were fully protected from challenge with the viruses of ME-SA and SEA topotypes and partially protected against challenge with the virus of CHY topotype at 28 days post-immunization. In contrast, the swine inoculated with the genetically modified vaccine were completely protected from the infection of viruses of the three topotypes.ConclusionsSome amino acid substitutions in the FMDV vaccine strain genome did not have an effect on the ability of viral replication in vitro. The vaccine prepared from genetically modified FMDV by reverse genetics significantly improved the protective efficacy to the variant of the CHY topotype, compared with the wild O/HN/CHA/93 virus. Thus, the full-length cDNA clone of FMDV can be a useful tool to develop genetically engineered FMDV vaccine candidates to help control porcinophilic FMD epidemics in China.

CpG oligodeoxynucleotide and montanide ISA 206 adjuvant combination augments the immune responses of a recombinant FMDV vaccine in cattle

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. To prevent the spread of FMDV, inactivated virus vaccines are used to immunize animals in developing countries. However, there are safety concerns. In addition, it is difficult to distinguish the vaccinated animals from those naturally infected ones. In our lab, we have developed a recombinant FMDV vaccine named A7. A7 contained multiple B cell and T cell epitopes, which reside in a capsid protein (VP1) of FMDV. To enhance its immunogenicity, A7 was formulated with CpG ODN RW03 in combination with Montanide ISA 206 (ISA), and the resultant vaccine (A7+ISA+CpG ODN RW03) was used to immunize mice and cattle. It was found that CpG ODN RW03 and ISA combination could facilitate A7 to induce a vigorous and long-lasting specific antibody response in mice and cattle. After FMDV challenge, 80% (4/5) of the calves immunized with A7+ISA+CpG ODN RW03 were protected, which was superior to those immunized with A7+ISA (25%, 1/4) or inactivated FMDV vaccine (50%, 2/4). These findings suggest that CpG ODN RW03 could be used with Montanide ISA 206 as a potent adjuvant for recombinant FMDV in cattle.

DNA immunization of pigs with foot-and-mouth disease virus minigenes: From partial protection to disease exacerbation

Despite several attempts to design new vaccines, there are as of yet no available alternatives to the conventional FMDV vaccines. Here, we present the divergent results obtained in pigs after immunization with two experimental DNA vaccines encoding one B and two T cell FMDV epitopes, either expressed alone (pCMV-BTT) or fused to a strong signal peptide (pCMV-spBTT). While all pigs vaccinated with pCMV-spBTT showed both a delay in the disease onset and reduced severity of signs and lesions after FMDV challenge, pigs immunized with pCMV-BTT showed an exacerbation of the disease and most of the pigs remained viremic at 10 days post-infection, the end-point of the experiment, thus opening concerns about FMDV-suboptimal immunization. Interestingly, only one of the four pigs vaccinated with pCMV-spBTT showed neutralizing antibodies before challenge, demonstrating that partial protection against FMDV could be afforded in the absence of preexisting neutralizing antibodies.

Whole Genome Sequencing of A Candidate Strain for FMDV Vaccine: Genomic Structure and Genetic Variation

Whole Genome Sequencing of A Candidate Strain for FMDV Vaccine: Genomic Structure and Genetic Variation

Whole Genome Sequencing of A Candidate Strain for FMDV Vaccine: Genomic Structure and Genetic Variation

Whole Genome Sequencing of A Candidate Strain for FMDV Vaccine: Genomic Structure and Genetic Variation

Ad5-vectored FMDV vaccine elicits antigen-specific CTL responses in swine. (43.10)

Abstract Foot-and-mouth disease virus (FMDV) is a highly infectious picornavirus that causes disease in cloven-hoofed animals. Neutralizing antibodies are thought to play a primary role in FMDV immunity, and their induction forms the basis for current vaccine approaches. However, the role of cytotoxic T-lymphocytes (CTLs) in recovery and protection has remained largely unexplored. Immune evasion strategies of FMDV are predicted to impair the development of functional CTL assays. Specifically, virus encoded proteases inhibit expression of Class I MHC molecules by infected cells. In order to overcome these difficulties, we have analyzed the response to a recombinant, human Adenovirus 5 (huAd5) vectored, FMDV vaccine. Swine homozygous for MHC were vaccinated with a replication-defective huAd5 expressing the unprocessed capsid polyprotein of FMDV, strain A24. An MHC-matched cell line was used for antigen stimulation and as a source of target cells in cellular cytotoxicity assays. PBMCs from vaccinated animals were incubated with Ad5-infected target cells for 3-5 days. CD6+ T cells were isolated and mixed with Ad5-FMDV infected target cells. Antigen-specific killing was detected relative to control target cells. This suggests that vaccination with adenovirus vectored vaccine constructs can induce a functional CTL response against epitopes of FMDV in a MHC restricted manner.

Effect of the extract made fromCochinchina momordicaseeds on the humoral immune responses of mice to a commercial foot-and-mouth disease vaccine (serotypes O and Asia 1)

The extract from ECMS was investigated for its effect on the humoral immune responses to foot-and-mouth disease vaccination. Fifty-six mice were randomly divided into seven groups with eight animals in each. Mice in groups 5 to 7 were subcutaneously (s.c.) injected with 0.5 mg DEX daily for 4 days to induce immunosuppression. The animals were then orally given ECMS (200 microg in 250 microl saline) in groups 3 and 6 or 250 microl saline in group 2, or s.c. injected with ECMS (50 microg in 100 microl saline) in groups 4 and 7 or 100 microl saline in group 5. After that, the animals in groups 2 to 7 were s.c. immunized twice with 100 microl of commercial oil-adjuvanted bivalent FMDV vaccine (serotypes O and Asia 1) at intervals of 21 days. Mice in group 1 received injection of 100 microl saline only. After 2 weeks, blood was sampled to determine FMDV-specific IgG and isotype IgG1, IgG2a, IgG2b and IgG3. Results indicated that oral administration or s.c. injection of ECMS augmented responses of specific IgG and most IgG isotypes. Giving ECMS tended to enhance serum-specific IgG and IgG isotype responses of mice immunosuppressed by s.c. injection of DEX. Considering the safety and immunomodulatory effect of ECMS in both normal and immunosuppressed mice demonstrated in the present study, this extract deserves further investigation to evaluate its potential in improving FMD vaccination in farm animals such as pigs, sheep and cattle.

Enhancement of the immune responses to vaccination against foot-and-mouth disease in mice by oral administration of an extract made from Rhizoma Atractylodis Macrocephalae (RAM)

This study was designed to evaluate the effects of oral administration of a water extract made from the Rhizoma Atractylodis Macrocephalae (RAM) on the immune responses in mice immunized with FMDV type O vaccine. Thirty-five ICR mice were randomly divided into five groups with seven animals in each group, and orally administered daily for 4 days at a dose equivalent to 0, 0.0625, 0.125, 0.25 or 0.5 g of dried RAM, respectively. After that, the animals were subcutaneously immunized twice with FMDV vaccine at 2-week intervals. Blood samples were collected 3 weeks after boosting for measurement of FMDV-specific IgG titers and the IgG subclasses, lymphocyte proliferation as well as production IL-5 and IFN-γ. Results indicated that serum FMDV-specific IgG titers and the IgG subclass responses were significantly enhanced in mice orally administered RAM at the dose of 0.25 or 0.5 g when compared with the control group ( P < 0.05). Splenocyte proliferation in response to Con A and LPS and production of IL-5 and IFN-γ by splenocytes were also significantly enhanced ( P < 0.05). Considering the immunomodulatory effect and safety of RAM demonstrated in this study, this herb deserves further investigation to evaluate its potential improvement of FMD vaccination in other animals such as pigs, goats and cattle.

Expression of Foot-and-Mouth Disease Virus Capsid Proteins in Silkworm-Baculovirus Expression System and Its Utilization as a Subunit Vaccine

BackgroundFoot-and-mouth disease (FMD) is a highly contagious disease of livestock that causes severe economic loss in susceptible cloven-hoofed animals. Although the traditional inactivated vaccine has been proved effective, it may lead to a new outbreak of FMD because of either incomplete inactivation of FMDV or the escape of live virus from vaccine production workshop. Thus, it is urgent to develop a novel FMDV vaccine that is safer, more effective and more economical than traditional vaccines.Methodology and Principal FindingsA recombinant silkworm baculovirus Bm-P12A3C which contained the intact P1-2A and 3C protease coding regions of FMDV Asia 1/HNK/CHA/05 was developed. Indirect immunofluorescence test and sandwich-ELISA were used to verify that Bm-P12A3C could express the target cassette. Expression products from silkworm were diluted to 30 folds and used as antigen to immunize cattle. Specific antibody was induced in all vaccinated animals. After challenge with virulent homologous virus, four of the five animals were completely protected, and clinical symptoms were alleviated and delayed in the remaining one. Furthermore, a PD50 (50% bovine protective dose) test was performed to assess the bovine potency of the subunit vaccine. The result showed the subunit vaccine could achieve 6.34 PD50 per dose.ConclusionThe results suggest that this strategy might be used to develop the new subunit FMDV vaccine.

Biochemical and structural studies with neutralizing antibodies raised against foot-and-mouth disease virus

The function of a loop exposed on the aphthovirus capsid (the G–H loop of protein VP1) has been explored by combining genetic and structural studies with viral mutants. The loop displays a dual function of receptor recognition and interaction with neutralizing antibodies. Remarkably, some amino acid residues play a critical role in both such disparate functions. Therefore residues subjected to antibody pressure for variation may nevertheless maintain a role in receptor recognition for which invariance is a requirement. Evolution of FMDV in cell culture may relax the requirements at this site and allow further increase of antigenic diversification. Essential residues at one stage of virus evolution may become dispensable at another not very distant point in the evolutionary landscape. Implications for FMDV evolution and vaccine design are discussed.

Application of monoclonal antibodies to quality control of foot-and-mouth disease vaccines

Panels of monoclonal antibodies (mAbs) produced against foot-and-mouth disease (FMD) virus types O, A and C were selected for cell culture neutralization titre (NT), mouse protection index (MPI), trypsin sensitivity (TS) and avidity to different epitopes. The selected sets were used to assay the antigen concentration and the fit between FMDV vaccine and challenge strains. It was observed that FMD vaccines protect more than 75% of vaccinated cattle when manufactured with antigens characterized by (1) a high degree of fit with the potency control virus, and (2) mean ELISA 50% titres ( T 50) > 28 for O, > 18 for A and > 75 for C types, respectively, using the corresponding mAb set.

Host cell modulation of foot-and-mouth disease virus procapsid synthesis

BHK 21 (clone 13S) cells of high (BHK-SH) and low (BHK-SL) passage number were infected with foot-and-mouth disease virus (FmDV) subtypes A 24, A 25 and C 3. While the amount of virus specific RNA produced in BHK-SH cells was 25% of that in BHK-SL cells and the virion production was 27% (C 3) to 53% (A 24) lower, the synthesis of viral proteins was comparable, associated with an accumulation of procapsids in BHK-SH cells. The results suggest that changes in viral infection pattern with increasing BHK 21 cell passage number should be considered in FMDV vaccine production.

Biotechnological approach to a new foot-and-mouth disease virus vaccine.

Major contributions towards the development of an absolutely safe FMDV vaccine are evident. With the identification of VP1 as the immunogenic protein, it is possible to manufacture a subunit vaccine via biotechnology. DNA sequences encoding the VP1 protein can be introduced into a bacterium with ease; under the appropriate conditions, large amounts of VP1 can be produced in a short time. The accumulation of amino acid sequences generated by recombinant DNA techniques allows identification of antigenic domains, which are the basis of variability among serotype and subtype viruses. As a result, vaccine production by chemical synthesis of short peptides corresponding to the antigenic determinants is greatly facilitated. At present, results from experimental vaccines employing genetically engineered or chemically synthesized VP1 antigens against homologous virus infection are encouraging. The current approach of preparing vaccine is to utilize the antigenic specificity of the virus. Since FMDV undergoes antigenic drift, variants not neutralized by type-specific serum will arise. An alternative approach is to prepare vaccines based on antigenic sites shared among all serotype and subtype viruses.