Stabilised FMDV capsids

Abstract

The present invention relates to the stabilisation of foot-and-mouth disease virus (FMDV) capsids, by specific substitution of amino acids in a specific region of FMDV VP2. The invention provides stabilised FMDV capsids and vaccines against FMD.

Claims

1. A foot-and-mouth disease virus (FMDV) VP2 protein mutant, that comprises an amino acid substitution of a tyrosine at position number 93 (93Y), wherein the position number is relative to the numbering of the amino acids presented in SEQ ID NO: 1.

2. An FMDV capsid comprising the FMDV VP2 protein mutant of claim 1.

3. An isolated host cell comprising the FMDV VP2 protein mutant of claim 1.

4. A vaccine against FMD comprising a pharmaceutically acceptable carrier and an FMDV capsid comprising the FMDV VP2 protein mutant of claim 1.

5. An isolated host cell comprising the FMDV VP2 protein mutant of claim 1 and an FMDV capsid comprising said FMDV VP2 protein mutant.

6. A vaccine against FMD comprising a pharmaceutically acceptable carrier and the FMDV VP2 protein mutant of claim 1.

7. A vaccine against FMD comprising a pharmaceutically acceptable carrier and an isolated host cell comprising the FMDV VP2 protein mutant of claim 1.

8. A vaccine against FMD comprising a pharmaceutically acceptable carrier and one or more of the following: a foot-and-mouth disease virus (FMDV) VP2 protein mutant that comprises an amino acid substitution of a tyrosine at position number 93 (93Y), an FMDV capsid comprising said FMDV VP2 protein mutant, and an isolated host cell comprising said FMDV VP2 protein mutant; wherein the position number is relative to the numbering of the amino acids presented in SEQ ID NO: 1.

9. A method for the vaccination against FMD of an animal susceptible for FMDV, comprising the step of inoculating the animal with the vaccine of claim 8.

Description

EXAMPLES

(1) 1. Methods And Materials

(2) 1.1. Cells and viruses

(3) Baby hamster kidney (BHK) clone 13 cells (strain 21; ATCC CCL-10) were maintained according to standard procedures. FMDV stocks were amplified and titrated in BHK-21 cells using standard procedures. Cultured BHK-21 cells were also used for RNA transfection and virus recovery. In addition, plaque assays were performed in either IB-RS-2 (Instituto Biologico renal suino) cells or Chinese hamster ovary (CHO) cells (strain K1; ATCC CCL-61), respectively propagated in RPMI medium (Sigma) and Ham's F-12 medium (Invitrogen) supplemented with 10% foetal calf serum (FCS, Delta Bioproducts).

(4) One-step growth kinetic analyses of FMDV virion capsids were carried out in BHK-21 cells. Briefly: BHK-21 cells were infected with the virus for 1 h at an m.o.i. of 2-4 pfu/cell, washed with MBS-buffer (25 mM morpholine-ethanesulfonic acid, 145 mM NaCl, pH 5.5). Following incubation at 37° C. for the indicated time intervals, the infected cells were harvested at 2, 4, 6, 8, 10, 12, 16 and 20 h post-infection (p.i.) and subsequently frozen at −70° C. Virus titres were determined and expressed as plaque forming units per millilitre (pfu/ml).

(5) Isolation of virus after infectious copy cloning was on primary pig kidney (PK) cells, as described in Maree et al. (2010, Virus Res., vol. 153, p. 82). Culture, passage and amplification of wildtype and of recombinant FMD viruses was done on BHK-21 cells; infected or transfected 35 mm BHK-21 cell monolayers were frozen and thawed, and 1/10th of the volume was used to inoculate a fresh BHK-21 monolayer. Following virus adsorption (with periodical rocking for 60 min at 37° C.), virus growth medium (VGM; Eagle's basal medium (BME) with 1 FCS, 1 HEPES and antibiotics) was added, and the culture was incubated for no longer than 48 h at 37° C., after which the infected cells were frozen for subsequent passaging of the viruses.

(6) 1.2. Infectious clones of FMDV

(7) The infectious clone technology was used as a convenient way of generating infectious virion capsids comprising a specific VP2 protein mutant. The procedures applied were essentially as described in Rieder et., al. (1994, J. Virol., vol.68, p. 7092). In short:

(8) 1.2.1. Construction:

(9) A genome-length cDNA copy of the SAT-2 vaccine strain, ZIM/7/83, was constructed following an exchange-cassette strategy using an FMDV A12 genome-length clone for a template, as described (Rieder et al., 1993, J. of Virol., vol. 67, p. 5139; Van Rensburg et al., 2002, Ann. N Y Acad. Sci., vol. 969, p. 83). This initial construct was used for the transfection of in vitro synthesized RNA transcripts, followed by the recovery of infectious viral particles. This was later optimized to allow direct transfection of BHK-21 cells with DNA, and the exchange of the outer capsid-coding region. This system was used to prepare synthetic RNA or plasmid DNA for transfection of BHK-21 cells, and generation of viable SAT-2 FMDV.

(10) 1.2.2. Site-Directed Mutagenesis:

(11) Site-directed mutagenesis of infectious clone plasmids was done using amplicon overlap-extension PCR and site-directed mutagenesis, using the QUICKCHANGE XLII™ mutagenesis kit (for easy modification of nucleotides; Clontech) according to the manufacturer's instructions (Papworth et al., 1996, Strategies, vol. 9, p. 4). Briefly, each of the PCR processes involved the use of two genome-specific overlapping (reverse-complemented) oligonucleotides, to introduce mutations into distinct PCR products. The mutagenic primers were designed to be between 40 and 49 nucleotides in length and encoded the desired mutation with about 15 to 20 nucleotides of overlapping sequence that matched the viral sequence on both sides of the mutation. The PCRs were performed with the PFU ULTRA TAQ™ polymerase high fidelity DNA polymerase) and cycling conditions using: 95° C. for 50 s, 60° C. for 60 s, and 68° C. for 6 min (18 cycles). The PCR amplicons were cut out with an appropriate restriction enzyme, and used to transfect ultra-competent XL10-Gold™ E. coli cells. Following confirmation of the introduced nucleotide mutations by sequencing, a DNA fragment containing the region encoding the VP2 protein mutant was inserted into a DNA cloning plasmid for further use.

(12) In Vitro RNA Synthesis, Transfection and Virus Recovery

(13) RNA was synthesized from linearized plasmid DNA templates with the MEGASCRIPT™ T7 kit (an ultra-high yield in vitro transcription kit; Ambion). The transcript RNAs were examined by agarose gel electrophoresis to evaluate their integrity and the RNA concentrations were determined spectrophotometrically. BHK-21 cell monolayers, in 35 mm cell culture wells (Nunc™), were transfected with the in vitro-generated RNA using LIPOFECTAMINE2000™ (a cationic liposome formulation; InVitrogen). The transfection medium was removed after 3-5 h. and replaced with viral growth medium, followed by incubation at 37° C. for up to 48 h with a 5% CO.sub.2 influx. After one freeze-thaw cycle, the transfection supernatants were used for serial passaging on BHK-21 cells. BHK-21 monolayers in 35 mm cell culture wells were infected using 1/10th of clarified infected supernatants and incubated for 48 h at 37° C. Viruses were subsequently harvested from infected cells by a freeze-thaw cycle and passaged four times on BHK-21 cells, using 10% of the supernatant from the previous passage. Following the recovery of viable (recombinant) viruses, the integrity of the viruses was verified once again with automated sequencing using the ABI PRISM™ BIGDYE Terminator Cycle Sequencing Ready Reaction Kit v3.0 (Perkin Elmer Applied Biosystems). Typically viruses were passaged four times before analysis.

(14) RNA Extraction, cDNA Synthesis, and PCR Amplification.

(15) RNA was extracted from infected cell lysates using either a standard guanidinium-based nucleic acid extraction method or TRIZOL™ reagent (a guanidinium thiocyanate reagent: Life Technologies) according to the manufacturer's instructions and used as template for cDNA synthesis. Viral cDNA was synthesised with SUPERSCRIPT III™ (a reverse transcriptase; Life Technologies), as described in Bastos et al. (2001, Arch. Virol., vol. 146, p. 1537).

(16) 1.3. Virus Neutralization Test

(17) The detection of a virus-neutralising antibody response in vaccinated animals was done by using the micro-neutralization test, essentially as described in the OIE Manual of Standards (2009). Reference cattle sera were prepared by two consecutive vaccinations (vaccinated on day 0, boosted on day 28, and bled on day 38) with FMDV that are the same as or equivalent to the virus against which the antibody response was to be detected. IB-RS-2 cells were used as the indicator system in the neutralization test. The end point titre of the serum against homologous and heterologous viruses was calculated as the reciprocal of the last dilution of serum to neutralise 100 TCID50 virus in 50% of the wells. One-way antigenic relationships (R1-values) of wildtype and engineered FMDV viruses relative to the reference sera were calculated and expressed as the ratio between the heterologous/homologous serum titre. All neutralization titre determinations were repeated at least twice.

(18) 1.4. Sucrose Density Gradient Purification

(19) Sucrose density gradient separation was used for purification of virion capsids, as well as of empty capsids, and at different stages of production, inactivation, or harvest. FMDV empty capsids from an expression system, such as from Vaccinia or Baculovirus, were harvested, loaded onto a 15-45% sucrose gradient and spun for 20 h at 22.000 rpm (SW41 rotor, Beckman) at 12° C. FMDV virion capsids from a cell-culture were harvested, clarified, concentrated with 8% (w/v) PEG 6000 at 4° C. The precipitate was solubilised in 50 mM HEPES (pH 8.0) with 200 mM NaCl and 1% NP40, and resolved on 10-50% (w/v) sucrose density gradients in HEPES/NaCl by rate zonal centrifugation at 36.000×g for 16 h at 4° C. Next, each gradient was fractionated, and fractions were analysed spectrophotometrically by measuring the absorbance at 260 nm. Fractions containing 146S virions were quantified based on absorption, and pooled for analysis. The presence of the outer capsid proteins was verified by agar-gel protein-electrophoresis using standard SDS-PAGE protocols, or Western blotting. The integrity of the RNA in virion capsids was verified by RT-PCR and sequencing of the VP1 coding region.
1.5. Baculovirus Expression System

(20) The Baculovirus/insect cell expression system (BVES) was used to express FMDV empty capsids with either parental VP2 or VP2 protein mutant. Procedures were essentially as described in (Porta et al., 2013, J. Virol. Methods, vol. 187, p. 406). In short: Sf9 cells were grown in Insect-XPRESS™ (an insect cell culture media; Lonza) supplemented with 2% FCS and antibiotics at 27.5° C. Transfer vector and AcMNPV bacmid KO1629 (0.5 μg of each) were mixed in the presence of 3 μl FUGENE™ (a nonliposomal transfection reagent; Roche) for 20 min at room temperature and used to transfect Sf9 cells at a density of 1.2×10{circumflex over ( )}6/well in a 6-well plate. Since Baculovirus DNA with a knockout of gene 1629 will not initiate an infection unless rescued by recombination with a Baculovirus transfer vector, the AcMNPV harvested in the culture supernatant after 5 days was 100% recombinant virus. Virus stocks were produced by infecting Sf9 cell monolayers at a confluence of 70% with 200 μl recombinant virus inoculum per 175 cm.sup.2 flask and harvested from culture supernatants after 5 days. Alternatively, adherent cell-culture in roller bottles, and 2 I suspension cultures were used. For the expression of empty capsids, Sf9 cells at a density of 1-2 10{circumflex over ( )}6/ml were infected with 1/10 volume of Baculovirus stock. After 3 days virus extraction was with 1% Triton X-100 in the presence of 5 μl/ml protease inhibitor cocktail (Sigma).
1.6. Vaccinia Expression System

(21) The Vaccinia virus expression system was used to express FMDV empty capsids, comprising either wildtype VP2 protein or VP2 protein mutant. Procedures were essentially as described in King et. al. (supra), these have been applied to serotypes O, SAT2 and A. In short the procedures, exemplified by A22 were as follows.

(22) 1.6.1. Vaccinia Virus Transfer Vectors

(23) An expression cassette based on the sequence of FMDV A22 Iraq was designed, synthesized de novo (Geneart™) and cloned into the Vaccinia virus transfer vector pBG200 downstream of the T7 promoter. Substitution of a BstEII-SpeI fragment with a sequence encoding the VP2 H93F mutation converted the pBG200-A22-wt plasmid to pBG200-A22-H93F.

(24) 1.6.2. Generation and Selection of Vaccinia Virus Recombinants

(25) Recombinant Vaccinia viruses were made by transfecting plasmids pBG200-A22-wt and pBG200-A22-H93F into CV-1 cells infected with Vaccinia virus (VV) strain WR. Recombinant VVs (with an interrupted thymidine kinase gene) were selected in HuTK-143 cells using 5-bromo-2-deoxyuridine. Three rounds of plaque purification in conjunction with screening by PCR using FMDV-specific primers were carried out to obtain stable recombinant VVs. These were amplified in RK13 cells and virus stocks titrated by plaque assay on BS-C-1 cells. All mammalian cells were grown at 37° C. in DMEM, supplemented with 10% FCS and appropriate antibiotics.

(26) 1.6.3. Sedimentation of Empty Capsids Produced via Vaccinia Virus Expression

(27) A 175 cm.sup.2 flask of RK13 cells was infected with either vA22-wt or vA22-H93F at an MOI 10 and vTF7.3 at an MOI 5. After 24 h cells were harvested by centrifugation at 2.000×g for 5 min at 4° C. and the pellet resuspended in 1 ml 0.5% IGEPAL™ (a nonionic detergent: Sigma) in 40 mM sodium phosphate, 100 mM NaCl pH 7.6. Samples were incubated on ice for 20 min, clarified, loaded onto a 15-45% sucrose gradient and spun for 20 h at 22.000 rpm (SW41 rotor, Beckman) at 12° C. Each gradient was fractionated into 12 fractions of 1 ml and aliquots were analysed by Western blotting.

(28) 1.7. Capsid Dissociation Assays

(29) Wild-type and VP2 protein mutant-containing FMDV capsids, present in cell culture supernatants or in samples that were purified by sucrose gradient, were taken up into standard TNE buffer, and subjected to conditions of different pH or temperature to assess their stability.

(30) 1.7.1. pH Stability of Virion Capsids:

(31) Briefly: 10% to 10{circumflex over ( )}7 pfu/ml of infectious FMDV virion capsids were mixed with TNE buffer (pH preferably above 7), at a ratio of 1:50 v/v respectively. The mixtures were subsequently incubated for 30 min. at room temperature. As a control, virus particles were also mixed with virus growth medium at the same ratio. The samples were subsequently neutralised with 1 M Tris (pH 7.4), 150 mM NaCl and titrated on BHK-21 cells. Alternatively, sucrose gradient purified particles with an approximate titre of 4-8×10{circumflex over ( )}6 pfu/ml were treated at pH 6.0, for different time intervals following a 1:50 dilution in TNE buffer.

(32) 1.7.2. Temperature Stability of Virion Capsids:

(33) Alternatively FMDV virion capsids in TNE buffer (pH 7.4) were treated at temperatures of 25, 37, 45 or 55° C. for 30 minutes in a water bath, after which the samples were cooled on ice and titrated. The 1:50 dilution of the sucrose gradient purified particles ensured that any stabilising effect of the sucrose was negligible. Also, sucrose gradient purified particles with an approximate titre of 4-8×10% pfu/ml were heated to 42° C. or 49° C., for different time intervals.

(34) The number of infectious FMDV virion capsids remaining after low pH- or high temperature treatment was determined by plaque titrations on BHK-21 cells. The respective logarithmic values of the virus titres at the different time points were linearly fitted and the slopes were determined. The percentage of remaining infectious particles was also calculated and plotted along with the exponential decline used to calculate the inactivation rate constant.

(35) 1.7.3. Stability Assays of Empty Capsids:

(36) A 200 μl aliquot of an empty capsid-containing fraction was diluted 1:3 either (i) with phosphate buffer pH 7.6 and incubated in a water bath at 56° C. for 2 h, or (ii) with 50 mM sodium acetate buffer pH 4.6, to give a final pH of 5.2 and incubated at room temperature for 15 min before neutralisation with NaOH. Treated samples were loaded onto 15-45% sucrose gradients, and centrifuged. Each fraction was precipitated with an equal volume of saturated ammonium sulphate overnight at 4° C. Precipitates were collected by centrifugation at 16.000×g for 15 min at 4° C. and analysed by western blot.

(37) 1.8. ELISA Assays

(38) ELISA was performed as described in Harmsen et al. (supra). In short this regards a double antibody sandwich ELISA for quantification of FMDV capsids, using one of two single-domain Llama derived antibody fragments that are specific for FMDV structures of either 146S virion capsid (antibody M170) or of 12S pentamer structures (antibody M3). Only O serotype strains could be detected in the 146S specific ELISA, whereas strains of most serotypes are detected in the 12S specific ELISA. However, the 146S concentration of serotypes A and Asia 1 FMDV strains could be measured indirectly using the 12S specific ELISA by prior conversion of 146S into 12S particles by heat treatment. Stability was determined by thermofluor assay.

(39) 1.9. Electron Microscopy:

(40) EM was used to study the level of intactness of virion capsids after chemical inactivation, and of empty capsids, after heat treatment.

(41) Purified chemically-inactivated virion capsids of wildtype and VP2 S93Y were examined by electron microscopy after a storage period of 10 days at 4° C. The samples were allowed to adhere on carbon coated formvar grids for 30 s, followed by two washes with water before staining with 1% uranyl acetate for 45 s. Excess stain was removed by blotting and the grids examined on a FEI T12 electron microscope operating at 80 KeV. For empty capsids, purified wildtype and VP2 S93F capsids were heated to 56° C. for 2 hours before examining by EM.

(42) 1.10. Thermofluor Assays

(43) The Thermofluor shift assay was performed as described (Walter et al., 2012, supra) to measure and compare temperature stability of infectious FMDV virion capsids; either VP2 protein mutant- or wildtype VP2 protein-comprising FMDV were used to test the capsid stability by detecting the release of the viral RNA, by monitoring the RNA-specific fluorescent dye SYBR GREEN™. A temperature gradient was applied from 25-95° C. and viral genome release was detected by increase in fluorescence as the capsids dissociated into pentamers.

(44) 1.11. FMDV Inactivation

(45) Mutant and wildtype FMDV were harvested from infected BHK-21 cell monolayers, and were inactivated with 5 mM binary ethyleneimine (BEI) for 26 h at 26° C. Next they were PEG concentrated and purified on a sucrose gradient. The BEI-inactivated, sucrose gradient-purified antigens were used for formulation into vaccines for animal vaccinations.

(46) 1.12. Animal Vaccination Experiments

(47) Vaccines prepared from FMDV capsids were tested in the standard FMDV serology model: the Guinea pig, to evaluate their immunogenic potency and duration of the antibody response, as a function of their antigen stability. Vaccine antigens were either wildtype FMDV capsids, or VP2 S93Y substitution mutant-comprising capsids of the wildtype strain. These were tested both as inactivated virion capsids and as empty capsids, and were tested either for SAT-2 serotype or for 0 serotype, respectively. All animal experiments were performed in full compliance with legal- and animal welfare regulations. Groups of 10 Guinea pigs received a 0.2 ml dose of a test- or control vaccine by intra-muscular inoculation, and were bled at day zero and at different time points during the course of the experiment. FMDV-neutralizing antibodies in the sera were analysed using the virus neutralisation (VN) test according to OIE guidelines.

(48) 2. Results and Conclusions

(49) 2.1. Temperature Stability Assays

(50) 2.1.1. Stability of Infectious Virion Capsids:

(51) Dissociation Kinetics:

(52) Infectious virion capsids were subjected to dissociation kinetics assays, comparing wildtype- and VP2 protein mutant-containing virion capsids. Virion capsids of both types at a titre of about 2-10×10{circumflex over ( )}5 pfu/ml were incubated at 49° C. for 2 hours. The number of infectious particles remaining intact after this treatment was determined. The temperature inactivation profiles of the particles followed linear kinetics and the lability of the different viruses was reflected by the inactivation rate constant values found; these were: wildtype SAT-2: 0.0155/min.; SAT-2 VP3 E198A: 0.0163/min.; and SAT-2 VP2 593Y: 0.0075/min. This showed that the inactivation rate of wildtype and of a (control) VP3 substitution mutant were essentially the same, whereas the inactivation rate for the VP2 S93Y mutant was significantly lower. This reflects the improved thermal stability of FMDV capsids comprising the VP2 protein mutant according to the invention, when compared to capsids with wildtype VP2 protein, or capsids containing VP2 protein with arbitrary substitutions.

(53) When the percentage of infectious FMDV virion capsids remaining after heat treatment was determined, it was found that about 15% of the SAT-2 VP2 S93Y virion capsids remained following a 2 hour incubation at 49° C., compared to only 1.4% remaining of the wildtype SAT-2 and the SAT-2 VP3 E198A substitution mutant virus.

(54) Thermofluor Assays:

(55) The thermofluor shift assay was used as a direct measurement of FMDV capsid stability, in response to temperature variation. The assay detects the release of RNA, monitored by an RNA-specific fluorescent dye that binds the viral genome, upon the dissociation of a capsid. The virions were purified by sucrose gradient, and inactivated with BEI. Virion samples had concentrations between 140 and 420 μg/ml. As a control, FMDV serotype A, isolate 24 virus was included.

(56) Because typically sharp clear peaks in the first derivative of fluorescence were observed under steady heating, the peak temperatures were taken as a direct indicator of the dissociation temperature for that virus construct. The values found were: SAT-2 VP2 S93Y dissociated at 53° C., SAT-2 VP2 S93H at 51° C., and the wildtype SAT-2 at 47° C., see FIG. 4. Control samples were: FMDV A24 virus, which had the overall highest dissociation temperature of 55° C., however this is only 2 degrees higher than the SAT-2 VP2 S93Y mutant. A SAT-2 VP2 S113G substitution-mutant virion capsid (not presented in FIG. 4) served as a negative control. This showed a decrease in dissociation temperature to 45° C., even a few degrees lower than the wildtype SAT-2 virus. The virion capsid SAT-2 VP2 S93Y therefore had the highest increase in dissociation temperature as compared to its wildtype virus of 6° C. This agrees well with the degree of stabilisation observed in the heat inactivation kinetic assay (supra).

(57) The stability of FMDV virion capsids of serotype O, isolate O1 Manisa was also determined using thermofluor analyses. These assays were run in a different buffer at pH 7.0, therefore their base level differs from that of FIG. 4. Results are presented in FIG. 10, divided over two panels, to prevent cluttering the image. The peak values found were: Panel A: wt O1M: 40° C.; 93F: 43° C.; 93Y: 44° C. Panel B: wt O1M: 40° C.; 93W: 42° C.; 97Q: 42° C.; 98F: 44° C.
2.1.2. Stability of Empty Capsids:

(58) Stability of FMDV empty capsids was tested in a variety of ways. First, the sucrose gradient purification already provided a reliable indication of capsid integrity: when capsids had disintegrated, no clear band could be obtained using sucrose gradient centrifugation. Next, thermostability was tested using gel-electrophoresis and Western blot: sucrose gradient purified empty capsids were heated to 45° C. for 1 h (FIG. 5, panel A), or to 56° C. for 2 h (FIG. 5, panel B). Next, samples were loaded onto a further 15-45% sucrose density gradient, centrifuged, and the gradient was collected into 12 fractions of 1 ml. From each fraction 500 μl was precipitated with 500 μl saturated ammonium-sulphate and the pellet was run on an SDS gel. Finally the gel was blotted and the blot incubated with an antibody against VP1. Empty capsids that remained intact after heat incubation gathered just below the middle of the sucrose gradient, ending up in fractions 4 and 5, and showed up in the gel-lanes corresponding to these fractions. Whereas (partly) disassembled capsids were less dense and remained near the top of the gradient, and were found in the gel-lanes corresponding to fractions 10 and 11.

(59) Results showed that empty capsids from wildtype FMDV of serotype O, subtype 1 Manisa rapidly disintegrated, and were found in fractions 10-11 for both temperatures. Surprisingly, VP2 protein mutant-containing capsids could much better resist the heat treatment: FMDV capsids with VP2 protein mutants with substitutions S93H and S93F were all totally intact after 1 h at 45° C. (FIG. 5, panel A). After 2 hours at 56° C. the mutant S93H was not stable (all material in fractions 10-11; FIG. 5, panel B); mutant S93Y was partly stable (about 10% was in fraction 10, rest in fraction 4); and S93F was completely stable (all in fraction 4).

(60) A sample from the VP2 S93F protein mutant comprising empty capsid material from the 2 h 56° C. treatment, was also studied by electron microscopy, which confirmed the gel-electrophoresis results: all these capsids were found to be intact and no pentamers could be seen (FIG. 6, see below).

(61) When FMDV empty capsids of serotype O, isolate O1 Manisa, were expressed via the baculovirus-/insect cell expression system, wildtype O1M empty capsids could not be obtained after the standard 5 day culturing conditions. However a mutant VP2 protein-containing empty capsid, having the S93F substitution could be produced in either roller bottles, or in 2 I. suspension cultures. This yielded adequate amounts of stabilised mutant capsids for analyses such as Western blot, sucrose gradient, ELISA, and EM, as well as for vaccination studies in Guinea pigs.

(62) 2.2. ELISA Assays

(63) ELISA assays were used to detect the level of dissociation of live infectious FMDV during incubation at 49° C. for 1 hour. Compared were wildtype serotype 0 FMDV (FIG. 7, panel A), and a VP2 protein mutant-containing serotype O FMDV, namely VP2 S93Y (FIG. 7, panel B). The lines correspond to the total amounts of 146S or 12S particles that were detected over time, next to a blank control sample. More 12S signal indicates higher capsid breakdown. For the wildtype FMDV, virion capsid levels decreased, and 12S levels steadily increased during the 1 h incubation. The blank sample remained at threshold level as expected. However for the VP2 protein mutant-containing virion capsids, no rise in the level of 12S particles was detected, indicating a much improved thermal stability as compared to the wildtype virion capsids.
2.3. Stability After Chemical Inactivation

(64) FMDV virion capsids were subjected to standard chemical inactivation with BEI and analysed by electron microscopy. This was done either directly after the chemical inactivation, or after a further 10 days storage at 4° C. The FMDV capsids tested were either wildtype FMDV of serotype O subtype 1 Manisa, or the corresponding VP2 protein mutant-containing virion capsids with substitution VP2 S93Y.

(65) The EM analyses showed that initially the amount of pentamers versus the amount of intact virion capsids was 10:90% for the wildtype virion capsids, and 0:100% for the VP2 protein mutant-containing capsids. After cold storage the difference became much larger; the wildtype material was 80:20% pentamers versus intact virion capsids (FIG. 8, panel A), as compared to 10:90% for the mutant VP2-containing virion capsids (FIG. 8, panel B).

(66) 2.4. Results From Animal Vaccination Studies

(67) 2.4.1. Vaccination with Empty Capsids:

(68) FMDV empty capsids of Serotype O, subtype 1 Manisa, were expressed in a Baculovirus/insect cell system; empty capsids were either of wildtype, or comprised VP2 protein mutant with the VP2 S93F substitution. Unfortunately, the parental serotype O1 M empty capsids were so unstable they could hardly be detected after insect cell expression (5 days at 27° C., using standard pH 6.5 insect cell culture medium). Therefore only VP2 protein mutant-containing empty capsids were used to vaccinate Guinea pigs. The capsid antigen was formulated in standard w/o/w emulsion, in doses equivalent to 5 or 20 μg 146S antigen per final vaccine dose. As a control, standard inactivated whole virus FMDV vaccine of type O1M was also inoculated.

(69) Virus neutralisation results at 3 and at 4 weeks post vaccination showed Guinea pig VN titres resulting from the S93Y mutant empty capsid vaccine was comparable to the titres obtained with classical FMDV vaccine. It was concluded that the potency of a vaccine based on in vitro expressed VP2 protein mutant-comprising FMDV empty capsids, had demonstrated proof of concept.

(70) 2.4.2. Vaccination with Inactivated Virion Capsids:

(71) Virion capsids of FMDV SAT-2 serotype from wildtype and from VP2 protein mutant-comprising virion capsids, carrying the VP2 S93Y substitution, were tested in Guinea pigs. Virion capsids had been inactivated with BEI. First the amount of intact virion capsid antigen was determined, and samples were diluted such that the final vaccine concentration was 5 μg/ml. Samples were formulated in standard w/o/w emulsion, and after formulation, the samples were stored for 1 month at 4° C. Two groups of 10 guinea pigs were immunised, and blood samples were taken at day zero, and at 1 and 6 months post-immunisation. IB-RS-2 cells were used as an indicator system and wildtype FMDV SAT-2/ZIM/7/83 was used as the reference virus. In this set-up Log 2 virus neutralisation titres of 5 and above are considered protective.

(72) No FMDV neutralising antibodies were detectable on the day of inoculation. The vaccinates receiving the wildtype FMDV virion capsids did not serorespond above protective levels, whereas all animals receiving virion capsids with the VP2 protein mutant showed protective level of seroresponse, both at 1 and at 6 months post vaccination. The difference in group mean neutralising antibody titres was significant (p>0.05) at both time points (error bars=SEM). Results are presented in FIG. 9.

(73) The fact that the vaccine of the wildtype FMDV virion capsids did not induce a protective seroresponse, was most likely caused by the instability of the native capsids after inactivation, and following the 1 month storage.

(74) However, the virion capsids comprising the VP2 protein mutant according to the invention were so stable they survived both the chemical inactivation and the storage, and still were able to induce solid protective humoral immunity, even after only a single vaccination, and even up to 6 months post vaccination. The inventors were surprised to find this was even possible for an FMDV vaccine of the SAT-2 serotype.

(75) 3. Ongoing and Planned Experiments:

(76) 3.1. Guinea Pig Vaccination Experiments

(77) A Guinea pig vaccination experiment is in preparation which will use a vaccine of FMDV empty capsids of serotype O1 Manisa, containing either parental empty capsids, or comprising the VP2 protein mutant with the VP2 S93Y substitution. Several test and control groups will be included; groups of 5 Guinea pigs each will receive the different capsid antigens (wildtype or mutant), formulated in a w/o emulsion adjuvated with light mineral oil.

(78) Further, a similar Guinea pig experiment is planned for testing a different adjuvant formulation: FMDV capsids of serotype O and possibly A will be formulated into a single oil-in-water emulsion, using a light paraffin oil.

(79) 3.2. Cattle Vaccination-Challenge Studies

(80) Two separate cattle vaccination-challenge studies are planned to be held in appropriate high-containment facilities. The first study will be a vaccination using inactivated virion capsids of SAT-2 serotype FMDV, with or without VP2 protein mutant VP2 S93Y. According to the protocol non-pregnant heifers will receive a single dose of vaccine, and will be challenged with a wildtype SAT-2 FMDV strain.

(81) We plan to divide 26 cattle into five groups: Group 1: 6 cattle will be vaccinated with wild type FMDV SAT-2 vaccine in commercial adjuvant; Group 2: 6 cattle will receive SAT-2 93Y VP2 protein mutant antigen in commercial adjuvant; Group 3: 6 cattle will receive SAT-2 93H VP2 protein mutant in commercial adjuvant; Group 4: 6 cattle will receive SAT-2 93H VP2 protein mutant with an alternative adjuvant. Group 5: 2 cattle receiving a mock vaccination.

(82) Sera will be collected at regular intervals, initially every 2 days, then weekly and monthly, and will be assessed using VN test. Once the VN titres start to drop the animals will be challenged, in conditions of high containment, with 10{circumflex over ( )}4 cattle adapted, live FMDV viral particles, intradermolingually.

(83) In a comparable experiment, cattle will be vaccinated according to a standard PD50 type protocol, as prescribed by the European Pharmacopeia, using FMDV empty capsids of serotype O1 Manisa, with or without VP2 protein mutant comprising the VP2 S93Y substitution. As control, one group will receive a standard serotype O FMDV vaccine. The protocol incorporates the vaccination, and subsequent challenge infection with a Serotype O wildtype FMDV strain. Experimental analysis will include full serology, as well as challenge virus re-isolation.

LEGEND TO THE FIGURES

(84) FIG. 1:

(85) Schematic structure of the icosahedral FMDV capsid. The VPs are indicated: 1=VP1, 2=VP2, and 3=VP3. A pentamer subunit is outlined by thick lines.

(86) The icosahedral symmetry axes are indicated: 5-fold: pentagon, 3-fold: triangle, and 2-fold: oval. (From: Mateo et al., 2003, J. Biol. Chem., vol. 278, p. 41019, FIG. 1, panel A.)

(87) FIG. 2: Multiple alignment of the amino acid sequence of the VP2 protein αA helix region for a number of representative FMDV isolates; at the top the numbering and the amino acid sequence are indicated from the FMDV serotype O isolate 1 BFS, which is represented in SEQ ID NO: 1 and individually as: O1BFS SEQ ID NO: 2 O1M 87 SEQ ID NO: 3 CS8c1 SEQ ID NO. 4 ASIA Bar2003 SEQ ID NO: 5 A22 Iraq 95 SEQ ID NO: 6 SAT1 bot SEQ ID NO: 7 SAT ZIM7 83 SEQ ID NO: 8 SAT3 KNP10 90 SEQ ID NO: 9

(88) FIG. 3:

(89) Graphical representation of a 3-dimensional model structure of the two-fold symmetry axis in an FMDV capsid, showing hydrophobic stacking interactions between two neighbouring VP2 protein mutants with a 93W substitution.

(90) FIG. 4:

(91) Illustrative results of a Thermofluor assay, using live FMDV virion capsids from serotype SAT-2. The peaks indicate the temperature at which maximal release of RNA, monitored by RNA-specific fluorescent dye, thus where the FMDV virion capsid fully dissociates. This marks the dissociation temperature.

(92) A24=wildtype FMDV serotype A, isolate 24; 93Y=FMDV SAT-2 VP2 S93Y; 93H=FMDV SAT-2 VP2 S93H; WT=wildtype FMDV SAT-2.

(93) FIG. 5:

(94) Western Blot analysis of heat stable FMDV empty capsids of serotype O, subtype 1 Manisa, which were heated to 45° C. for 1 hour (panel A), or 56° C. for 2 hours (panel B) and then loaded onto a 15-45% sucrose density gradient. FMDV empty capsids with VP2 S93F or VP2 S93H VP2 protein mutant remained intact at 45° C. (panel A) and migrated through the gradient to fractions 4 and 5 whereas the wild-type VP2 protein containing capsids broke apart on heat treatment, remaining near the top of the gradient in fractions 10 and 11. When the experiment was repeated, empty capsids containing S93F VP2 protein mutant were remarkably stable even after 2 hat 56° C.; VP2 593Y containing capsids had about 10% degradation, and VP2 S93H containing capsids were unstable.

(95) FIG. 6:

(96) Electron micrograph of heat treated empty FMDV capsids containing VP2 protein mutant with S93F substitution. The mutant capsids were incubated at 56° C. for 2 hours and the samples examined by electron microscopy. The capsids were found completely intact. Bars indicate a size reference.

(97) FIG. 7:

(98) Results of ELISA assay, detecting the level of dissociation of infectious FMDV virions during incubation at 49° C. for 1 hour. Compared were wildtype serotype O FMDV (panel A), and a VP2 protein mutant-containing serotype O FMDV, namely VP2 S93Y (panel B). The lines correspond to the amounts of 146S or 12S particles that were detected over time, next to a blank control sample.

(99) FIG. 8:

(100) Electron micrographs of chemically inactivated FMDV virion capsids of serotype O subtype 1 Manisa that had been stored after inactivation for 10 days at 4° C. Panel A shows a sample of wildtype FMDV and panel B shows a sample of a similar initial amount of the corresponding virion capsids that comprised a VP2 protein mutant, with substitution S93Y. About 80% of wildtype inactivated capsids were found to be dissociated into pentamers. However inactivated VP2 S93Y mutant capsids were about 90% intact.

(101) FIG. 9:

(102) Graphical representation of results from animal vaccination trial with inactivated SAT-2 FMDV virion capsids comprising VP2 protein mutant with VP2 S93Y substitution. Plotted are the mean Log 2 virus neutralisation titres of groups of 10 Guinea pigs, at 1 month and at 6 months post vaccination. Error bars indicate s.e.m. The vaccine was based on inactivated SAT2 serotype FMDV virion capsids of either wildtype SAT-2, or VP2 protein mutant-comprising virion capsids with a VP2 S93Y substitution. Log 2 virus neutralisation titres of 5 and above are considered protective. SAT 593Y=SAT-2 VP2 S93Y; SAT wt=wildtype SAT-2.

(103) FIG. 10:

(104) Results of thermofluor analyses with FMDV virion capsids of Serotype O, isolate O1 Manisa. The graphs are divided over two panels, to prevent cluttering the image. These assays were run in a buffer at pH 7.0.

(105) WT=wildtype FMDV serotype O, isolate 1 Manisa; 93Y=FMDV serotype O, isolate 1 Manisa VP2 S93Y; 93F=FMDV serotype O, isolate 1 Manisa VP2 S93F; 93W=FMDV serotype O, isolate 1 Manisa VP2 S93W; 97Q=FMDV serotype O, isolate 1 Manisa VP2 S97Q; 98F=FMDV serotype O, isolate 1 Manisa VP2 Y98F.