Institute for Animal Health, Pirbright Laboratory, Pirbright, Surrey GU24 0NF, UK1
INRA Virology et Immunologie moléculaires, INRA, 78350 Jouy-en-josas, France2
Author for correspondence: Leticia Cedillo-Barrón. Present address: Centro de Investigacion y de Estudios Avanzados del IPN, Departamento de Biomedicina Molecular, Av. IPN #2508 Col. Zacatenco, 07360 México DF. Fax +52 5 747 7134. e-mail lcedillo{at}mail.cinvestav.mx
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Abstract |
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Introduction |
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Currently, control of the disease includes slaughter of infected and exposed animals and vaccination with a chemically inactivated virus vaccine. Such conventional vaccines, although generally effective, do have some disadvantages. These include thermal instability, the short-term nature of protection and extra cost due to the high-security containment necessary for their preparation. A particularly serious problem is that several outbreaks in Europe have been attributed to incomplete inactivation of the virus or to the escape of live virus from vaccine production laboratories. Thus, the development of recombinant FMDV vaccines, including peptide (DiMarchi et al., 1986 ; Geysen et al., 1984
; Francis et al., 1987
) and DNA vaccines (Ward et al., 1997
; Chinsangaram et al., 1998
), as potentially safe alternatives has been explored. Vaccination with DNA encoding protective antigens offers the potential to manipulate the induced immune response through the simultaneous delivery of adjuvants, cytokines and other proteins, enhancing the vaccination efficacy (Kim et al., 1997
; Somasundaram et al., 1999
; Tuteja et al., 2000
). In this study, we have taken advantage of the fact that natural empty particles, lacking nucleic acid and containing VP1, VP3 and VP0 (the uncleaved precursor form), are able to induce an antibody response similar to that induced by infectious FMDV particles in guinea pigs (Rowlands et al., 1975
). Significantly, these empty capsids induce neutralizing antibody of the same specificity as the whole virus. This is presumably because these particles resemble natural virus particles antigenically including, importantly, the major target of neutralizing antibodies, the GH loop of VP1 (Rweyemamu et al., 1979
; Grubman et al., 1985
; Geysen et al., 1984
; Francis et al., 1985
). In principle, therefore, empty capsids appear to be a good candidate for the development of a recombinant vaccine.
Although protection against FMDV generally correlates with neutralizing antibodies (van Bekkum, 1969 ), a T cell response is quite clearly necessary for effective immunity. While it is true that FMDV structural proteins do stimulate T cell responses (Garcia-Valcarcel et al., 1996
), we have shown recently that the non-structural proteins 3D and 2B are potent stimulators of cellular and humoral specific immune responses in the natural host (Foster-Cuevas, 1996
; Foster et al., 1998
; Collen et al., 1998
). Furthermore, these non-structural proteins also induce high proliferative T cell and delayed-type hypersensitivity (DTH) responses when administered as DNA vaccines (L. Cedillo-Barrón and M. Foster-Cuevas, unpublished data).
Taking this into account, we have constructed a plasmid (pcDNA3.1/P12A3C3D) that contains all of the structural and non-structural proteins necessary for the formation of FMDV empty capsids, i.e. the P1 sequence plus the 2Apro and 3Cpro proteases necessary for processing of the P1 polyprotein into VP1, VP3 and VP0. In addition, the 3D RNA polymerase was included as a potent source of T cell epitopes. This construct, when tested in pigs, induced neutralizing antibody and protection. Finally, we have shown that co-administration of a plasmid encoding porcine granulocytemacrophage colony-stimulating factor (GM-CSF) can improve the antibody responses to the virus significantly.
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Methods |
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FMDV O1Kaufbeuren (O1K) was prepared by infecting confluent monolayers of baby hamster kidney (BHK) cells. The infected cultures were incubated on a rocking platform until cell lysis was observed (1824 h) and the lysate was then clarified by centrifugation (3000 r.p.m., 20 min).
Construction of recombinant plasmid.
The P12A3C3D cassette, which contains FMDV serotype O1K sequences, includes the complete P12A, 3C and 3D coding sequences with the addition of an ATG initiation codon. It was subcloned into pcDNA3.1 downstream of cytomegalovirus (CMV) and T7 promoters. Plasmid pCAK (Belsham et al., 1990 ) carries the P12A region from FMDV O1K. The P12A sequence was excised by using EcoRI and XhoI endonucleases. At the same time, the 3C and 3D coding sequences were excised from pT7S3 (which contains the full-length infectious cDNA of FMDV O1K; Ellard et al. 1999
) by digestion with HpaI, blunt-ended and then digested with XhoI. Both fragments were then ligated into the pcDNA3.1 vector that had been digested with ApaI, blunt-ended and then cut with EcoRI. The resultant plasmid was called pcDNA3.1/P12A3C3D.
Expression of transfected recombinant plasmid.
Expression of the FMDV cassette in pcDNA3/P12A3C3D driven by the CMV promoter was verified by transfection of COS-7 cells (in 35 mm wells) using Fu-GENE 6 reagent (Boehringer Mannheim). Two days after transfection, cells were analysed for expression of FMDV proteins by labelling with [35S]methionine/[35S]cysteine and immunoprecipitation and the result was confirmed by indirect immunofluorescence. For immunofluorescence, monolayers of cells were grown on cover slips and, after transfection with pcDNA3.1/P12A3C3D, were fixed in 4% paraformaldehyde in PBS (1 h at room temperature). Samples were then blocked with 10% normal goat serum (NGS) (1 h, room temperature) and then stained with MAb 2B against VP1 (provided by E. Brocchi, Brescia, Italy) followed by fluorescein-conjugated goat anti-mouse serum. For [35S]methionine cell labelling, the cells were washed 48 h after transfection and labelled with 0·1 mCi [35S]Promix (Amersham) (methionine and cysteine) in met/cys-free supplemented medium and then lysed with RIPA buffer (100 mM TrisHCl, pH 8·38·6, 2% Triton X-100, 150 mM NaCl, 0·6 M KCl, 5 mM EDTA, 1% aprotinin, 3 mM PMSF, 1 µg/ml leupeptin and 5 µg/ml soybean trypsin inhibitor). For immunoprecipitation reactions, the lysate was pre-cleared with fixed cells of Staphylococcus aureus Cowan strain (1 h at 4 °C). After centrifugation, the labelled FMDV proteins were immunoprecipitated from the lysate with a hyperimmune guinea pig anti-FMDV antibody immobilized on Protein ASepharose beads (Pharmacia). The bound radioactive proteins were then detected by SDSPAGE and autoradiography. Expression driven by the T7 promoter was analysed in COS-7 cells infected with the recombinant vaccinia virus vTF7-3 (Fuerst et al., 1986 ) and then transfected with pcDNA3.1/P12A3C3D. After 24 h, the cells were labelled with [35S]met/cys and cell extracts were prepared and immunoprecipitated as described above.
Animals.
Thirty-two Large White cross-bred Landrace pigs weighing 2025 kg were used for the study. All animal infectivity experiments were performed within a high-containment isolation compound.
Vaccines and vaccination.
Inactivated FMDV vaccine was prepared from type O1 Lausanne FMDV antigen concentrate kept in the International Vaccine Bank at Pirbright. Vaccine was prepared as a water-in-oil-in-water emulsion using Montanide ISA 206 (Seppic) and contained 2·4 µg 146S antigen per 2 ml dose. Pigs were given a single dose by the intramuscular route, immediately caudal to the ear (Salt et al., 1998 ).
For DNA vaccination, recombinant plasmids were purified from Escherichia coli JM109 using the Qiagen plasmid purification Giga kit. DNA was diluted to the required concentration in endotoxin-free Dulbeccos PBS (DPBS, Sigma). Pigs were injected three times at 34 week intervals with 300 µg recombinant plasmid pcDNA3.1/P12A3C3D and, when specified, 200 µg porcine pcDNA3/GM-CSF diluted in 2·5 ml DPBS. Aliquots (1 ml) were injected, by using a 27 gauge needle, into the tibialis cranialis muscle of both legs and 0·25 ml aliquots were administered intradermally into the dorsal surface of the ears. Each injection was preceded by intramuscular administration of 10 mM carditoxin (Latoxan) in 0·5 ml endotoxin-free DPBS.
The experiments consisted of four groups, each containing four pigs: group 1, conventional FMDV vaccine; group 2, plasmid pcDNA3/GM-CSF; group 3, plasmid pcDNA3.1/P12A3C3D; group 4, plasmids pcDNA3.1/P12A3C3D and pcDNA3/GM-CSF. Serum samples were taken at regular intervals and assayed for antibodies against intact FMDV and FMDV 3D polymerase by ELISA and for neutralizing antibody as described previously (Golding et al., 1976 ).
Challenge of animals.
Two experiments were performed to determine the ability of the plasmid pcDNA3.1/P12A3C3D to induce an immune response against FMDV proteins. Vaccination and experimental infection of pigs were performed in an identical manner in each experiment, as described above. Between 15 and 18 days after the third immunization with DNA, all of the pigs were challenged with live virus. Challenge virus was prepared by serial passage of FMDV O1 Lausanne in pigs. Virulent FMDV was recovered from lesions and titrated on bovine thyroid (BTY) cells. Test animals were injected in the heel bulb with 105·5 TCID50 in 0·1 ml M25 saline. The FMDV O1 Lausanne and O1K isolates are quite closely related in terms of antigenic properties and P1 nucleotide sequence, and therefore efficient protection against the virus challenge occurs. After the virus challenge in experiment A, the animals from different groups were isolated in different rooms, whereas, in the second experiment (B), all groups were housed in the same room in order to expose the vaccinated animals to high levels of virus-containing aerosol from the infected, unvaccinated animals. Animals were examined daily and protection was scored by time of appearance and severity of lesions. Total protection was defined as complete absence of lesions. Animals were classified as partially protected against FMDV if the development of FMDV lesions was delayed and lesions were restricted in distribution. As animals were inoculated with FMDV in one rear foot, the development of a single foot lesion was considered to be a significant level of protection. The four groups included control animal groups vaccinated with conventional inactivated FMDV vaccine and unprotected animals similarly immunized with an unrelated plasmid (pcDNA3/GM-CSF).
Detection of anti-3D antibodies.
Detection of serum antibodies to the FMDV 3D protein was performed by ELISA using polyvinyl plates (Nunc) coated with purified recombinant GST3D (2·5 µg/ml) in 0·1 M carbonate/bicarbonate buffer, pH 9·6, overnight at 4 °C. After blocking with 0·05% Tween 20/3% skimmed milk/1% NGS in PBS, plates were incubated with duplicate double serial dilutions of test sera for 1 h at 37 °C. Goat anti-porcine Ig peroxidase conjugate (Sigma) at 1:5000 dilution was then added for 1 h, followed by the substrate (0·01% hydrogen peroxide in phosphate/citrate buffer). Absorbances were determined at 492 nm. Recombinant GST3D fusion protein was purified from bacterial inclusion bodies by preparative SDSPAGE (10%) as described previously by Katrak et al. (1992) .
Detection of antibodies to FMDV.
Ninety-six well flat-bottomed plates (Nunc Immunoplate II) were coated with rabbit anti-O1 Lausanne FMDV antiserum in 0·1 M carbonate/bicarbonate buffer, pH 9·6, overnight at 4 °C. Plates were washed three times and 50 µl clarified tissue culture fluid containing FMDV was added to each well. The plates were incubated for 60 min at 37 °C and then serial dilutions of test sera followed by peroxidase-labelled goat-anti-porcine IgG (Sigma) and substrate were applied sequentially as described above.
Detection of neutralizing antibodies.
Sera taken from all vaccinated pigs on day 0, before immunization, and on day 63, after the last DNA injection but before virus challenge, were analysed for neutralizing antibody titres by using a micro-neutralization assay with monolayers of primary BTY cells. Double dilutions of antibody were reacted with 0·5 log10 dilution of virus for 1 h. Cells were then added as indicators of residual infectivity and microplates were incubated at 37 °C for 3 days prior to fixing and staining (Golding et al., 1976 ). The end-point titres were calculated as the reciprocal of the last serum dilution to neutralize 100 TCID50 homologous FMDV in 50% of the wells.
Delayed-type hypersensitivity (DTH) reaction.
T cell stimulation in vivo was analysed by the DTH test (Foster et al., 1998 ; Bautista & Molitor, 1997
). Pigs were injected intradermally in the ventral abdomen with 7·5 µg purified inactivated O1 FMDV in 0·1 ml PBS or 20 µg purified GST3D RNA polymerase in 0·1 ml PBS. Purified recombinant GST and PBS were injected as negative controls. Skin induration was measured 48 h after antigen injection.
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Results |
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Effect of co-administration of a plasmid encoding porcine GM-CSF on anti-virus antibody production
Results obtained when the plasmid encoding porcine GM-CSF was co-administered with pcDNA3.1/P12A3C3D were generally inconclusive (see above). There was, however, a significant positive effect on the stimulation of antibodies against virus proteins. Statistical comparisons of antibody titres against whole virus for two groups of eight pigs, one group vaccinated with pcDNA3.1/P12A3C3D and the other group vaccinated with pcDNA3.1/P12A3C3D plus pcDNA3/GM-CSF, before immunization (day 0) and after the first (day 17), second (day 28) and third (day 56) DNA immunizations showed that the group of animals that received pcDNA3.1/P12A3C3D plus pcDNA3/GM-CSF had statistically significant higher antibody levels against FMDV after the second (P=0·008) and third (P=0·04) DNA immunizations (Fig. 6).
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Discussion |
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Recently, the use of a DNA vaccine for FMDV serotype A has been reported. The plasmid encoded the capsid precursor together with protein 3C, but yielded variable protection and antibodies (Ward et al., 1997 ; Chinsangaram et al., 1998
). In this study, we report experiments that extend this approach and suggest that DNA vaccination based on expression and processing of FMDV capsid precursors may be an alternative to the conventional inactivated whole-virus vaccine (Belsham, 1993
; Roosien et al., 1990
; Lewis et al., 1991
; Saiz et al., 1994
; Sanz-Parra et al., 1999
; Mayr et al., 1999
). The plasmid constructed (pcDNA3.1/P12A3C3D) contained all of the virus sequences required for correct production and processing of the FMDV serotype O1 capsid proteins. Protein 3C was included in the construct to achieve cleavage of protein precursor P12A and 3D. In addition to the T cell and B cell epitopes present within the P12A precursor and 3C protease (Collen, 1994
; Rodriguez et al., 1994
), the 3D polymerase was included in the construct as an additional potent source of T cell epitopes (Foster-Cuevas, 1996
; Collen et al., 1997). Although we have only demonstrated appropriate synthesis and processing of the capsid (and 3D) polypeptide chains from the transfected constructs in this particular study, and have not formally demonstrated assembly of empty capsids in vivo, previous electron microscopy observations with a similar construct (Abrams et al., 1995
) did demonstrate formation of empty capsids. Thus, it seems probable that these were also produced in vivo in these studies as a result of the DNA vaccination. Consistent with this, animals vaccinated with pcDNA3.1/P12A3C3D alone induced antibody responses against the whole virus and against the 3D polymerase, and promising results were obtained in the protection experiments.
Although three doses of DNA were administered, vaccination of pigs with plasmid pcDNA3.1/P12A3C3D alone gave promising results, particularly considering that the animals were challenged with a high dose (105·5 TCID50) of FMDV O1 Lausanne. This virus isolate, despite being related to O1K in terms of antigenic properties and P1 nucleotide sequence, gives a more acute infection with clearer clinical symptoms than O1K (J. Salt, personal communication). Thus, in experiment A, with the different groups housed separately, 2/4 animals were totally protected against high doses of virus challenge and in experiment B, where all animals were housed together, 2/4 were partially protected. In spite of the small number of pigs, the result of the second experiment (experiment B) becomes even more significant as one of the control pigs vaccinated with conventional vaccine developed a small lesion in the snout, presumably due to the high prevailing level of virus challenge encountered by all the animals.
Despite some exceptions (Sanz-Parra et al., 1999 ), there is generally a correlation between T cell-dependent virus-neutralizing antibodies and protection (McCullough et al., 1992
; Francis et al., 1987
; Collen et al., 1989
) and, indeed, there was a correlation between protection and levels of neutralizing antibodies in our experiments. In contrast, however, levels of anti-virus antibodies detected by ELISA did not correlate consistently with neutralizing antibody levels and, consequently, with protection. Additionally, some animals with efficient anti-3D responses by ELISA were not protected.
Despite the fact that this limited number of experiments has not established a clear correlation between DTH and protective immunity, it is clear from the observed synthesis of antibodies and the necessary concomitant generation of memory B cells that the DNA vaccination has stimulated T cell help. All of the conventionally vaccinated animals gave positive DTH responses to the virus, which indicates a Th1-type response, but this does not necessarily preclude a Th2 response. On the other hand, only 1/4 of the animals vaccinated with the DNA construct tested positive for DTH to the virus and there was no difference between the two experimental designs (experiments A and B).
A significant result in this study was the stimulatory effect of co-administration of a plasmid encoding porcine GM-CSF. Statistical analysis of the results showed that there was a significant increase (P=0·008) in anti-virus antibody production in those animals that received P12A3C3D plus GM-CSF. The increase in anti-FMDV antibody was statistically significant after the second and third DNA inoculations. In summary, this study suggests that DNA vaccination is a feasible strategy for stimulating porcine T cell and B cell responses against FMDV; its efficiency may be improved by co-administration of GM-CSF, perhaps through enhancing antigen presentation. Further work may improve the efficiency of this approach, with a reduction in the number of vaccinations required being particularly desirable.
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Acknowledgments |
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Footnotes |
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c Present address: Gulbenkian Institute for Science, Rua da Quinta Grande 6, Apartado 14, P-2781 Oeiras Codex, Portugal.
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References |
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Received 31 October 2000;
accepted 30 March 2001.