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1 Appendix 60 Significantly enhanced immune responses induced by a FMD DNA vaccine in swine using a protein antigen Yanmin Li *, Catrina Stirling, Haru Takamatsu and Paul Barnett FMD vaccine group, Porcine Immunology Group, Pirbright Laboratory, Institute for Animal Health, Ash Road, Woking, Surrey, GU4 0NF, UK. Abstract: Introduction: It has previously been shown that a FMD DNA vaccine containing the empty capsid cassette-structural protein precursor P and the non-structural proteins A, C and D (pcdna./p-acd, P) combined with an adjuvant plasmid expressing porcine granulocyte macrophage colony stimulating factor (pogm-csf) induced neutralising antibodies to FMDV and conferred partial protection against live virus challenge in swine. Current studies are aimed at enhancing the immune responses from this DNA vaccine further in swine by incorporating a prime/ vaccination strategy. Increasing the priming dose of FMD DNA vaccine (600 µg) and GM- CSF (400 µg) and combining with a protein induced an average anti-fmdv antibody titre which was up to 0 times higher than that following conventional vaccination. Materials and Methods: Groups of pigs were immunised with P and pogm-csf plasmids via the intramuscular/ intradermal route (i.m./i.d) once, twice or three times which was followed weeks later by a protein of inactivated FMDV antigen and FMDV D protein via the i.m. or i.d. route. Results: FMDV specific immune responses were significantly increased following the protein antigen of the P DNA vaccinated pigs. Conclusion: FMDV P DNA vaccination followed by an inactivated FMDV antigen/d could be a more efficient vaccination strategy in this model. Introduction: Vaccination with a DNA plasmid by various routes has been shown to elicit protective immune responses to the encoded antigen in a variety of animal models (Ulmer et al. 99; Somasundaram et al. 999; Lodmell et al. 998). This novel approach is particularly attractive for several reasons: the antigen is endogenously synthesised and processed and therefore more closely mimics natural infection. This results in the antigen being presented via both MHC class I and class II pathways generating both humoral and cellular immune responses. The use of plasmid DNA as a vaccine can also trigger innate immunity in the host as an effect of the unmethylated CpG motifs in the bacterial plasmid backbone (Yankaucka et al., 99; Wolff et al., 99; Klinman et al., 004). Additionally, DNA vaccines are non-infectious, easy to prepare, inexpensive, and are stable at room temperature reducing cold chain requirements (Babiuk et al., 000; Gurunathan et al., 000; Cichutek 000). DNA vaccines have the potential to provide a more effective and cheaper vaccine for economically important domestic animals such as cattle and pigs and are particularly advantageous over the conventional FMD vaccine because they do not require high-security containment facilities for manufacture, and are easy to manipulate for incorporation of marker genes or covering against various serotypes and field isolates in an outbreak. The structural proteins of FMDV VP0, VP and VP were produced when the P-A precursor was cleaved by the viral protease C. One of each of these proteins can form into protomers and five protomers assemble into a pentamer. An icosahedral capsid particle is then assembled with twelve pentamers. When this capsid particle lacks the RNA genome, they are called empty capsids (Yafal and Palma, 979; Rombaut et al., 99; Abrams et al., 995). It was found that empty capsid particles are capable of inducing antibody responses at a similar level to that induced by the whole virus (Rowlands et al., 975; Grubman et al., 985; Francis et al., 985). Taking this observation together with the finding that the non-structural protein D stimulates a strong humoral and cellular immune response in the host (Foster et al., 998), a P FMDV DNA vaccine was constructed containing an empty capsid gene cassette-p-a, C and D. Partial protection against homologous O Lausanne virus challenge was induced in pigs after three immunisations of this P plasmid. The antibody responses induced by this FMD DNA vaccine was improved by co-administration of a plasmid encoding porcine granulocyte macrophage colony stimulating factor (GM-CSF) (Cedillo-Barron et al., 00). Furthermore, it has been found that increasing the amount of P plasmids and pogm-csf DNA plasmids from 00 µg and 00 µg each to 600 µg and 400 µg respectively improved the immune response to FMDV in vaccinated pigs in a recent study performed in our group (unpublished data). This study was aimed at optimising this vaccination protocol to enhance the antibody and cellular responses induced by FMDV DNA immunisation of pigs by employing the prime/ strategy, and simplifying the DNA vaccination protocol by reducing the injection intervals without decreasing the specific immune responses in vaccinated animals. 77

2 Materials and Methods: Animal experiment large white cross-bred Landrace pigs, weighing 0-5 Kg, were housed as four groups of three. Each animal received 600µg of P plasmids and 400µg of adjuvant plasmid pogmcsf dissolved in sterile saline via two ml shots in each hind leg muscle followed by administration of the remaining ml, which was equally split intradermally (i.d.) into the dorsal surface of either ear. The vaccination regime for different groups were as outlined below. Briefly, Pigs of groups, and were vaccinated with DNA plasmids once, twice or three times at weeks interval, respectively, and this was followed by a protein antigen with 7.5 µg of O Lausanne inactivated antigen and 0 µg of FMDV D protein via the i.d. route. The group 4 pigs received two DNA immunisations weeks apart followed by a protein antigen, as other groups but via the i.m. route. Serum samples were taken regularly on a weekly basis after each vaccination until the experiment was terminated at 7 days (group ) or days (groups, and 4) post protein antigen. Samples prior to vaccinations were also collected for background level assessment.. pcdna./p-acd (600µgms) + GMCSF (400 µgms) times + i.d. protein. pcdna./p-acd (600µgms) + GMCSF (400 µgms) once + i.d. protein. pcdna./p-acd (600µgms) + GMCSF (400 µgms) times + i.d. protein 4. pcdna./p-acd (600µgms) + GMCSF (400 µgms) times + i.m. protein Serum samples from single conventional O Lausanne vaccinated pigs (each pig receiving 6.5 µg of antigen per dose) using vaccine supplied from the International Vaccine Bank, Pirbright Laboratory, IAH, UK were also used for testing the FMDV specific antibody and neutralising antibody responses, and the results were compared to those obtained in the DNA vaccinated pigs. Detection of FMDV specific antibody responses The antibody responses to FMDV in serum were analysed by an indirect sandwich ELISA using inactivated OKaufbeuren (OK) virus. The antibody in serum samples from P DNA vaccinated pigs was detected using rabbit anti-porcine antibodies HRP conjugate (DAKO). Antibody titres were expressed as the reciprocal of the highest serum dilution with an OD value at least two times that of the serum samples at 0 day. Neutralising antibodies to FMDV in serum samples were detected by microneutralisation assay using porcine kidney RSB cells and FMDV O Kaufbeuren virus (Golding et al., 976). The neutralising antibody titres were calculated as the log 0 of the reciprocal antibody dilution required for 50% neutralisation of 00TCID 50 virus. Delayed type hypersensitivity (DTH) test To investigate the existence of any T cell mediated immunity following P DNA vaccination, all four groups pigs were administered with O Lausanne inactivated antigen and/or recombinant protein FMDV D, days following the last DNA vaccination. Each of P vaccinated pigs received either 7.5 µg/0.ml unpurified antigen or 0 µg/0.ml D protein (diluted in endotoxin free PBS) via the intradermal route on one side of the abdomen. PBS alone was used as a control. The pigs were monitored daily and the skin, measured as the induration (diameter of raised skin or inflammation) at the site of intradermal injection, days later. A measurement greater than a mm increase in skin thickness/swelling was considered as a positive response. Results FMDV antibody responses and neutralising antibody detection Antibodies to FMDV were demonstrated after the second DNA vaccination and increased significantly at 7 days post protein antigen which were then maintained until the end of the experiment in all animals representing groups, and 4 (Figure a, c and d). The highest anti-fmdv antibody titre (animal VB57 in group ) was which is about 64 times higher than that observed in single conventional vaccinated pigs (Figure a). Antibody responses to FMDV were induced following protein antigen in all group animals lasted for 5 weeks (Figure b). The highest antibody titre from the group pigs was the same as that observed in group pigs before the protein antigen (Figure c) or the single conventional vaccinated pigs (Figure a), but was about 64 times lower than that observed in group pigs. There was no significant difference in the antibody responses to FMDV following the protein antigen, either i.d. or i.m. among groups, and 4 (Table ), although the antibody responses induced in three times P DNA vaccinated pigs (group ) were greater than those in pigs vaccinated twice with the P DNA construct (groups and 4) prior to the protein antigen Neutralising antibody responses were demonstrated as early as days post primary DNA vaccination in one animal from each of groups and 4. All animals in groups, and 4 produced neutralizing antibodies after the second DNA vaccination, and their ability to neutralize FMDV O Kaufbeuren virus was significantly enhanced after the protein antigen (Figure a, c and d). The highest 78

3 neutralizing antibody titre was recorded in group and was about one log 0 higher than that observed in any of the single conventional vaccinated pigs (Figure b). The neutralising antibodies induced in group pigs (single vaccination) were also elevated after the protein antigen, however the highest titre was more than one log 0 lower than that obtained in groups, and 4 pigs. There was no significant difference in the neutralizing antibody responses among groups, and 4 after either i.d. or i.m protein antigen.(table ), although group gave the best neutralizing antibody responses prior to the protein antigen. Delayed type hypersensitivity (DTH) test When a protein antigen was administered via the i.d. route, as a DTH test, the skin reaction was recorded. The results are summarised in Table. Two animals from each of groups and showed clear DTH reactions to FMDV antigen. One animal from each of groups, and gave a weak response to FMDV antigen. Two animals from group and one animal from group showed DTH responses to FMDV D protein. In general, animals from group displayed stronger DTH responses than those from group. Discussion A number of FMD DNA vaccines have been developed and partial protection against virus challenge was induced in DNA vaccinated animals (Ward et al., 997; Huang et al., 999; Benvenisti et al., 00; Wong et al., 000; 00). However, the induced neutralising antibodies, which are a vital for vaccine efficacy, was slower and at a lower level than that from a conventional vaccine (Ward et al., 997; Huang et al., 999). The enhancement of immune responses using various DNA based prime strategies has also been documented (Hanke et al., 998; Gonzalo et al., 00; Robinson, 00; Moore and Hill, 004) and demonstrated to be effective for FMDV in mice when using a plasmid encoding VP followed by a VP peptide (Shieh et al., 00). To improve the efficacy of a FMD DNA vaccine previously constructed (Cedillo-Barron et al., 00), we have examined several vaccination parameters including incorporation of protein antigen strategies to induce stronger protective immunity in pigs. The results in this study show that antibody responses to FMDV were generated after the secondary P vaccination and increased if pigs received the tertiary vaccination. However, both FMDV specific antibody responses and neutralizing antibody responses were significantly enhanced following the i.d. or i.m. antigen in all vaccinated animals (Figure and ). There was no difference in either total antibody or neutralizing antibody responses after protein following two or three DNA vaccinations. The route of protein antigen administration i.d. or i.m. as a showed no significant difference in either specific antibody responses or neutralizing antibody responses. The average total FMDV antibody titre after the i.m. antigen in the twice P DNA vaccinated pigs was about 4 times higher and the neutralizing antibody titre was about one log 0 higher than those observed in the single conventional vaccinated pigs (Table ). This result is important in considering the practical application of this scheme in the field as a single i.m. is far more practical to undertake. Results from animals given a single P DNA vaccination followed by a protein antigen suggest that this relatively simple vaccination regime can induce similar levels of both FMDV specific and neutralising antibody in animals to those induced by a single dose of conventional vaccine. Although much further work is required to examine longevity of such a response, protection from challenge with live virus and the degree of sterile immunity conferred, these results support the theory that a prime regime combining a DNA prime and protein could potentially be an effective new approach to FMDV vaccination. Results from the DTH test suggest that the more times an animal has been vaccinated with DNA plasmid the lower the DTH response observed. Animals vaccinated only once with plasmid showed a marked response to the antigen, however those vaccinated times showed little or no reaction at all. This suggests that repeated DNA vaccination can cause desensitization to the antigen in this system. These data also suggest that repeated DNA vaccination to achieve high antibody titres may have an adverse effect on the cellular response supporting the theory that the prime/ regime of or vaccinations with DNA followed by a protein is a much more effective regime for optimizing both humoral and cellular responses. Overall, the best and simplest vaccination procedure observed was immunizations at week intervals with 600µg P-ACD plasmid combined with 400µg of the GM-CSF plasmid adjuvant via i.m. / i.d delivery each time followed weeks later by an i.m. of 0 µg recombinant D and 7.5 µg of O Lausanne vaccine antigen. An effective protective immune response encompassing both the cellular and humoral arms of the immune system combined with a practical immunization regime is vital in the development of any vaccine which could be used in the field. We have confirmed the efficacy of a prime regime for FMD DNA vaccination in at least one natural target host, pigs. 79

4 Such encouraging results suggest that a prime strategy is worthy of further investigation and a good candidate approach in the development and application of new generation FMD vaccines. Conclusions: FMDV DNA (P) vaccination followed by an inactivated FMDV antigen and protein D may be a more effective vaccination strategy in swine. Specific immune responses to FMDV were significantly improved in pigs receiving two P DNA vaccinations and protein antigen than a single DNA vaccination followed by protein antigen. Recommendations: Further assessment on DNA vaccination strategies and regimes that incorporate prime/ regimes that explore the potential for further improvement and/or refinement. Acknowledgements: This work has been financially supported by EU (QLRT ). References: Abrams, C. C., King, A. M. Q. & Belsham, G. J Assembly of foot-and mouth disease virus empty capsids synthesized by a vaccinia virus expression system. J. Gen. Virol., 76: Babiuk, L. A., van Drunen Littel-van den Hurk S., Loehr B. I. & Uwiera R Veterinary applications of DNA vaccines. Dev. Biol. (Basel), 04: 7-8. Benvenisti, L., Rogel, A., Kuznetzova, L., Bujanove,r S., Becker, Y. & Stram, Y. 00. Gene gun-mediate DNA vaccination against foot-and-mouth disease virus. Vaccine, 9: Cedillo-Barrόn, L., Foster-Cuevas, M., Belsham, G. J., Lefevre F. & Parkhouse R. M. E. 00. Induction of a protective response in swine vaccinated with DNA encoding foot-and-mouth disease virus empty capsid proteins and the D RNA polymerase. J. Gen. Virol., 8: Cichutek, K DNA vaccines: development, standardization and regulation. Intervirology, 4: -8. Collen, T., Baron, J., Childerstone, A., Corteyn, A., Doel, T. R., Flint, M., Garcia-Valcarcel, M., Parkhouse, R. M. E. & Ryan, M. D Hetrotypic recognition of recombinant FMDV proteins by bovine T-cells: the polymerase (PD pol ) as an immunodominant T-cell immunogen. Virus Res., 56: 5-. Foster, M., Cook, A., Cedillo, L. & parkhouse, R. M. E Serological and cellular immune responses to non-structural proteins in animals infected with FMDV. Vet. Quart., 0(Suppl.): S8- S0. Francis, M. J., Fry, C. M., Rowlands, D. J., Brown, F., Bittle, J. L., Houghten, R. A., & Lerner, R. A Immunological priming with synthetic peptides of foot-and-mouth disease virus. J. Gen. Virol., 66: Golding, S. M., Hedger, R. S. & Talbot P Radial immno-diffusion and serum neutralisation techniques for the assay of antibodies to swine vescular disease. Res. Vet. Sci., 0: Gonzalo, R. M., del Real, G., Rodriguez, J. R., Rodriguez, D., Heljasvaara, R., Lucas, P., Larraga, V. & Esteban, M. 00. A heterologous prime- regime using DNA and recombinant vaccinia virus expressing the Leishmania infantum P6/LACK antigen protects BALB/c mice from cutaneous leishmaniasis. Vaccine, 0: 6-. Grubman, M. J., Morgan, D. O., Kendal, J. & Baxt, B. 985, Capsid intermediates assembled in a foot-and-mouth disease virus genome RNA-programmed cell-free translation system and infected cells. J. Virol. 56: 0-6. Gurunathan, S., Klinman, D. M. & Seder, R. A DNA vaccines: immunology, application, and optimization. Annu. Rev. Immunol., 8: Hanke, T., Blanchard, T. J., Schneider, J., Hannan, C. M., Becker, M., Gilbert, S. C., Hill, A. V., Smith, G. L. & McMichael, A Enhancement of MHC class I-restricted peptide-specific T cell induction by a DNA prime/mva vaccination regime. Vaccine, 6:

5 Huang, H., Yang,,Z., Xu, Q., Sheng, Z., Xie, Y., Yan, W., You, Y., Sun, L. & Zheng, Z Recombinant fusion protein and DNA vaccines against foot and mouth disease virus infection in guinea pig and swine. Viral Immunol, : -8. Klinman, D.M., Currie, D., Gursel, I. & Verthelyi, D Use of CpG oligodeoxynucleotides as immune adjuvants. Immunol. Reviews, 99: 0-6. Lodmell, D. L., Ray, N. B. & Ewalt, L. C Gene gun particle-mediated vaccination with plasmid DNA confers protective immunity against rabies virus infection. Vaccine, 6: 5-8. Moore, A.C. & Hill, A.V Progress in DNA-based heterologous prime- immunization strategies for malaria. Immunol Rev., 99: 6-4. Robinson, H.L. 00. Prime vaccines power up in people. Nat. Med, 9: Rombaut, B., Foriers, A. & Boeye, A. 99. In vitro assembly of poliovirus 4S subunits: identification of the assembly promoting activity of infected cell extracts. Virology, 80(): Rowlands, D. J., Sangar, D. V. & Brown, F A comparative chemical and serological study of the full and empty particles of foot-and-mouth disease virus. J. Gen. Virol., 6: 7-8. Shieh, J. J., Liang, C. M., Chen, C. Y., Lee, F., Jong, M. H., Lai, S. S. & Liang, S. M. 00. Enhancement of the immunity to foot-and-mouth disease virus by DNA priming and protein ing immunization. Vaccine, 9: Somasundaram, C., Takamatsu, H., Andreoni, C., Audonnet, J. C., Fisher, L., Lefèvre, F. & Charley, B Enhanced protective response and immuno-adjuvant effects od porcine GM-CSF on DNA vaccination of pigs against Aujeszky s disease virus. Vet. Immunol. Immunopathol., 70: Ulmer, J. B., Donnelly, J. J., Parker, S. E., Rhodes, G. H., Felgner, P. L., Dwarki, V. J., Gromkowaski, S. H., Deck, R. R., De Witt, D. M., Friedman, A., Hawe, L. A., Leaner, K. R., Martinez, D., Perry, H. C., Shiver, J. W., Montgomery, D. C. & Liu, M. A. 99. Heterologous protection against influenza by injection of DNA encoding a viral protein. Science, 59: Ward, G., Rieder, E. & Mason, P. W Plasmid DNA encoding replicating foot-and-mouth disease virus genomes induces antiviral immune responses in swine. J. Virol., 7: Wolff, J. A., Ludtke, J.J., Acsadi, G., Williams, P. & Jani, A. 99. Long-term persistence of plasmid DNA and foreign gene expression in mouse muscle. Hum. Mol. Genetics, : Wong, H. T., Cheng, S. C., Chan, E. W., Sheng, Z. T., Yan, W. Y., Zheng, Z. X. & Xie Y Plasmids encoding foot-and-mouth disease virus VP epitopes elicited immune responses in mice and swine and protected swine against viral infection. Virology, 78: 7-5. Wong, H. T., Cheng, S. C., Sin, F. W., Chan, E. W., Sheng, Z. T. & Xie, Y. 00. A DNA vaccine against foot-and-mouth disease elicits an immune response in swine which is enhanced by coadministration with interleukin-. Vaccine, 0: Yafal, A. G. & Palma, E. L Morphogenesis of foot-and-mouth disease virus. I. Role of procapsids as virion precursors. J. Virol., 0: Yankaucka, M. A., Morrow, J. E., Parker, S. E., Abai, A., Rhodes, G.H., Dwarki, V.J. & Gromkowski, S. H. 99. Long-term anti-nucleoprotein cellular and humoral immunity is induced by intramuscular injection of plasmid DNA containing the NP gene. DNA Cell Biol., :

6 a) Group Antibody titre before DTH VB56 VB57 VB Antibody titre after DTH 7II 5II II III 7III 5III III Animal reference b) Group after 00 Antibody titre after II 5II II 8II 6II VB58 VB60 VB6 c) Group Antibody titre before DTH UX7 UX8 UX Antibody titre after DTH 7II 4II II III 7III 4III III 8III Animal reference d) Antibody titre before Ag Group 4 VB65 VB66 VB Antibody titre after Ag 7II 5II II III 7III 5III III Animal reference Figure. FMDV specific antibody titres in P vaccinated pigs. a, b, c and d represent results from groups,, and 4, respectively. Columns represent antibody titres before the tertiary P vaccination in figure c or DTH/ antigen in figure a and d using the left hand Y axis scale, while lines represent antibody titres obtained after tertiary P vaccination or DTH/ antigen accordingly using the right hand Y axis scale. II: days post the secondary vaccination in groups, and 4, but DTH in group ; III: days post the tertiary vaccination in group, or the DTH/antigen in groups and 4. 8

7 a) Group.5 VNT Titre II 5II II 7III 5III III VB56 VB57 VB59 b) Group VNT Titre II 5II II 8II 6II VB58 VB60 VB6 c) Group.5 VNT titre II 4II II 7III 4III III 8III UX7 UX8 UX9 d) Group 4.5 VNT Titre II 5II II 7III 5III III VB65 VB66 VB67 Figure. Neutralising antibody titres in P vaccinated pigs. a, b, c and d represent results from groups,, and 4, respectively. II: days post the secondary vaccination in groups, and 4, but DTH in group ; III: days post the tertiary vaccination in group, or the DTH/antigen in groups and 4. 8

8 a) Antibody titre O Lausanne vaccinated pigs 4 7 9& UC8 UC8 UC74 UC77 b) O Lausanne vaccinated pigs 0 7 9& UC77 UC8 UC8 Figure. Immune responses induced in O Lausanne single vaccinated pigs. a: FMDV specific antibody titres. b: neutralising O Kaufbeuren antibody titres. Table. Comparation of average antibody titres among four groups of P vaccinated pigs Groups of P-ACD vaccinated P vaccination (times) FMDV protein FMDV antibody titres 0 days post protein 7 days post protein Neutralising antibody titres 0 days post protein 7 days post protein Twice i.d Once i.d Three i.d Twice i.m O lausanne vaccinated once Table. DTH responses in P vaccinated pigs Groups Animals FMDV FMDV PBS D antigen VB VB VB VB VB VB6 0 0 UX UX UX