Appendix 60

Appendix 60

Significantly enhanced immune responses induced by a FMD DNA vaccine in swine using a protein antigen boost Yanmin Li1*, Catrina Stirling2, Haru Takamatsu2 and Paul Barnett1 1FMD vaccine group, 2Porcine Immunology Group, Pirbright Laboratory, Institute for Animal Health, Ash Road, Woking, Surrey, GU24 0NF, UK.

Abstract:

Introduction: It has previously been shown that a FMD DNA vaccine containing the “empty capsid” cassette-structural protein precursor P1 and the non-structural proteins 2A, 3C and 3D (pCDNA3.1/P1-2A3C3D, P1) 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/boost vaccination strategy. Increasing the priming dose of FMD DNA vaccine (600 µg) and GMCSF (400 µg) and combining with a protein boost induced an average anti-FMDV antibody titre which was up to 30 times higher than that following conventional vaccination. Materials and Methods: Groups of pigs were immunised with P1 and poGM-CSF plasmids via the intramuscular/ intradermal route (i.m./i.d) once, twice or three times which was followed 3 weeks later by a protein boost of inactivated FMDV antigen and FMDV 3D protein via the i.m. or i.d. route. Results: FMDV specific immune responses were significantly increased following the protein antigen boost of the P1 DNA vaccinated pigs. Conclusion: FMDV P1 DNA vaccination followed by an inactivated FMDV antigen/3D boost 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. 1993; Somasundaram et al. 1999; Lodmell et al. 1998). 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., 1993; Wolff et al., 1992; Klinman et al., 2004). Additionally, DNA vaccines are non-infectious, easy to prepare, inexpensive, and are stable at room temperature reducing cold chain requirements (Babiuk et al., 2000; Gurunathan et al., 2000; Cichutek 2000). 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, VP3 and VP1 were produced when the P1-2A precursor was cleaved by the viral protease 3C. 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, 1979; Rombaut et al., 1991; Abrams et al., 1995). 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., 1975; Grubman et al., 1985; Francis et al., 1985). Taking this observation together with the finding that the non-structural protein 3D stimulates a strong humoral and cellular immune response in the host (Foster et al., 1998), a P1 FMDV DNA vaccine was constructed containing an “empty capsid” gene cassette-P1-2A, 3C and 3D. Partial protection against homologous O1 Lausanne virus challenge was induced in pigs after three immunisations of this P1 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., 2001). Furthermore, it has been found that increasing the amount of P1 plasmids and poGM-CSF DNA plasmids from 300 µg and 200 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/boost strategy, and simplifying the DNA vaccination protocol by reducing the injection intervals without decreasing the specific immune responses in vaccinated animals.

Materials and Methods: Animal experiment

12 large white cross-bred Landrace pigs, weighing 20-25 Kg, were housed as four groups of three. Each animal received 600µg of P1 plasmids and 400µg of adjuvant plasmid poGMCSF dissolved in sterile saline via two 1 ml shots in each hind leg muscle followed by administration of the remaining 0.5 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 2, 1 and 3 were vaccinated with DNA plasmids once, twice or three times at 3 weeks interval, respectively, and this was followed by a protein antigen boost with 7.5 µg of O1 Lausanne inactivated antigen and 20 µg of FMDV 3D protein via the i.d. route. The group 4 pigs received two DNA immunisations 3 weeks apart followed by a protein antigen boost, 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 3) or 21 days (groups 1, 2 and 4) post protein antigen boost. Samples prior to vaccinations were also collected for background level assessment.

1. pcDNA3.1/P1-2A3C3D (600µgms) + GMCSF (400 µgms) 2 times + i.d. protein boost 2. pcDNA3.1/P1-2A3C3D (600µgms) + GMCSF (400 µgms) once + i.d. protein boost 3. pcDNA3.1/P1-2A3C3D (600µgms) + GMCSF (400 µgms) 3 times + i.d. protein boost 4. pcDNA3.1/P1-2A3C3D (600µgms) + GMCSF (400 µgms) 2 times + i.m. protein boost

Serum samples from single conventional O1 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 O1Kaufbeuren (O1K) virus. The antibody in serum samples from P1 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 O1 Kaufbeuren virus (Golding et al., 1976). The neutralising antibody titres were calculated as the log10 of the reciprocal antibody dilution required for 50% neutralisation of 100TCID50 virus.

Delayed type hypersensitivity (DTH) test

To investigate the existence of any T cell mediated immunity following P1 DNA vaccination, all four groups pigs were administered with O1 Lausanne inactivated antigen and/or recombinant protein FMDV 3D, 21 days following the last DNA vaccination. Each of P1 vaccinated pigs received either 7.5 µg/0.1ml unpurified antigen or 20 µg/0.1ml 3D 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, 2 days later. A measurement greater than a 2mm 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 boost which were then maintained until the end of the experiment in all animals representing groups 1, 3 and 4 (Figure 1a, c and d). The highest anti-FMDV antibody titre (animal VB57 in group 1) was 204800 which is about 64 times higher than that observed in single conventional vaccinated pigs (Figure 3a). Antibody responses to FMDV were induced following protein antigen boost in all group 2 animals lasted for 5 weeks (Figure 1b). The highest antibody titre from the group 2 pigs was the same as that observed in group 3 pigs before the protein antigen boost (Figure 1c) or the single conventional vaccinated pigs (Figure 3a), but was about 64 times lower than that observed in group 1 pigs. There was no significant difference in the antibody responses to FMDV following the protein antigen boost, either i.d. or i.m. among groups 1, 3 and 4 (Table 1), although the antibody responses induced in three times P1 DNA vaccinated pigs (group 3) were greater than those in pigs vaccinated twice with the P1 DNA construct (groups 1 and 4) prior to the protein antigen boost

Neutralising antibody responses were demonstrated as early as 21 days post primary DNA vaccination in one animal from each of groups 2 and 4. All animals in groups 1, 3 and 4 produced neutralizing antibodies after the second DNA vaccination, and their ability to neutralize FMDV O1 Kaufbeuren virus was significantly enhanced after the protein antigen boost (Figure 2a, c and d). The highest

neutralizing antibody titre was recorded in group3 and was about one log10 higher than that observed in any of the single conventional vaccinated pigs (Figure 3b). The neutralising antibodies induced in group 2 pigs (single vaccination) were also elevated after the protein antigen boost, however the highest titre was more than one log10 lower than that obtained in groups 1, 3 and 4 pigs. There was no significant difference in the neutralizing antibody responses among groups 1, 3 and 4 after either i.d. or i.m protein antigen boost.(Table 1), although group1 gave the best neutralizing antibody responses prior to the protein antigen boost.

Delayed type hypersensitivity (DTH) test

When a protein antigen boost was administered via the i.d. route, as a DTH test, the skin reaction was recorded. The results are summarised in Table 2. Two animals from each of groups 1 and 2 showed clear DTH reactions to FMDV antigen. One animal from each of groups 1, 2 and 3 gave a weak response to FMDV antigen. Two animals from group 2 and one animal from group 3 showed DTH responses to FMDV 3D protein. In general, animals from group 2 displayed stronger DTH responses than those from group 3.

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., 1997; Huang et al., 1999; Benvenisti et al., 2001; Wong et al., 2000; 2002). 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., 1997; Huang et al., 1999). The enhancement of immune responses using various DNA based prime boost strategies has also been documented (Hanke et al., 1998; Gonzalo et al., 2002; Robinson, 2003; Moore and Hill, 2004) and demonstrated to be effective for FMDV in mice when using a plasmid encoding VP1 followed by a VP1 peptide (Shieh et al., 2001). To improve the efficacy of a FMD DNA vaccine previously constructed (Cedillo-Barron et al., 2001), we have examined several vaccination parameters including incorporation of protein antigen boost strategies to induce stronger protective immunity in pigs.

The results in this study show that antibody responses to FMDV were generated after the secondary P1 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 boost in all vaccinated animals (Figure 1 and 2). There was no difference in either total antibody or neutralizing antibody responses after protein boost following two or three DNA vaccinations. The route of protein antigen administration i.d. or i.m. as a boost showed no significant difference in either specific antibody responses or neutralizing antibody responses. The average total FMDV antibody titre after the i.m. antigen boost in the twice P1 DNA vaccinated pigs was about 14 times higher and the neutralizing antibody titre was about one log10 higher than those observed in the single conventional vaccinated pigs (Table 1). This result is important in considering the practical application of this scheme in the field as a single i.m. boost is far more practical to undertake. Results from animals given a single P1 DNA vaccination followed by a protein antigen boost 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 boost regime combining a DNA prime and protein boost 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 3 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/boost regime of 1 or 2 vaccinations with DNA followed by a protein boost is a much more effective regime for optimizing both humoral and cellular responses.

Overall, the best and simplest vaccination procedure observed was 2 immunizations at 3 week intervals with 600µg P1-2A3C3D plasmid combined with 400µg of the GM-CSF plasmid ‘adjuvant’ via i.m. / i.d delivery each time followed 3 weeks later by an i.m. boost of 20 µg recombinant 3D and 7.5 µg of O1 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 boost regime for FMD DNA vaccination in at least one natural target host, pigs.

Such encouraging results suggest that a prime boost strategy is worthy of further investigation and a good candidate approach in the development and application of new generation FMD vaccines.

Conclusions:

• FMDV DNA (P1) vaccination followed by an inactivated FMDV antigen and protein 3D boost may be a more effective vaccination strategy in swine. • Specific immune responses to FMDV were significantly improved in pigs receiving two P1 DNA vaccinations and protein antigen boost than a single DNA vaccination followed by protein antigen boost.

Recommendations:

• Further assessment on DNA vaccination strategies and regimes that incorporate prime/boost regimes that explore the potential for further improvement and/or refinement.

Acknowledgements:

This work has been financially supported by EU (QLRT-2001-01304).

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a)

b)

c)

d)

Antibody titre before DTH

3300 2900 2500 2100 1700 1300 900 500 100

Group 1

VB56 VB57 VB59

Animal reference

203000 183000 163000 143000 123000 103000 83000 63000 43000 23000 3000

Antibody titre after DTH

7II 15II 21II 3III 7III 15III 21III

Antibody titre after boost

3300 2900 2500 2100 1700 1300 900 500 100

Antibody titre before DTH

3300 2900 2500 2100 1700 1300 900 500 100

Antibody titre before Ag boost

3300 2900 2500 2100 1700 1300 900 500 100

Group 2 after boost

7II 15II 21II 28II 36II days post vaccination

Group 3

UX37 UX38 UX39

Animal reference

203000 183000 163000 143000 123000 103000 83000 63000 43000

Antibody titre after

23000 3000

DTH

Group 4

VB65 VB66 VB67

Animal reference

203000 43000 63000 83000 103000 123000 143000 163000 183000Antibody titre after 23000 3000

Ag boost

VB58 VB60 VB61

7II 14II 21II 3III 7III 14III 21III 28III

7II 15II 21II 3III 7III 15III 21III

Figure 1. FMDV specific antibody titres in P1 vaccinated pigs. a, b, c and d represent results from groups 1, 2, 3 and 4, respectively. Columns represent antibody titres before the tertiary P1 vaccination in figure 1c or DTH/ antigen boost in figure 1a and d using the left hand Y axis scale, while lines represent antibody titres obtained after tertiary P1 vaccination or DTH/ antigen boost accordingly using the right hand Y axis scale. II: days post the secondary vaccination in groups 1, 3 and 4, but DTH in group 2; III: days post the tertiary vaccination in group 3, or the DTH/antigen boost in groups 1 and 4.

a)

VNT Titre

3.5 3 2.5 2 1.5 1 0.5 0

Group 1

0 21 7II 15II 21II 7III 15III 21III

days post vaccination

VB56 VB57 VB59

b)

c)

VNT Titre

3.5 3 2.5 2 1.5 1 0.5 0

Group 2

0 21 7II 15II 21II 28II 36II

days post vaccination

VNT titre

3.5 3 2.5 2 1.5 1 0.5 0

Group 3

0 21 7II 14II 21II 7III 14III 21III 28III

days post vaccination

VB58 VB60 VB61

UX37 UX38 UX39

d)

VNT Titre

3.5 3 2.5 2 1.5 1 0.5 0

Group 4

0 21 7II 15II 21II 7III 15III 21III

days post vaccination

VB65 VB66 VB67

Figure 2. Neutralising antibody titres in P1 vaccinated pigs. a, b, c and d represent results from groups 1, 2, 3 and 4, respectively. II: days post the secondary vaccination in groups 1, 3 and 4, but DTH in group 2; III: days post the tertiary vaccination in group 3, or the DTH/antigen boost in groups 1 and 4.

a)

b)

Antibody titre

3300 2900 2500 2100 1700 1300 900 500 100

O1 Lausanne vaccinated pigs

4 7 9&14 28 43 56 71

days post vaccination

UC81 UC82 UC74 UC77

2.5

2.0

1.5

1.0

0.5

0.0

O1 Lausanne vaccinated pigs

0 7 9&14 28 43 56 71

days post vaccination

UC77 UC81 UC82

Figure 3. Immune responses induced in O1 Lausanne single vaccinated pigs. a: FMDV specific antibody titres. b: neutralising O1 Kaufbeuren antibody titres.

Table 1. Comparation of average antibody titres among four groups of P1 vaccinated pigs

Groups of FMDV antibody titres Neutralising antibody titres P1-2A3C3D vaccinated P1 vaccination (times) FMDV protein boost 0 days post protein boost 7 days post protein boost 0 days post protein boost 7 days post protein boost 1 Twice i.d. 367 72533 1.05 2.71 2 Once i.d. - 1367 0.5 1.45 3 Three i.d. 2400 42667 0.55 3.05 4 Twice i.m. 333 34133 0.85 2.8 O1 lausanne vaccinated once 2400 1.9

Table 2. DTH responses in P1 vaccinated pigs

Groups Animals FMDV 3D FMDV antigen PBS

1 VB56 2 30 0 VB57 0 40 0

2

3 VB59 2 5 0 VB58 13 20 0 VB60 0 5 0 VB61 13 20 0 UX37 10 5 0 UX38 0 0 0 UX39 5 0 0