The Global control of FMD - Tools, ideas and ideals Erice, Italy October 2008

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1 Appendix 13 INTRADERMAL VACCINATION WITH 1/10 DOSE AGAINST FMDV PROTECTS PIGS AS WELL AGAINST CLINICAL DISEASE AND SUBCLINICAL VIRUS SHEDDING AS INTRAMUSCOLAR VACCINATION WITH A FULL DOSE. P. Eblé *, K. Weerdmeester, F. van Hemert-Kluitenberg and A. Dekker Central Veterinary Institute of Wageningen UR (CVI), P.O. Box 65, 8200 AB Lelystad, The Netherlands ABSTRACT The aim of this study was to investigate whether intradermal (ID) vaccination against foot-andmouth disease (FMD) is suitable as an alternative for the usually used intramuscular (IM) route. We used vaccines containing a normal or 10-fold dose antigen and compared groups of pigs that were vaccinated ID with either 0.2ml or 4x0.2ml with groups of pigs that were vaccinated IM with a standard 2ml, 0.2ml or 4x0.2ml dose. As compared with the 2ml IM vaccinated pigs, the pigs vaccinated ID with 0.2ml of the same vaccine were equally protected against clinical disease and subclinical virus shedding. Moreover, although VN-titres at 28 days post vaccination (dpv) of the 0.2ml ID vaccinated group were lower as compared to the IM vaccinated 2ml group, logistic regression analysis of the virus neutralising antibody (VN) titres at 28 dpv, the number of virus shedding pigs and application method showed that the ID vaccinated pigs needed significantly lower VN-titres as compared to the IM vaccinated pigs in order to be protected against virus shedding. We conclude that the ID route might be used as an alternative for IM application of FMD vaccine. ID application might induce more efficient immunity against FMD and, moreover, because the dose required in the ID route is lower compared to the IM route, ID application may reduce the cost of FMD vaccination markedly. 1. INTRODUCTION Despite all effort to eradicate the disease, FMD is still present in large parts of the world. The disease is endemic in large parts of Africa, Asia and South America. In some endemic areas, vaccination is applied as tool to eradicate the disease, but costs of vaccination are high. After a successful vaccination program in Western Europe, the European Union adopted a non-prophylactic vaccination strategy in Since then, the control policy for outbreaks in the EU has been primarily based on stamping out, in combination with movement restrictions and hygienic measures. Emergency vaccination during an outbreak was usually not applied because of the adverse economic consequences of vaccination as compared to slaughter of infected and in-contact animals. Since the European FMD outbreak in 2001, in which huge numbers of animals were killed, the OIE and the EU (Anonymous, 2005; Anonymous, 2003) have amended their regulations, and the option to use emergency vaccination (as a vaccinate-to-live policy) during an outbreak of FMD, has become more favourable (Anonymous, 2005). All registered FMD emergency vaccines are based on inactivated virus particles in an adjuvant and usually are administered intramuscularly in doses of 2ml. If the dose used per animal could be reduced, the total number of animals that can be vaccinated from one formulated (emergency-) vaccine batch is higher which would be an advantage in an outbreak situation in the EU. Not only for emergency vaccination, but also for vaccination in endemic regions reduction of vaccine dose would have benefit because the costs per vaccine dose could then be reduced. Reduction of vaccine dose might be accomplished by using another application method such as intra-dermal (ID) vaccination. Studies with hepatitis B, rabies and influenza vaccines suggest that intradermal vaccination has potential greater immunogenicity, because the skin is populated with dendritic cells, which are efficient and potent antigen-presenting cells for induction of protective immunity, and can result in dose-sparing (Glenn and Kenney, 2006). Moreover, intradermal vaccination has also other advantages compared to intramuscular vaccination. Intradermal application of a vaccine is less painful than IM application, ID application reduces lesions of edible tissue and, when a needle-less device is used, iatrogenic transfer of blood related agents cannot take place. In the present study we used a needle-less device for intradermal vaccination of pigs against FMD. This 87

2 device is already applied e.g. for vaccination against Aujezsky's disease (Visser et al., 1994) and PRRS (Martelli et al., 2007). In the presented study, we compared the efficacy of ID vaccination of pigs against FMD with that of the usually used intramuscular route. 2. MATERIALS AND METHODS 2.1 Animals and experimental design Three animal experiments were performed using conventionally reared 6-week old piglets. In the first experiment, 12 piglets were randomly allocated to 4 groups of 3 pigs each. Pigs in group 1 were not vaccinated, pigs in group 2 were vaccinated intramuscularly (IM) at 28 days before inoculation (-28 dpi) with a single dose double-oil-in-water emulsion (DOE) vaccine that contained 3µg of O Taiwan (O TAW 3/97) 146S antigen per 2 ml dose (vaccine A). Pigs in group 3 were vaccinated with the same vaccine, applied intradermally (ID) with 0.2ml (0.3µg, 1/10 dose) at 28 dpi using a needle-less device especially designed for this purpose (Intradermal Application Liquids (IDAL) Injector, Intervet International B.V., Boxmeer, The Netherlands). Pigs in group 4 were also vaccinated ID with 0.2ml vaccine, but with a vaccine that contained 10 times more antigen than usual (vaccine B) and thus received 3µg antigen, analogous to the IM vaccinated group. All vaccinations were given in the neck of the left-hand side of the pig. Before challenge, the pigs were moved to the high-containment unit and the groups were housed in separate pens. After an acclimatization period, challenge of the pigs was performed at 28 days post vaccination by intradermal inoculation in the bulb of the heel of the left hind-foot with 0.1 ml of FMD virus type O TAW 3/97 containing 10 5 TCID 50 /ml. In the second experiment, 25 pigs were randomly allocated to 5 groups of 5 pigs each. Pigs in group 1 were not vaccinated, pigs in group 2 were vaccinated IM with 2 ml of DOE vaccine that contained 3µg of FMDV O Taiwan 146S antigen per 2ml dose (vaccine C), pigs in group 3 were vaccinated IM with 2 ml of DOE vaccine that contained 30 µg of FMDV O Taiwan 146S antigen per 2ml dose (vaccine D), pigs in group 4 were vaccinated ID with 0.2 ml of vaccine C and thus received 0.3µg 146S antigen and pigs in group 5 were vaccinated ID with 0.2 ml of vaccine D and thus received 3µg 146S antigen. Housing, inoculation etc. were identical as in the first experiment. The experiment was ended at 7dpi. In the third experiment, 48 pigs were randomly allocated to 6 groups of 8 pigs each. Pigs in group 1 received 0.2ml PBS ID (non-vaccinated group), pigs in group 2 were vaccinated IM with 2 ml of DOE vaccine that contained 3 µg of O Taiwan 146S antigen per 2ml dose (vaccine E); pigs in group 3 were vaccinated IM with 0.2 ml of vaccine E and thus received 0.3µg 146S antigen; pigs in group 4 were vaccinated IM with 4x0.2 ml of vaccine E and thus received 1.2µg 146S antigen; pigs in group 5 were vaccinated ID with 0.2 ml of vaccine E and thus received 0.3µg 146S antigen and pigs in group 6 were vaccinated ID with 4x0.2 ml of the vaccine and thus received 1.2µg 146S antigen. The pigs that were vaccinated four times were vaccinated four times in the neck of the left-hand side of the pig. Of each group, 3 pigs were removed for other research purposes at 1, 3 and 7 dpv respectively. At 28 dpv, the remaining 5 pigs of each group were challenged with FMDV. Housing, inoculation etc. were identical as in the first experiment. The experiment was ended at 14dpi. 2.2 Data collection and sample preparation After vaccination, the pigs were inspected daily (exp. 1, 2 and 3) and the location at which the vaccination was given was examined every day for local reactions (exp. 3 only). Serum samples were collected twice a week. After challenge, clinical signs (rectal temperature and vesicle score for feet, nose and mouth) of the pigs were recorded daily. OPF was collected daily after challenge from 0-7 dpi (exp. 1 and 2) or 0-14 dpi (exp. 3) using cotton mouth swabs. In the laboratory, the swabs were incubated for 30 minutes in 4 ml EMEM and then centrifuged. Half of each sample was stored at 20 C until ELISAs were performed. To the other part of each sample 5% FBS and 10% antibiotics was added and stored at -70 C for virus isolation and RT-PCR. Serum samples were centrifuged and serum was stored at 20 C. At the end of the experiment 3, examination of the location of vaccination was performed. 2.3 Laboratory tests Challenge virus and OPF samples were assayed for the presence of virus by plaque titration on monolayers of secondary pig-kidney cells (De Leeuw et al., 1979). Virus titres were expressed as 10 log plaque forming units (pfu) per ml. 88

3 Neutralising antibody titres (VN-titres) against FMDV O Taiwan in serum samples were measured using the neutralisation assay as described previously (De Leeuw et al., 1979). End-point titres were calculated as the reciprocal of the final serum dilution that neutralised 100 TCID 50 of FMDV in 50% of the wells. The titres after infection where compared with the titres at the day of challenge. Antibodies against non-structural proteins of FMDV in serum samples were determined using a commercially available ELISA (Ceditest FMDV-NS) used according to the instructions of the manufacturer. 2.4 Statistical methods The results of the experimental groups with the same treatment in experiments 1, 2 and 3 were pooled. Differences in number of pigs with generalized FMD and virus excretion between the nonvaccinated and all the vaccinated groups, the IM 3 µg vaccinated and the other vaccinated groups and between the ID 0.3 µg vaccinated and the other vaccinated groups were statistically analysed using the Fisher Exact test (StatXact -5). Differences in VN-titres at 28 days post vaccination between the non-vaccinated and all vaccinated groups, the IM 3 µg vaccinated and the other vaccinated groups and the ID 0.3 µg and the other vaccinated groups were statistically analysed using the non-parametric Kruskal Wallis test (StatXact -5). A non-parametric permutation test (StatXact -5) was used for pair-wise comparison between groups if the Kruskal-Wallis test gave a significant result. Using logistic regression (R for Windows, version 2.6.2) we examined whether or not application method (IM versus ID) had influence on the relation between VN-titre and protection against virus shedding. For this, of all vaccinated pigs, VN-titres at 28 dpv, application method (IM or ID) and virus shedding (+ or -) were included in the model. First, the relation between VN-titre and protection against virus shedding was modelled. Then, the models with and without inclusion of application method were compared using a likelihood ratio test. All significance levels were set at p< RESULTS 3.1 Clinical signs and virus shedding After vaccination, no systemic reactions were observed in any of the vaccinated pigs. In the IM vaccinated pigs no local reactions could be seen at the location of vaccination. In the ID vaccinated pigs, a swelling at the location of vaccination could be observed with a diameter that differed from approximately 2-5cm. After challenge, all pigs of the non-vaccinated groups showed signs of generalized FMD (vesicles at another site than the inoculated foot), all but one showed fever and all shed virus from day 1-2 post inoculation. None of the pigs vaccinated IM with the standard 3 µg dose developed generalized FMD after challenge, although in some of the pigs vesicles at the inoculated foot were observed. Two pigs shed virus, although virus shedding was brief and titres were low as compared to the non-vaccinated groups. All pigs IM vaccinated with the vaccine that contained 10 times more antigen than usual were clinically protected against challenge and did not shed virus. In the group vaccinated IM with a 1/10 dose, also in none of the pigs generalized FMD was observed after challenge, although in one pig vesicles at the inoculated foot and fever was observed. Three pigs of this group shed virus after challenge. The pigs that were vaccinated IM four times with a 1/10 dose all were protected against challenge and no virus shedding was observed in this group. Of the intradermally vaccinated pigs with the 0.3 µg dose, in three pigs lesions on the inoculated foot were observed and one pig shed virus subclinically. In one pig, generalized FMD was observed although no virus was detected in OPF samples from this pig. Also in the group intradermally vaccinated with the vaccine that contained 10 times more antigen than usual (ID 3 µg vaccinated pigs) one pig showed generalized FMD without virus shedding. In this group, in five pigs lesions at the inoculated foot were observed of which one pig also showed fever. Three pigs shed virus subclinically from which two only briefly and at low titres. Of the ID vaccinated group that was vaccinated four times with the 0.3µg, all pigs were protected against clinical disease and no virus shedding was observed in this group (Table 1). Table 1; Results of all experiments 89

4 Clinical signs VI OPF VN-titer 28dpv NS-ELISA Non-vaccinated exp 1 3/3 3/3 <0.3 3/3 exp 2 5/5 5/5 <0.3 0/5 a exp 3 5/5 5/5 <0.3 0/5 b total 13/13 13/13 <0.3 IM vaccinated (3µg) exp 1 0/3 0/ /3 exp 2 0/5 (2LH c ) 2/ /5 a exp 3 0/5 0/ /5 total 0/13 2/ IM vaccinated (30µg) exp 2 0/5 0/ /5 a IM vaccinated (0.3µg) exp 3 0/5 (1LH) 3/ /5 IM vaccinated (4x0.3µg) exp 3 0/5 0/ /5 ID vaccinated (0.3µg) exp 1 0/3 0/ /3 exp 2 1/5 (2LH) 1/ /5 a exp 3 0/5 (1LH) 0/ /5 total 1/13 1/ ID vaccinated (3µg) exp 1 1/3 (2LH) 1/ /3 exp 2 0/5 (3LH) 2/ /5 a total 1/8 3/8 1.4 ID vaccinated (4x0.3µg) exp 3 0/5 0/ /5 a experiment ended at 7 dpi; b early euthanasia; c left hindfoot All vaccinated groups differed significantly from the non-vaccinated group (p<0.01) with regard to generalization of FMD. Between the IM 3 µg vaccinated groups and the other vaccinated groups, no significant differences could be detected (p>0.05). Also between the ID 0.3 µg vaccinated and the other vaccinated groups, no significant differences could be detected (p>0.05). Regarding the number of pigs that shed virus, all vaccinated groups differed significantly from the non-vaccinated group (p<0.01), except the IM 0.3µg vaccinated group (p=0.07). Between the IM 3 µg vaccinated groups and the other vaccinated groups, no significant differences could be detected (p>0.05). Between the ID 0.3 µg vaccinated and the other vaccinated groups, only the IM 0.3µg vaccinated group (p=0.04) differed significantly in the number of pigs shedding virus, but the other vaccinated groups did not (p>0.05). In the 3 µg IM vaccinated pigs, at the location of vaccination, intramuscularly, reactions to the vaccine of on average 5x4x3 cm that consisted of granulation tissue and necrosis were observed. Also the other IM vaccinated groups had similar reactions to the vaccine. In the ID vaccinated pigs, intradermally, reactions to the vaccine of on average 1-2 cm diameter consisting of granulation tissue and necrosis were observed. 3.2 Serological responses All vaccinated pigs developed neutralising antibodies against FMDV after vaccination. The mean virus neutralising antibody titre at 28 dpv of the vaccinated pigs is shown in Table 1. Increase of the antigen-payload (vaccines B and D) had a positive effect on the VN titres of the IM vaccinated groups, but resulted in no to almost the opposite effect for the ID vaccinated groups. Variation of the administered volume gave a dose-effect response in both the IM vaccinated groups (2ml>4x0.2ml>0.2ml) as the ID vaccinated groups (4x0.2ml>0.2ml). The titres of the IM vaccinated pigs were in general higher than those of the ID vaccinated pigs, except for the 4x0.3 µg ID vaccinated group that had a very good VN response (Figure 1). Statistically, at 28 days post vaccination the VN-titres of the pigs of all vaccinated groups differed significantly from the non-vaccinated group. The IM 3 µg vaccinated group differed significantly from the ID 3 µg (p=0.02) and the IM 0.3 µg vaccinated groups (p=0.01), but not from the other vaccinated groups. The ID 0.3 µg vaccinated group differed significantly from the IM 30 µg vaccinated group (p=0.04) and the ID 3 µg vaccinated group (p=0.02), but not from the other vaccinated groups. 90

5 At a group level, a clear boost in VN-titre post challenge could be detected in the non-vaccinated and the ID 3 µg group of exp. 1 (Figure 1). VN-titre at time of challenge had a significant relation with protection against virus shedding (p<0.001). Moreover, the application method plus interaction between application method and VNtitre fitted significantly better than the model without inclusion of application method (p=0.002). Based on this statistical model it can be concluded that the IM vaccinated pigs needed significantly higher VN-titres as compared to the ID vaccinated pigs in order to be protected against virus shedding. Positive reactions in the NS-ELISA were seen in all pigs of the non-vaccinated and the ID 3 µg vaccinated group of exp.1. Of exp. 2, all pigs tested negative, probably due to early euthanasia of these pigs. In exp. 3 only pig 9761 of the IM 0.3 µg tested positive in the NS-ELISA (Table 1). Figure 1; VNT responses VNT responses Exp. 1 VNT responses Exp log VN-titre non-vac 1 IM 3µg ID 0.3µg 0.5 ID 3µg days post vaccination 10 log VN-titre non-vac 2 IM 3µg IM 30µg 1.5 ID 0.3µg 1 ID 3µg days post vaccination VNT responses Exp log VN-titre non-vac IM 3 µg IM 0.3 µg IM 4x0.3 µg ID 0.3 µg ID 4x0.3 µg days post vaccination Group means of virus neutralising antibody responses 4. DISCUSSION We compared the efficacy of ID vaccination of pigs against FMD with the normally used intramuscular route at varying antigen doses. In the study presented in this paper, ID vaccination with 0.2ml vaccine was as effective as IM vaccination with the full 2ml dose. Effectiveness was established both as the number of pigs that were protected against generalization of FMD after challenge but also as number of pigs that were protected against (subclinical) virus shedding. ID inoculation has been investigated before as a means of vaccinating humans, laboratory animals and domestic farm animals (Hunsaker et al., 2001). For humans, effective ID vaccination is described for e.g. influenza (Auewarakul et al., 2007; Kenney et al., 2004; Belshe et al., 2004), rabies (Warrell et al., 1985; Warrell et al., 2008; Chutivongse et al., 1990) and hepatitis B (Karahocagil et al., 2006, Sangfelt et al., 2008) vaccination. Successful ID vaccination of pigs has been reported for Aujeszky's disease (Visser et al., 1994; Vanderpooten et al., 1997; Vannier and Cariolet, 1991; Mikulska-Skupien et al., 2004) and PRRS (Martelli et al., 2007). To our knowledge, our study is the first in which intra-dermal vaccination of pigs against FMD was assessed. ID 91

6 vaccination was effective even when a 1/10 vaccine dose was used. Using a lower vaccine dose will not only reduce the costs of vaccination but also could be essential to achieve a high vaccination coverage during an emergency vaccination in an FMD epidemic. Moreover, ID vaccination is less painful and reduces lesions in tissue that later might be used for consumption and, if delivered by a needle-less device, also avoids unintentional transfer of blood related agents. Therefore, ID vaccination might be a good alternative for the routinely used IM application of FMD vaccine. Even though we used a vaccine that is optimised for IM administration, ID vaccination with 1/10 vaccine dose resulted in a similar (slightly higher) VN-titre and was more successful for protection against virus shedding after challenge with FMDV as compared to IM vaccination with 1/10 vaccine dose. Therefore, ID application of FMD vaccine may result in a better immunological response as compared to IM vaccination with the same vaccine dose. Moreover, although VN-titres of the 0.2ml ID vaccinated group were lower as compared to the IM vaccinated 2ml group at time of challenge, protection against clinical disease and subclinical virus shedding of both groups were comparable and our results show that the ID vaccinated pigs needed significantly lower VN-titres as compared to the IM vaccinated pigs in order to be protected against virus shedding. In cattle, it is well established that protection against challenge at 3 weeks after vaccination is correlated with induced VN-antibody response caused by vaccination (Pay and Hingley, 1987). For pigs (Haas, 1999) this correlation is less clear but this is probably due to paucity of data. Recently, we demonstrated that in pigs reduction of virus shedding after challenge was correlated with vaccine induced VN-titres (Eble et al., 2007). However, the results of the present study show that after ID vaccination protection against challenge is accomplished at a lower VN-titre and thus suggest that ID application, as compared to IM application, might induce other immune mechanisms that contribute to protection. In the present study, we used several control groups in order to compare the vaccination methods with respect to total administered volume and/or antigen dose. For both IM and ID administration, for the vaccines that contained a normal dose antigen (vaccines A, C and E), a dose effect response was observed. Thus, the more total volume was administered, the better the neutralising antibody response. The vaccines with a 10-fold antigen payload induced a higher neutralising antibody response than the 'normal' vaccine when administered IM. Similar findings have been reported for cattle (Cox et al., 2006), sheep (Barnett et al., 2004) and pigs (Eble et al., 2007). However, if the vaccines that contained a 10-fold dose antigen were administered ID the results were relatively poor. This was probably due to the properties of the device that we used, which was optimised for use with adjuvated vaccine of the manufacturer. The vaccines (B, D) which we prepared with a 10-fold antigen load became very viscous because of the high protein content it contained. The IDAL device had difficulty in coping with these vaccines and probably delivered less than 0.2ml per dose, which is probably the cause of the disappointing results of these vaccines delivered ID. Although the pigs that were ID vaccinated with 1/10 of the normally used vaccine dose were equally protected against clinical disease and subclinical virus shedding as the pigs that were vaccinated IM with a 2 ml dose, still some of them showed clinical signs of FMDV (albeit merely locally at the site of challenge) and (subclinical) virus shedding after challenge. Therefore, the vaccination regime that we used could be further optimised. An improvement was already found in the group that was vaccinated ID 4x with 0.2ml. In this group, all pigs were completely protected against clinical signs and virus shedding and the mean VN-titre post vaccination was remarkably higher as compared to the 1x 0.2ml ID vaccinated group. As described previously, the height of VN-titre is correlated with protection against infection (Eble et al., 2007). In the 4x vaccinated groups, all vaccinations were given at the left side of the neck. Maybe less vaccination spots but separated from each other at locations with different draining lymph nodes would be as effective, as has been described for rabies vaccination (World Health Organisation, 2007). We conclude that ID vaccination with FMD vaccine seems to work very well in pigs. However, before ID vaccination against FMD can be used in field circumstances, more research should be carried out with respect to optimization of the used device, vaccine composition, administration strategy and efficacy in other species. 5. ACKNOWLEDGMENTS The authors wish to thank the laboratory assistants and staff of the animal isolation units that participated in the described research for their assistance. We thank Intervet International BV, Boxmeer, and The Netherlands for providing the IDAL device. This work was supported financially by the Netherlands Ministry of Agriculture, Nature and Food Quality. 6. REFERENCES 92

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