Vaccine 32 (2014) 5221–5227
Contents lists available at ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
Enhanced immune responses of foot-and-mouth disease vaccine using new oil/gel adjuvant mixtures in pigs and goats Min-Eun Park a,c , Seo-Yong Lee a,c , Rae-Hyung Kim a , Mi-Kyeong Ko a , Kwang-Nyeong Lee a , Su-Mi Kim a , Byoung-Kwan Kim b , Jong-Soo Lee c , Byounghan Kim a , Jong-Hyeon Park a,∗ a
Animal and Plant Quarantine Agency, 175 Anyang-ro, Manangu, Anyang city, Gyeonggido 430-757, Republic of Korea MJ Biologics, 1961 Premier Drive Suite 402, Mankato, MN 56001, USA c Veterinary College, Chungnam National University, Yuseonggu, Daejeon 305-764, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 1 April 2014 Received in revised form 9 June 2014 Accepted 9 July 2014 Available online 25 July 2014 Keywords: Foot-and-mouth disease Adjuvant Immune response Vaccine Protection
a b s t r a c t The immunity and protective capability produced by vaccines can vary remarkably according to the kinds of adjuvants being used. In the case of foot-and-mouth disease (FMD) vaccines in pigs, only oil-adjuvant vaccines have been used, and these tend to show lower immunity in pigs than in cattle. New adjuvants for these vaccines are therefore needed. We made different experimental FMD vaccines using new adjuvants (ISA 201, Carbigen, Emulsigen-D) and well-known adjuvants (ISA 206, aluminum hydroxide gel) and then conducted tests to compare the enhancement in pig immunity. More effective immune responses and protection against challenge were observed with the new adjuvants Emulsigen-D and ISA 201 compared to existing adjuvants. In the case of dairy goats, a mixture of Emulsigen-D and aluminum hydroxide gel produced rapid neutralizing antibody responses that were similar to results from tests conducted with pigs. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Foot-and-mouth disease (FMD) is an acute infectious disease that infects cloven-hoofed mammals, such as pigs, cattle, sheep, and goats. This is an important disease of livestock that reduces productivity by forming blisters in mouths and on hooves [1]. Largescale outbreaks of this disease occurred in pigs in Taiwan in 1997 [2], in cattle and pigs in the United Kingdom in 2001 [3], and in cattle and pigs in Korea and Japan in 2010–2011 [4–6]. Efforts directed at the eradication and prevention of FMD centering on stampingout policies are controversial, and the prevention and control of the disease using vaccines have become areas of extreme interest. The large-scale occurrences of FMD type O in Korea in 2010–2011 led to the stamping out of approximately 3.48 million ungulates [7]. Consequently, the Korean government vaccinated all susceptible domestic animals to prevent additional occurrences of FMD and is still implementing prevention policies using vaccination, although there has been no additional occurrence. The World Reference Laboratory for FMD (Pirbright, UK) has recommended vaccine virus strains that have the widest antigen
∗ Corresponding author. Tel.: +82 31 467 1719; fax: +82 31 463 4516. E-mail addresses: [email protected], [email protected] (J.-H. Park). http://dx.doi.org/10.1016/j.vaccine.2014.07.040 0264-410X/© 2014 Elsevier Ltd. All rights reserved.
spectrum, and these are currently referred to and used in various countries, including countries where FMD exists and in countries where FMD is likely to occur. However, a rapid response to viruses that swiftly mutate in areas around the region of occurrence is not easy, and quickly developing vaccines using recent field strains is essential for disease control in the relevant country and for susceptible surrounding countries. Traditionally, FMD vaccines have been developed and evaluated mainly based on cattle. The World Organization for Animal Health (OIE) has standardized experimental methods using vaccinated bovine serum for vaccine-matching tests, but experimental methods for goats and pigs have not been standardized [8]. The FMD that occurred in 2010–2011 in Korea infected both cattle and pigs, but it occurred more in pigs than in cattle so that 3.318 million heads of pigs were stamped out while 0.151 million heads of cattle were stamped out. Inactivated FMD vaccines are commonly produced as gel or oil adjuvants depending on the serotype. Gel vaccines are only used in cattle or small ruminants and are not suitable for pigs due to their short duration of immunity [9]. Oil-adjuvanted vaccines are known to significantly increase humoral immunity and have superior antibody formation. In this study we compared the effects of experimental vaccines using various kinds of adjuvants in order to select vaccines having
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M.-E. Park et al. / Vaccine 32 (2014) 5221–5227
optimum antigenicity, immunogenicity, and protection. We used the O/Andong/SKR/2010 virus that was isolated in Korea in 2010 as the vaccine strain. To develop vaccines optimized for pigs, pigs were inoculated with vaccines, and immunological response and protection were evaluated. We also tested the effect of vaccines on dairy goats as one of the susceptible ruminant animals of FMD. Currently, double-oil-emulsion-type oil adjuvants or vaccines containing ISA 206 are used in Korea. Immunity and protection experiments were conducted in pigs and dairy goats to compare the effects of Carbigen and Emulsigen-D in addition to ISA 201 [1,10], which was identified as being effective in cattle and pigs in recent experiments. In addition, aluminum hydroxide, which helps protein absorption and stabilization, was included to observe if the effects of the vaccines were enhanced. 2. Materials and methods 2.1. Virus purification and inactivation FMD virus O/Andong/SKR/2010 was used for antigen preparation in the BHK21 cell line. For virus infection, culture medium was replaced by serum-free Dulbecco’s Modified Eagle Medium (Cellgro, VA, USA), which was then inoculated with the viruses. After 1 h incubation at 37 ◦ C and 5% CO2 , extracellular virus was removed. Twenty-four hours after infection, the viruses were inactivated by 0.003 N of BEI for 24 h in a shaking incubator and concentrated with polyethylene glycol (PEG) 6000 (81260; Sigma Aldrich, WI, USA). After centrifugation, the resuspended virus was layered on 15–45% sucrose density gradients and centrifuged [7]. After ultracentrifugation, the bottom of the centrifuge tube was punctured, and 1-ml fractions were collected. A sample of each fraction was tested for detection of the virus particle by using the Lateral Flow Device (BioSignTM FMDV Ag; Princeton BioMeditech Corporation, NJ, USA) for the FMD virus (FMDV) protein. The fractions having the FMDV particles were selected and confirmed by transmission electron microscopy. 2.2. Preparation of experimental vaccines The concentrated antigen was diluted in Tris–NaCl buffer (pH 7.6) and added to each of the following adjuvants: Carbigen (CARBIGENTM ; MVP Technologies, NE, USA), Emulsigen-D (EMULSIGEN® -D; MVP Technologies, NE, USA), ISA 201 (MONTANIDETM ISA 201 VG; SEPPIC, France), ISA 206 (MONTANIDETM ISA 206 VG; SEPPIC, France), and Emulsigen-D with aluminum hydroxide gel (Rehydragel® HPA; General Chemical, NJ, USA). The ratio of adjuvant to total volume was 20:80 for Carbigen and Emulsigen-D, and was 50:50 for ISA 201 and ISA 206 (volume [v]/v). For the oil/gel adjuvant mixture, we added 10% aluminum hydroxide gel. The mixture was stirred at 300 rpm for 10 min at 30 ◦ C in a water incubator to form a water-in-oil-in-water blend. The stability of the vaccines was tested by the dropping method [11]. The amount of antigen was prediluted from the concentrate before mixing with the adjuvant to maintain the same amount of antigen per dose of vaccine. 2.3. Immunization and FMDV challenge in pigs and dairy goats For the first study, 22 female pigs (8 weeks old) were divided into five test groups (n = 4 each group; Carbigen, Emulsigen-D, ISA 201, ISA 206, Emulsigen-D with aluminum hydroxide [ED + AL]) and an unvaccinated control group (n = 2). All the pigs were inoculated with an experimental vaccine containing 10 g of antigen per dose (2 ml). The serum of the pigs was collected into serumseparating tubes 0, 7, 14, 21, and 28 days after vaccination, and
neutralizing antibodies were measured. At 28 day postvaccination, test pigs were challenged with 105.0 TCID50 /0.1 ml of FMDV O/Andong/SKR/2010 from blisters of infected pigs by intradermally injecting to the foot pad; animals were then observed for about 2 weeks postchallenge. A clinical score was determined by summing points as follows: (i) an elevated body temperature of 40 ◦ C (score of 1), >40.5 (score of 2), or >41 (score of 3); (ii) reduced appetite (1 point) or no food intake and food left over from the day before (2 points); (iii) lameness (1 point) or reluctance to stand (2 points); (iv) presence of heat and pain after palpation of the coronary band (1 point) or not standing on the affected foot (2 points); (v) vesicles on the feet, dependent on the number of feet affected and with a maximum of 4 points; and (vi) visible mouth lesions on the tongue (1 point), gums or lips (1 point), or snout (1 point), with a maximum of 3 points [12]. For the second study, three dairy goats were vaccinated with ED + AL, and two goats were used as control. Dairy goats were inoculated with 1 ml (half the dose for pigs) of a vaccine made using the same vaccination method as commercial vaccines. The blood was collected 0, 7, 14, 21, and 28 days later. At 28 day postvaccination, the goats were also intradermally challenged with 105.0 ID50 /0.1 ml into the dorsal tongue. Animals were treated in accordance with the ethical guidelines of the animal welfare committee of the Animal and Plant Quarantine Agency (QIA). 2.4. Virus neutralization test and non-structural protein ELISA The neutralizing antibody titers in the serum were measured using the virus neutralization test (VNT) as specified in the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals of the World Animal Health Organization. Serum samples were collected from the animals after vaccination and virus challenge. The sera were heat inactivated at 56 ◦ C for 30 min. Following 1 h incubation in serial-diluted sera and virus suspension, LF-BK cells were added to the plate and incubated for a period of 2–3 days. The neutralizing antibody titers were calculated as the log10 of the reciprocal antibody dilution to neutralize 100 TCID50 of virus [13]. PrioCHECK FMDV NSP (Prionics AG, Schlieren-Zurich, Switzerland), an ELISA kit for the detection of FMDV non-structural protein (NSP) antibodies in serum samples of pigs and goats, was employed to detect NSP antibodies. 2.5. Analysis of FMDV replication in animals Real-time RT-PCR was carried out on sera and swab samples of the experimental animals. Swab samples were gathered from the mouth and nose using cotton swabs. Total cellular RNA was extracted using the MagNa pure 96 system (Roche, Germany) according to the manufacturer’s protocol. Real-time RT-PCR was conducted using the one-step Primescript RT-PCR kit (TAKARA, Otsu, Japan) according to the manufacturer’s instructions. Primers targeting the FMDV 3D region were sense 5 -GGAACYGGGTTTTAYAAACCTGTRAT-3 and antisense 5 -CCTCTCCTTTGCACGCCGTGGGA-3 . The probe was 5 CCCADCGCAGGTAAAGYGATCTGTA-3 ; its 5 end was labeled with 6-FAM, and the 3 end was labeled with TAMRA. The CFX96 TouchTM Real-Time PCR Detection system (Bio-Rad, CA, USA) was used for virus quantification [14]. 2.6. Statistical analysis Student’s t-tests were conducted using GraphPad Instat® ver 3.05 (GraphPad Software, CA, USA) for the analysis of immunogenicity and protection capability.
M.-E. Park et al. / Vaccine 32 (2014) 5221–5227
3. Results 3.1. Immune responses and protection in pigs Low titers of neutralizing antibodies were detected in the group vaccinated with Carbigen until 28 days postvaccination. Although the highest titers of neutralizing antibodies were detected in the ISA 201-vaccinated group, the titers reached the maximum value 3 weeks after vaccination, indicating that antibody response was relatively slow. Neutralizing antibodies were formed fastest in the Emulsigen-D group. High levels of neutralizing antibodies were formed starting 2 weeks after vaccination in the group inoculated with a mixture of Emulsigen-D and ED + AL, and the level of antibody response produced in this group was similar to that produced by ISA 206, which is an adjuvant widely used in commercial FMD vaccines (Table 1). NSP ELISA and VNT were conducted using serum samples collected on day 10 postchallenge. As shown in Table 1, high neutralizing antibody titers were induced in the group vaccinated with Emulsigen-D and the group vaccinated with ED + AL. Positive antibodies against NSP were only detected in the nonvaccinated control group and the Carbigen-vaccinated group. Based on these results, we selected ED + AL as an adjuvant for the nextgeneration vaccines and conducted experiments to determine if this combination also showed excellent immune response and protection in dairy goats. Severe clinical signs appeared in the non-vaccinated group and Carbigen-vaccinated group, and high titers of viruses in the Carbigen-vaccinated groups were detected from serum and nasal swab samples, indicating low efficacy of vaccination (Fig. 1A). In the case of animals vaccinated with Emulsigen-D, some virus was secreted into nasal mucus, but no symptoms were apparent other than lesions at the site of virus challenge (Fig. 1B). The amount of virus shedding was lower and protection was better in animals where ED-AL was used in combination compared to animals receiving only Emulsigen-D-adjuvanted vaccine (Fig. 1C). In the case of pigs receiving ISA 201-adjuvanted vaccine, no clinical signs were apparent other than lesions at the site of virus challenge, and only very small quantities of virus over a short period were detected in the nasal swab samples (Fig. 1D). In the case of ISA 206, which is an adjuvant widely used in commercial vaccines, protection was not induced in one animal, which showed moderate clinical signs and viremia (Fig. 1E). Table 2 summarizes the virus secretion and lesions in the regions of inoculation after vaccination using five types of adjuvants followed by virus challenge. The ED + AL group showed relatively excellent protection for onset time of lesions at the inoculated site or clinical scoring. The quantities of excreted viruses in the ED + AL group were lower than in any other experimental group. 3.2. Immune response and protection in dairy goats Based on the results of the pig experiments, dairy goats were inoculated with ED + AL-adjuvanted vaccine and challenged 28 days after vaccination. The VNT showed that high titers of antibodies were detected in three vaccinated goats starting 7 days after vaccination, and the titers continuously increased until days 21–28 (Table 3). NSP ELISA and VNT were also conducted using serum samples collected on day 8 postchallenge with the virus. High levels of neutralizing antibodies were induced in goats vaccinated with ED + AL-adjuvanted vaccine, but NSP antibodies were not detected in the vaccinated group until 8 days after virus challenge. The dairy goats were challenged with the FMD virus on day 28 after vaccination, after which viremia and virus secretion were observed until day 8 postchallenge. High levels of viremia were observed until 7 days after challenge in the unvaccinated group. However, the virus was not detected in the serum samples from the
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vaccinated group, indicating protection against FMD (Fig. 2A and B). Remarkably, lower quantities of virus were detected in the vaccinated group, and the duration was shorter than the unvaccinated group (Table 4). No clinical signs were apparent in the goats.
4. Discussion and conclusions The control of FMD is dependent on the vaccination of susceptible animal species with inactivated whole virus vaccines [15]. Vaccination with good quality FMD vaccines helps in the prevention of livestock production losses and reduces the overall incidence of the disease [16]. Hence, efforts are focused on developing adjuvants that can promote protective immunity through induction of enhanced and more durable antibody responses [1]. Currently, the double-oil emulsion vaccines are preferred for FMD prevention as they can be used to protect all susceptible species, particularly during FMD outbreak situations. Also, oil-adjuvant vaccines induce higher and longer lasting immune responses and show less interference from maternal antibodies compared to aqueous vaccines [17]. The SEPPIC company (France) has developed a new adjuvant ISA 201 and claims that this adjuvant induces better immune responses (particularly cell-mediated immunity) [18]. For example, ISA 201 (PD50 = 15.59) showed better protection in pigs when compared to ISA 206 (PD50 = 10.05) [10]. Similar results can be observed in cattle; ISA 201 showed higher lymphoproliferative response and IgG2 response and lower quantities of virus shedding detected after FMDV challenge compared to ISA 206 [1]. ISA 201 in the present study also showed a similar enhancement of protection. Emulsigen-D is a unique oil-in-water emulsion and contains uniformly dispersed micron-size oil droplets, which ensure maximum emulsion stability and decreased viscosity. Micron-size oil droplets also increase the surface area available to antigens, reducing the quantity of oil required in the final vaccine. Emulsigen-D reduces the undesirable side effects associated with other oil-in-water or water-in-oil adjuvants while eliciting a rapid and strong immune response [19]. Emulsigen-D as an adjuvant produces increased immunogenicity because it incorporates dimethyl-dioctadecyl ammonium bromide (DDA), which is a Tcell immune stimulator in Emulsigen. Its efficacy as an adjuvant was proved in Toxoplasmia gondii and rabies [20,21]. According to Kaur et al., the DDA contained in Emulsigen-D induces the enhancement of immune responses by increasing the surface area of antigens in oil-in-water emulsions so that antigens spread slowly. Therefore, protection against Aujeszky’s disease virus is increased when infected animals have been vaccinated with Emulsigen plus DDA. In addition, aluminum compounds have been known to be the most frequently used adjuvant in veterinary vaccines [22]. These compounds have been found to induce memory cell responses and long-lasting protection when animals have been inoculated with vaccines, thereby enhancing immune reactions [23]. Among them, aluminum phosphate and aluminum hydroxide are the only adjuvants approved for routine use in humans because of their relatively low toxicity [24]. Carbigen is a carbomer-based adjuvant suspension containing a proprietary emulsified component. Its milky white appearance creates a smooth uniform mixture [25]. Carbigen is a stabilizing adjuvant that stabilizes epitope conformation and is mainly used for intranasal application [26]. In our study, the group inoculated with the mixture of adjuvant ED + AL showed the best immunogenicity and production of antibody response. The titer of virus detected in the serum and oral swabs after challenge was lowest in the group inoculated with ED + AL compared to other groups in which other adjuvants were used, indicating that ED + AL effectively suppressed viral replication
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Table 1 Immunity and protective response of the pigs after FMD vaccination with various adjuvants. Vaccinated groups by adjuvants with FMD antigen AD-P, 10 g
Pig no.
Virus neutralizing antibody titers (log)
Viremia
7dpv
14dpv
21dpv
28dpv
10dpcc
93 94 95 96
Contents lists available at ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
Enhanced immune responses of foot-and-mouth disease vaccine using new oil/gel adjuvant mixtures in pigs and goats Min-Eun Park a,c , Seo-Yong Lee a,c , Rae-Hyung Kim a , Mi-Kyeong Ko a , Kwang-Nyeong Lee a , Su-Mi Kim a , Byoung-Kwan Kim b , Jong-Soo Lee c , Byounghan Kim a , Jong-Hyeon Park a,∗ a
Animal and Plant Quarantine Agency, 175 Anyang-ro, Manangu, Anyang city, Gyeonggido 430-757, Republic of Korea MJ Biologics, 1961 Premier Drive Suite 402, Mankato, MN 56001, USA c Veterinary College, Chungnam National University, Yuseonggu, Daejeon 305-764, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 1 April 2014 Received in revised form 9 June 2014 Accepted 9 July 2014 Available online 25 July 2014 Keywords: Foot-and-mouth disease Adjuvant Immune response Vaccine Protection
a b s t r a c t The immunity and protective capability produced by vaccines can vary remarkably according to the kinds of adjuvants being used. In the case of foot-and-mouth disease (FMD) vaccines in pigs, only oil-adjuvant vaccines have been used, and these tend to show lower immunity in pigs than in cattle. New adjuvants for these vaccines are therefore needed. We made different experimental FMD vaccines using new adjuvants (ISA 201, Carbigen, Emulsigen-D) and well-known adjuvants (ISA 206, aluminum hydroxide gel) and then conducted tests to compare the enhancement in pig immunity. More effective immune responses and protection against challenge were observed with the new adjuvants Emulsigen-D and ISA 201 compared to existing adjuvants. In the case of dairy goats, a mixture of Emulsigen-D and aluminum hydroxide gel produced rapid neutralizing antibody responses that were similar to results from tests conducted with pigs. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Foot-and-mouth disease (FMD) is an acute infectious disease that infects cloven-hoofed mammals, such as pigs, cattle, sheep, and goats. This is an important disease of livestock that reduces productivity by forming blisters in mouths and on hooves [1]. Largescale outbreaks of this disease occurred in pigs in Taiwan in 1997 [2], in cattle and pigs in the United Kingdom in 2001 [3], and in cattle and pigs in Korea and Japan in 2010–2011 [4–6]. Efforts directed at the eradication and prevention of FMD centering on stampingout policies are controversial, and the prevention and control of the disease using vaccines have become areas of extreme interest. The large-scale occurrences of FMD type O in Korea in 2010–2011 led to the stamping out of approximately 3.48 million ungulates [7]. Consequently, the Korean government vaccinated all susceptible domestic animals to prevent additional occurrences of FMD and is still implementing prevention policies using vaccination, although there has been no additional occurrence. The World Reference Laboratory for FMD (Pirbright, UK) has recommended vaccine virus strains that have the widest antigen
∗ Corresponding author. Tel.: +82 31 467 1719; fax: +82 31 463 4516. E-mail addresses: [email protected], [email protected] (J.-H. Park). http://dx.doi.org/10.1016/j.vaccine.2014.07.040 0264-410X/© 2014 Elsevier Ltd. All rights reserved.
spectrum, and these are currently referred to and used in various countries, including countries where FMD exists and in countries where FMD is likely to occur. However, a rapid response to viruses that swiftly mutate in areas around the region of occurrence is not easy, and quickly developing vaccines using recent field strains is essential for disease control in the relevant country and for susceptible surrounding countries. Traditionally, FMD vaccines have been developed and evaluated mainly based on cattle. The World Organization for Animal Health (OIE) has standardized experimental methods using vaccinated bovine serum for vaccine-matching tests, but experimental methods for goats and pigs have not been standardized [8]. The FMD that occurred in 2010–2011 in Korea infected both cattle and pigs, but it occurred more in pigs than in cattle so that 3.318 million heads of pigs were stamped out while 0.151 million heads of cattle were stamped out. Inactivated FMD vaccines are commonly produced as gel or oil adjuvants depending on the serotype. Gel vaccines are only used in cattle or small ruminants and are not suitable for pigs due to their short duration of immunity [9]. Oil-adjuvanted vaccines are known to significantly increase humoral immunity and have superior antibody formation. In this study we compared the effects of experimental vaccines using various kinds of adjuvants in order to select vaccines having
5222
M.-E. Park et al. / Vaccine 32 (2014) 5221–5227
optimum antigenicity, immunogenicity, and protection. We used the O/Andong/SKR/2010 virus that was isolated in Korea in 2010 as the vaccine strain. To develop vaccines optimized for pigs, pigs were inoculated with vaccines, and immunological response and protection were evaluated. We also tested the effect of vaccines on dairy goats as one of the susceptible ruminant animals of FMD. Currently, double-oil-emulsion-type oil adjuvants or vaccines containing ISA 206 are used in Korea. Immunity and protection experiments were conducted in pigs and dairy goats to compare the effects of Carbigen and Emulsigen-D in addition to ISA 201 [1,10], which was identified as being effective in cattle and pigs in recent experiments. In addition, aluminum hydroxide, which helps protein absorption and stabilization, was included to observe if the effects of the vaccines were enhanced. 2. Materials and methods 2.1. Virus purification and inactivation FMD virus O/Andong/SKR/2010 was used for antigen preparation in the BHK21 cell line. For virus infection, culture medium was replaced by serum-free Dulbecco’s Modified Eagle Medium (Cellgro, VA, USA), which was then inoculated with the viruses. After 1 h incubation at 37 ◦ C and 5% CO2 , extracellular virus was removed. Twenty-four hours after infection, the viruses were inactivated by 0.003 N of BEI for 24 h in a shaking incubator and concentrated with polyethylene glycol (PEG) 6000 (81260; Sigma Aldrich, WI, USA). After centrifugation, the resuspended virus was layered on 15–45% sucrose density gradients and centrifuged [7]. After ultracentrifugation, the bottom of the centrifuge tube was punctured, and 1-ml fractions were collected. A sample of each fraction was tested for detection of the virus particle by using the Lateral Flow Device (BioSignTM FMDV Ag; Princeton BioMeditech Corporation, NJ, USA) for the FMD virus (FMDV) protein. The fractions having the FMDV particles were selected and confirmed by transmission electron microscopy. 2.2. Preparation of experimental vaccines The concentrated antigen was diluted in Tris–NaCl buffer (pH 7.6) and added to each of the following adjuvants: Carbigen (CARBIGENTM ; MVP Technologies, NE, USA), Emulsigen-D (EMULSIGEN® -D; MVP Technologies, NE, USA), ISA 201 (MONTANIDETM ISA 201 VG; SEPPIC, France), ISA 206 (MONTANIDETM ISA 206 VG; SEPPIC, France), and Emulsigen-D with aluminum hydroxide gel (Rehydragel® HPA; General Chemical, NJ, USA). The ratio of adjuvant to total volume was 20:80 for Carbigen and Emulsigen-D, and was 50:50 for ISA 201 and ISA 206 (volume [v]/v). For the oil/gel adjuvant mixture, we added 10% aluminum hydroxide gel. The mixture was stirred at 300 rpm for 10 min at 30 ◦ C in a water incubator to form a water-in-oil-in-water blend. The stability of the vaccines was tested by the dropping method [11]. The amount of antigen was prediluted from the concentrate before mixing with the adjuvant to maintain the same amount of antigen per dose of vaccine. 2.3. Immunization and FMDV challenge in pigs and dairy goats For the first study, 22 female pigs (8 weeks old) were divided into five test groups (n = 4 each group; Carbigen, Emulsigen-D, ISA 201, ISA 206, Emulsigen-D with aluminum hydroxide [ED + AL]) and an unvaccinated control group (n = 2). All the pigs were inoculated with an experimental vaccine containing 10 g of antigen per dose (2 ml). The serum of the pigs was collected into serumseparating tubes 0, 7, 14, 21, and 28 days after vaccination, and
neutralizing antibodies were measured. At 28 day postvaccination, test pigs were challenged with 105.0 TCID50 /0.1 ml of FMDV O/Andong/SKR/2010 from blisters of infected pigs by intradermally injecting to the foot pad; animals were then observed for about 2 weeks postchallenge. A clinical score was determined by summing points as follows: (i) an elevated body temperature of 40 ◦ C (score of 1), >40.5 (score of 2), or >41 (score of 3); (ii) reduced appetite (1 point) or no food intake and food left over from the day before (2 points); (iii) lameness (1 point) or reluctance to stand (2 points); (iv) presence of heat and pain after palpation of the coronary band (1 point) or not standing on the affected foot (2 points); (v) vesicles on the feet, dependent on the number of feet affected and with a maximum of 4 points; and (vi) visible mouth lesions on the tongue (1 point), gums or lips (1 point), or snout (1 point), with a maximum of 3 points [12]. For the second study, three dairy goats were vaccinated with ED + AL, and two goats were used as control. Dairy goats were inoculated with 1 ml (half the dose for pigs) of a vaccine made using the same vaccination method as commercial vaccines. The blood was collected 0, 7, 14, 21, and 28 days later. At 28 day postvaccination, the goats were also intradermally challenged with 105.0 ID50 /0.1 ml into the dorsal tongue. Animals were treated in accordance with the ethical guidelines of the animal welfare committee of the Animal and Plant Quarantine Agency (QIA). 2.4. Virus neutralization test and non-structural protein ELISA The neutralizing antibody titers in the serum were measured using the virus neutralization test (VNT) as specified in the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals of the World Animal Health Organization. Serum samples were collected from the animals after vaccination and virus challenge. The sera were heat inactivated at 56 ◦ C for 30 min. Following 1 h incubation in serial-diluted sera and virus suspension, LF-BK cells were added to the plate and incubated for a period of 2–3 days. The neutralizing antibody titers were calculated as the log10 of the reciprocal antibody dilution to neutralize 100 TCID50 of virus [13]. PrioCHECK FMDV NSP (Prionics AG, Schlieren-Zurich, Switzerland), an ELISA kit for the detection of FMDV non-structural protein (NSP) antibodies in serum samples of pigs and goats, was employed to detect NSP antibodies. 2.5. Analysis of FMDV replication in animals Real-time RT-PCR was carried out on sera and swab samples of the experimental animals. Swab samples were gathered from the mouth and nose using cotton swabs. Total cellular RNA was extracted using the MagNa pure 96 system (Roche, Germany) according to the manufacturer’s protocol. Real-time RT-PCR was conducted using the one-step Primescript RT-PCR kit (TAKARA, Otsu, Japan) according to the manufacturer’s instructions. Primers targeting the FMDV 3D region were sense 5 -GGAACYGGGTTTTAYAAACCTGTRAT-3 and antisense 5 -CCTCTCCTTTGCACGCCGTGGGA-3 . The probe was 5 CCCADCGCAGGTAAAGYGATCTGTA-3 ; its 5 end was labeled with 6-FAM, and the 3 end was labeled with TAMRA. The CFX96 TouchTM Real-Time PCR Detection system (Bio-Rad, CA, USA) was used for virus quantification [14]. 2.6. Statistical analysis Student’s t-tests were conducted using GraphPad Instat® ver 3.05 (GraphPad Software, CA, USA) for the analysis of immunogenicity and protection capability.
M.-E. Park et al. / Vaccine 32 (2014) 5221–5227
3. Results 3.1. Immune responses and protection in pigs Low titers of neutralizing antibodies were detected in the group vaccinated with Carbigen until 28 days postvaccination. Although the highest titers of neutralizing antibodies were detected in the ISA 201-vaccinated group, the titers reached the maximum value 3 weeks after vaccination, indicating that antibody response was relatively slow. Neutralizing antibodies were formed fastest in the Emulsigen-D group. High levels of neutralizing antibodies were formed starting 2 weeks after vaccination in the group inoculated with a mixture of Emulsigen-D and ED + AL, and the level of antibody response produced in this group was similar to that produced by ISA 206, which is an adjuvant widely used in commercial FMD vaccines (Table 1). NSP ELISA and VNT were conducted using serum samples collected on day 10 postchallenge. As shown in Table 1, high neutralizing antibody titers were induced in the group vaccinated with Emulsigen-D and the group vaccinated with ED + AL. Positive antibodies against NSP were only detected in the nonvaccinated control group and the Carbigen-vaccinated group. Based on these results, we selected ED + AL as an adjuvant for the nextgeneration vaccines and conducted experiments to determine if this combination also showed excellent immune response and protection in dairy goats. Severe clinical signs appeared in the non-vaccinated group and Carbigen-vaccinated group, and high titers of viruses in the Carbigen-vaccinated groups were detected from serum and nasal swab samples, indicating low efficacy of vaccination (Fig. 1A). In the case of animals vaccinated with Emulsigen-D, some virus was secreted into nasal mucus, but no symptoms were apparent other than lesions at the site of virus challenge (Fig. 1B). The amount of virus shedding was lower and protection was better in animals where ED-AL was used in combination compared to animals receiving only Emulsigen-D-adjuvanted vaccine (Fig. 1C). In the case of pigs receiving ISA 201-adjuvanted vaccine, no clinical signs were apparent other than lesions at the site of virus challenge, and only very small quantities of virus over a short period were detected in the nasal swab samples (Fig. 1D). In the case of ISA 206, which is an adjuvant widely used in commercial vaccines, protection was not induced in one animal, which showed moderate clinical signs and viremia (Fig. 1E). Table 2 summarizes the virus secretion and lesions in the regions of inoculation after vaccination using five types of adjuvants followed by virus challenge. The ED + AL group showed relatively excellent protection for onset time of lesions at the inoculated site or clinical scoring. The quantities of excreted viruses in the ED + AL group were lower than in any other experimental group. 3.2. Immune response and protection in dairy goats Based on the results of the pig experiments, dairy goats were inoculated with ED + AL-adjuvanted vaccine and challenged 28 days after vaccination. The VNT showed that high titers of antibodies were detected in three vaccinated goats starting 7 days after vaccination, and the titers continuously increased until days 21–28 (Table 3). NSP ELISA and VNT were also conducted using serum samples collected on day 8 postchallenge with the virus. High levels of neutralizing antibodies were induced in goats vaccinated with ED + AL-adjuvanted vaccine, but NSP antibodies were not detected in the vaccinated group until 8 days after virus challenge. The dairy goats were challenged with the FMD virus on day 28 after vaccination, after which viremia and virus secretion were observed until day 8 postchallenge. High levels of viremia were observed until 7 days after challenge in the unvaccinated group. However, the virus was not detected in the serum samples from the
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vaccinated group, indicating protection against FMD (Fig. 2A and B). Remarkably, lower quantities of virus were detected in the vaccinated group, and the duration was shorter than the unvaccinated group (Table 4). No clinical signs were apparent in the goats.
4. Discussion and conclusions The control of FMD is dependent on the vaccination of susceptible animal species with inactivated whole virus vaccines [15]. Vaccination with good quality FMD vaccines helps in the prevention of livestock production losses and reduces the overall incidence of the disease [16]. Hence, efforts are focused on developing adjuvants that can promote protective immunity through induction of enhanced and more durable antibody responses [1]. Currently, the double-oil emulsion vaccines are preferred for FMD prevention as they can be used to protect all susceptible species, particularly during FMD outbreak situations. Also, oil-adjuvant vaccines induce higher and longer lasting immune responses and show less interference from maternal antibodies compared to aqueous vaccines [17]. The SEPPIC company (France) has developed a new adjuvant ISA 201 and claims that this adjuvant induces better immune responses (particularly cell-mediated immunity) [18]. For example, ISA 201 (PD50 = 15.59) showed better protection in pigs when compared to ISA 206 (PD50 = 10.05) [10]. Similar results can be observed in cattle; ISA 201 showed higher lymphoproliferative response and IgG2 response and lower quantities of virus shedding detected after FMDV challenge compared to ISA 206 [1]. ISA 201 in the present study also showed a similar enhancement of protection. Emulsigen-D is a unique oil-in-water emulsion and contains uniformly dispersed micron-size oil droplets, which ensure maximum emulsion stability and decreased viscosity. Micron-size oil droplets also increase the surface area available to antigens, reducing the quantity of oil required in the final vaccine. Emulsigen-D reduces the undesirable side effects associated with other oil-in-water or water-in-oil adjuvants while eliciting a rapid and strong immune response [19]. Emulsigen-D as an adjuvant produces increased immunogenicity because it incorporates dimethyl-dioctadecyl ammonium bromide (DDA), which is a Tcell immune stimulator in Emulsigen. Its efficacy as an adjuvant was proved in Toxoplasmia gondii and rabies [20,21]. According to Kaur et al., the DDA contained in Emulsigen-D induces the enhancement of immune responses by increasing the surface area of antigens in oil-in-water emulsions so that antigens spread slowly. Therefore, protection against Aujeszky’s disease virus is increased when infected animals have been vaccinated with Emulsigen plus DDA. In addition, aluminum compounds have been known to be the most frequently used adjuvant in veterinary vaccines [22]. These compounds have been found to induce memory cell responses and long-lasting protection when animals have been inoculated with vaccines, thereby enhancing immune reactions [23]. Among them, aluminum phosphate and aluminum hydroxide are the only adjuvants approved for routine use in humans because of their relatively low toxicity [24]. Carbigen is a carbomer-based adjuvant suspension containing a proprietary emulsified component. Its milky white appearance creates a smooth uniform mixture [25]. Carbigen is a stabilizing adjuvant that stabilizes epitope conformation and is mainly used for intranasal application [26]. In our study, the group inoculated with the mixture of adjuvant ED + AL showed the best immunogenicity and production of antibody response. The titer of virus detected in the serum and oral swabs after challenge was lowest in the group inoculated with ED + AL compared to other groups in which other adjuvants were used, indicating that ED + AL effectively suppressed viral replication
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Table 1 Immunity and protective response of the pigs after FMD vaccination with various adjuvants. Vaccinated groups by adjuvants with FMD antigen AD-P, 10 g
Pig no.
Virus neutralizing antibody titers (log)
Viremia
7dpv
14dpv
21dpv
28dpv
10dpcc
93 94 95 96
