DNA prime-protein boost strategy with replicase-based DNA vaccine against foot-and-mouth disease in bovine calves

Veterinary Microbiology 163 (2013) 62–70

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DNA prime-protein boost strategy with replicase-based DNA vaccine against foot-and-mouth disease in bovine calves Pervaiz A. Dar a, Veluvarthy S. Suryanaryana a, G. Nagarajan b, Golla R. Reddy a, Hosur J. Dechamma a, Ganesh Kondabattula a,* a b

FMD Research Center, Indian Veterinary Research Institute, Banguluru 560024, India National Research Centre on Camel, Jorbeer, Bikaner 334001, Rajasthan, India

A R T I C L E I N F O

A B S T R A C T

Article history: Received 11 July 2012 Received in revised form 13 December 2012 Accepted 17 December 2012

The limited efficacy of DNA vaccines against foot-and-mouth disease (FMD) in cattle and other natural hosts has prompted a search for a more effective vaccination regimen. In this study we tested a DNA prime-protein boost vaccination strategy against FMD in bovine calves. We used purified recombinant FMDV specific multi-epitope protein (rMEG990) and an optimized sindbis virus replicase-based DNA vaccine expressing this protein (pSinCMV-Vac-MEG990). We demonstrate that vaccination with a low dose of pSinCMVVac-MEG990 (10 mg/animal) and subsequently boosting with rMEG990 resulted in induction of neutralizing antibodies, IFN-g production and protection against homologous virus challenge. However, vaccination with a high dose of pSinCMV-Vac-MEG990 (100 mg/ animal) and boosting with rMEG990 resulted in significantly lower immune responses and more severity to the challenge test. Additionally, we show that the post-vaccinal IFN-g levels in animals correlated positively to their protection against FMDV challenge. These findings suggest that a replicase-based DNA vaccine in proper prime-boost combination may offer an efficient vaccine strategy against FMDV and that IFN-g could be used as an additional immune parameter to predict protection against FMDV infection. ß 2012 Elsevier B.V. All rights reserved.

Keywords: Foot-and-mouth disease Replicase-based DNA vaccine Multivalent epitope vaccine Prime-boost vaccination regime Immune response Cattle

1. Introduction Foot-and-mouth disease (FMD) is an acute, highly contagious viral disease affecting cloven-hoofed animals, including cattle, swine, sheep, goats, wild pigs, wild ruminants and buffaloes (Grubman and Baxt, 2004). The disease is of considerable economic significance due to losses in animal productivity, mortality of young ones and export trade restrictions imposed on affected countries (Perry and Rich, 2007). The etiological agent, FMD virus (FMDV), is a single stranded RNA virus of the Aphthovirus genus, family Picornaviridae occurring in seven serotypes (O, A, C, SAT1, SAT2, SAT3 and Asia1) and more than 65

* Corresponding author. Tel.: +91 080 23410908; mobile: +91 9741582057. E-mail address: [email protected] (G. Kondabattula). 0378-1135/$ – see front matter ß 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2012.12.017

subtypes (Kitching et al., 1989). The wide variability is mainly due to a lack of accurate proof reading activity by viral RNA polymerase (3Dpol) resulting in a high mutation rate and quasi-species dynamics (Domingo et al., 1992, 2003). This diverse nature of FMDV complicates its control in a way that vaccination or natural infection with one serotype does not cross-protect against other serotypes and may also fail to protect against other subtypes of the same serotype (Kitching et al., 1989). Currently available FMD vaccines are mainly based on inactivated viral antigens (killed virus) formulated with various proprietary adjuvants (Rodriguez and Grubman, 2009). Although these vaccines have proven effective in reducing clinical disease in animals, they suffer from numerous shortcomings. Handling of live virus during the production process, the lack of cell-mediated and/or sterile immunity, and the problem with serological differentiation of infected from vaccinated animals (DIVA principle)

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have made vaccination strategies extremely challenging (reviewed in Rodriguez and Gay, 2011; Rodriguez and Grubman, 2009). In recent years, there has been an increasing interest in using plasmid DNAs as an effective and safe alternative vaccine for FMD (Chinsangaram et al., 1998; Ward et al., 1997; Wong et al., 2000). However, DNA vaccines generally tend to be less effective in inducing protective immune responses in natural hosts (Beard et al., 1999; Huang et al., 1999; Shieh et al., 2001). A large number of approaches are being used to improve the efficacy of DNA vaccines (reviewed in Leitner et al., 1999). Incorporation of alpha-virus replicase into the plasmid DNAs has been shown to improve their immunogenicity and biosafety (Berglund et al., 1998; Leitner et al., 2000). These replicase-based DNA vaccines, often referred as selfreplicating or suicidal DNA vaccines, have been reported effective against cancer (Haupt et al., 2002; Leitner et al., 2000; Quetglas et al., 2010; Ying et al., 1999) and many viral infections (Deshpande et al., 2002; Hariharan et al., 1998; Kamrud et al., 1999; Kirman et al., 2003). The enhanced efficacy of replicase-based DNA vaccines were initially attributed to greater levels of antigen expression but it is clear now that these vaccines effectively activate innate immune mechanisms (Leitner et al., 2003), especially the type I interferon system (Wu et al., 2010), apoptosis and activation of antigen presenting cells (APCs) (Leitner et al., 2004). The activity of replicase in the host cell is similar to viral infection producing dsRNA intermediates, which activate two major antiviral pathways; the 2-5A synthetase/RNaseL and the PKR pathway (Castelli et al., 1997; Der et al., 1997). The activation of these pathways predisposes the cell to death by caspasedependent apoptosis. dsRNA is also recognized by Toll-like receptor 3 on dendritic cells (DCs) and induces production of various immunostimulatory cytokines (Alexopoulou et al., 2001). These activated DCs then uptake antigen loaded apoptotic bodies and cross-present antigens in context of major histocompatibility complex class I (Albert et al., 1998). DNA vaccine-induced immune responses can be enhanced by boosting with a recombinant protein or viral antigens. This ‘‘prime-boost’’ strategy has been demonstrated to be effective against FMD in mice (Shieh et al., 2001), pigs (Li et al., 2008) and in a principle target host, cattle (Jin et al., 2005). The boosting effect may be due to the enhanced IFN-g secretion phenotypes/Th1 bias (Ramshaw and Ramsay, 2000) or the ability to avoid induction of anti-vector responses (Hanke et al., 1999) or generation of high levels of T-cell memory (Ramshaw and Ramsay, 2000). In fact, the initial priming events are imprinted on the immune system particularly T-cells and during the boost there is a selective increase in the numbers of these antigen specific T-cells (Seder and Hill, 2000). Previously, we demonstrated that a replicase-based DNA vaccine for FMD, pSinCMV-Vac-MEG990, was immunogenic and induced protective responses in guinea pigs (Dar et al., 2012). In this report, we tested the efficacy of pSinCMV-Vac-MEG990 in a DNA prime-protein boost regimen in bovine calves. Additionally, we show that antigen-specific IFN-g responses in vaccinated calves correlated to their protection against virus challenge.

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2. Materials and methods 2.1. Cell lines and virus strains The cell lines and virus strain used in this study were maintained and available at the FMD Research Center, Indian Veterinary Research Institute (IVRI), Bangalore, India. A Baby Hamster kidney (BHK)-21 clone 13 (Glasgow) cell line was grown at 37 8C under 5% CO2 in Dulbecco’s modified minimum essential medium (Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA), 25 mM HEPES (Sigma) and antibiotics. Vaccine virus strain ‘‘FMDV O/IND/R2/75’’ grown in BHK cells was used for inactivated antigen preparation and virus neutralization assays. A bovine adapted ‘FMDV O/IND/R2/75’ available as 10% homogenate of tongue lesion material in PBS was used for challenge virus preparation. 2.2. Production of plasmid DNAs and protein The construction of pSinCMV-Vac-MEG990 and its immunogenicity has been described earlier (Dar et al., 2012). The construct encoded multivalent epitope gene (MEG990) that represents the C-terminal halves of VP1 (residues 127–220 aa) of FMDV serotypes ‘A’, ‘Asia-1 and ‘O’, tandemly linked by viral 2A sequence. For the production of DNA plasmids, Escherichia coli DH5a cells (Invitrogen, Carlsbad, CA, USA) were electro-transformed with pSinCMV-Vac-MEG990 or pSinCMV-Vac and allowed to grow overnight in 250 ml Luria Bertani (LB) broth with 100 mg/ml ampicillin. The plasmid DNAs were purified by using Endo-Free plasmid maxi kit (Qiagen, USA) as per the manufacturer’s instructions. The plasmid DNAs were formulated in phosphate buffer saline (PBS, pH 7.4) and stored at 70 8C until use. For production of protein, the pET-32a expression plasmid encoding multivalent epitope gene (pET-MEG990) (Nagarajan et al., 2008) was transformed into E. coli BL21 (DE3) pLysS expression stain and induced for expression. Briefly, pET-MEG990 transformed E. coli BL21 were grown overnight at 37 8C in 5 ml LB broth containing 100 mg/ml ampicillin. 100 ml of this overnight culture was diluted 50fold into fresh LB broth without ampicillin and incubated at 37 8C until the culture reached an optical density of 0.4– 0.6 at A600 nm (4 h incubation). The bacterial cells were harvested and transferred into fresh 25 ml LB broth and induced by 1 mM isopropyl b-D-1 thiogalactopyranoside (IPTG) at 30 8C in an orbital shaker (300 rpm) for 6 h. The expression of recombinant protein was monitored by sodiumdodecyl polyacrylamide gel electrophoresis (SDSPAGE) analysis. The rMEG990 protein was purified by affinity purification using Ni-NTA Agarose as described earlier (Ratish et al., 1999). Further, the specificity of the protein was confirmed by blotting the protein onto nitrocellulose membrane and probing the blot using guinea pigs sera raised against the recombinant C-terminal half of VP1 of FMDV ‘O’ as described earlier (Suryanarayana et al., 1999). The recombinant protein was further passed

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through Amicon ultrafiltration using a 50 kDa cut off membrane and exchanged with Tris–NaCl buffer (25 mM Tris–HCl, 200 mM NaCl, pH 8.0). The yield and purity of recombinant protein was determined by the Lowry method (Lowry et al., 1951) and SDS-PAGE analysis respectively. The purified rMEG990 protein was emulsified in Montanide ISA 206 adjuvant (Seppic, France) as per manufacturer’s instructions to form a double oil emulsion formulation. The final vaccine formulation contained 0.5 mg of rMEM990 protein per ml and was stored at 4 8C until use.

inactivated at 56 8C for 30 min in water bath for use in virus neutralization test. Following the challenge of calves, we collected oropharyngeal fluid (OPF) on 0, 10 and 21 dpc for viral RNA detection and sera samples on 0 and 21 dpc for nonstructural protein (NSP) serology. The OPF samples were collected by sterilized cotton tampons and transported to laboratory on ice. A 200 ml of OPF was added to 1 ml TRIzol1 Reagent (Sigma) and stored at 70 8C until use.

2.3. Vaccination and challenge of calves

The post-vaccination sera of the calves were analyzed for anti-FMDV serotype ‘O’ neutralizing antibodies by virus neutralization assay in BHK-21 cells. Two fold serial dilutions of the test sera (50 ml) in duplicate were prepared in DMEM in a 96-well Tissue Culture plates. The virus suspension of 100 TCID50 in 50 ml volume was dispensed to each well and the mixture was incubated at 37 8C for 1 h. Thereafter a 50 ml suspension of the BHK-21 cells (0.5  105 cells) was added to each well and further incubated at 37 8C with 5% CO2. Appropriate controls with respect to test serum, challenge virus, cells and virus titration were included in the test. The cell monolayers were observed for CPE after 48 h and 50% serum neutralization end-point were calculated by the method of Reed and Muench (Reed and Reed, 1938). Virus neutralizing antibody titers were represented as log of the reciprocal of the last serum dilution that neutralized the virus activity by 50%.

2.5. Virus neutralization assay

Animal experimentation was carried out at Animal Isolation Unit, Indian Veterinary Research Institute, Bangalore, India in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forests, Government of India. Twenty three male calves of Hallikar breed (Bos indicus) aged between 6 and 8 months were used for this experiment. The animals were dewormed and screened for anti-FMDV antibodies prior to use in experiment. Animals were separated into groups and vaccinated as per the regime outlined in Table 1. Four weeks post vaccination, all the animals along with the two unvaccinated controls were needle challenged on tongue with 10,000 BID50 FMDV type ‘‘O’’ virus. The animals were monitored for the signs of FMD for ten days. The appearances of lesions were noted and animals were assigned clinical scores and protection status at the end of observation period.

2.6. Antigen specific interferon gamma assay Antigen specific IFN-g release in vaccinated calves was measured by a whole blood IFN-g release assay (Parida et al., 2006). Earlier optimization in our laboratory showed that a volume of 1 ml whole blood stimulated with 10 mg of FMDV antigen for 48 h at 37 8C resulted in the optimum antigen specific IFN-g release (unpublished data). Heparinized blood from each animal was dispensed in four 1 ml aliquots in a 24-well tissue culture plate. 100 ml volume of Concavallin A (10 mg/ml in PBS), FMDV ‘O’ antigen (10 mg/ ml) in duplicate and BHK-21 cell lysate (10 mg total

2.4. Sampling Heparinized and clotted blood samples were collected on 0, 9, 18 and 28 dpv using Vacutainer system (Greiner Bio-one, Austria). The heparinized blood samples were transported to the laboratory at ambient temperature (22  5 8C) and used for interferon gamma (IFN-g) assay within 5 h after collection. The clotted blood samples were processed for serum separation and the sera were heat Table 1 Vaccination schedule of calves. Group designation

No. of calves

Vaccination regimen

High dose prime

5

Priming: 100 mg pSinCMV-VacMEG990 per animal in 0.2 ml PBS on day 0 Boosting: 1 mg rMEG990 per animal in 2 ml oil adjuvanta on day 18

Low dose prime

5

Priming: 10 mg pSinCMV-VacMEG990 per animal in 0.2 ml PBS on day 0 Boosting: 1 mg rMEG990 per animal in 2 ml oil adjuvanta on day 18

Protein control

3

Priming and boosting: 1 mg/rMEG990 per animal in 2 ml oil adjuvanta on day 0 and day 18 respectively

Inactivated viral vaccine

5

Single dose of inactivated FMDV ‘O’ antigen (20 mg) per animal in 2 ml oil adjuvanta on day 0

Vector control

3

Single dose of 100 mg pSinCMV-Vac per animal in 0.2 ml PBS on day 0

Unvaccinated control

2

Nil

All the vaccinations were performed by deep intramuscular injection into deltoid muscle. a Montanide ISA 206 (Seppic, France).

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protein/ml) was then added to the wells, respectively. The blood cultures were incubated in a humidified atmosphere under 5% CO2 for 48 h at 37 8C, and then supernatant plasma were harvested from each well and stored at 20 8C until used for IFN-g quantitation. The IFN-g levels in plasma samples were quantified using Bovine IFN-g ELISA kit (Mabtech, Sweden) as per the manufacturer’s protocol. The plasmas were used undiluted in 100 ml volume along with the known concentrations of IFN-g standards in each ELISA plate. The plates were developed for 20 min at room temperature (RT) using ophenylenediamine dihydrochloride (Sigma, cat # P8287) and read at optical density (OD) of 450 nm in iMark Microplate Reader (Biorad, California, USA). Antigenic specific IFN-g release is defined as the mean of an IFN-g level in FMDV antigen stimulated supernatant after subtraction of IFN-g level of a mock antigen stimulated supernatant for that animal. 2.7. RT-PCR assay for viral RNA detection Total RNA was extracted from 200 ml of OPF samples using TRI Reagent (Sigma, USA) according to the manufacturer’s recommendations. RNA was resuspended in 10 ml of nuclease free water and a 5 ml of it was converted into first strand complementary DNA (cDNA) in 20 ml reaction volume using random hexamers and SuperScript1 III Reverse Transcriptase (Invitrogen) as per manufacturer’s instructions. For PCR amplification, 5 ml of cDNA was used in 50 ml final reaction volume using a universal primers pair for the 50 UTR region of FMDV genome (50 -GCCTG-GTCTT-TCCAGG-TCT-30 /50 -CCAGTCCCCTTCTCAGATC-30 ) (Reid et al., 2000). The PCR amplification condition included an initial denaturation phase at 94 8C for 5 min, cycling phase at 94 8C/1 min, 55 8C/1 min, 72 8C/2 min for 30 cycles and a final extension phase at 72 8C for 10 min. The PCR product was analyzed by agarose gel electrophoresis in 1.5% gel. A positive result was indicated by the presence of a 328 bp band corresponding to FMDV sequence in the 50 UTR region of the genome. 2.8. FMDV non-structural protein serology The presence of antibodies against 3AB and 3Dpol in post-challenge sera was determined by NSP-ELISA developed in our laboratory. Briefly, ELISA plates were coated overnight at 4 8C either with yeast expressed 3AB protein (500 ng/ml) or insect expressed 3Dpol protein (900 ng/ml). Sera samples (diluted 1:20 in blocking buffer) were added to the wells and incubated for 1 h at RT. The wells were washed with PBS-Tween (0.5%) and incubated further for 1 h at RT with an anti-bovine HRP conjugate. The plates were washed and substrate buffer solution containing 1 mg/ml OPD (Sigma) and 0.01% H2O2 was added for developing at RT. After significant development of color (20 min), the reaction was stopped by adding 1 M H2SO4, and the absorbance was recorded at 492 nm in a microplate reader. The results were expressed as absorbance of 21 dpc sera minus the absorbance of 0 dpc sera in NSP-ELISA.

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2.9. Statistical analysis The data were analyzed with the aid of GraphPad Prism 3.0 software (San Diego, CA). The SEM was used as the error value and an unpaired t-test was used to measure the difference of parameters between groups. The difference was reported significant if p < 0.05. 3. Results 3.1. Expression and purification of FMDV specific multivalent-epitope protein The recombinant FMDV specific multivalent-epitope protein (rMEG990) used in this study is depicted in Fig. 1. The pET-MEG990 transformed E. coli BL21 (DE3) pLysS cells showed expression of 57 kDa rMEG990 protein as against un-induced and vector transformed bacteria (Fig. 1A) and was recognized in Western blot by antiVP1 FMDV ‘O’ sera. The rMEG990 protein was purified by a Ni-NTA agarose column under denaturing conditions and the purity was assessed by SDS-PAGE analysis (Fig. 1B). The yield of purified rMEG990 protein was determined to be 13 mg/100 ml of bacterial culture. 3.2. Anti-FMDV neutralizing antibody response in vaccinated animals In both groups of the prime-boost calves the neutralizing antibody response was detected, although not always statistically significant as compared to unvaccinated control (Fig. 2). In the high dose prime calves the antibody titers were higher before the boost (1.2  0.09, p < 0.05, ttest) and decreased after the boost (0.9  0.13, p > 0.05, ttest). On the contrary, in low dose prime calves the neutralizing titers increased following the boost from a mean titer of 0.66  0.14 to 1.08  0.22 (p < 0.05). In the protein control group, the neutralizing antibody titers were non-significant at all points of observation as compared to unvaccinated control and vector control. The neutralizing antibody response in inactivated vaccine group were significantly higher at all point post-vaccination than the control groups (p < 0.05, t-test) with peak titers observed on day 18 (1.56  0.33). 3.3. Antigen specific IFN-g responses following vaccination The mean IFN-g release in different groups of calves following vaccination is shown in Fig. 3. The IFN-g response in both of the prime-boost groups were nonsignificant except that low dose primed calves showed higher levels on 28 dpv (m = 0.91  0.4, p < 0.05) as compared to unvaccinated controls. Interestingly, the high dose prime group showed lower post-boost IFN-g release as compared to their pre-boost levels. In the protein group and vector control groups IFN-g releases were nonsignificant at all the time points. In the inactivated virus vaccine group, the post-vaccinal IFN-g responses were significantly higher than unvaccinated control (p < 0.05, ttest) at all the time points, with peak response on 9 dpv (m = 3.08  0.92).

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Fig. 1. Expression and purification of foot-and-mouth disease virus specific recombinant multivalent epitope protein (rMEG990). (A) E. coli BL21 (DE3) pLysS cells were transformed with pET-MEG990 plasmid and induced for the protein expression by isopropyl b-D-1 thiogalactopyranoside. Lanes: M, standard protein MW marker; 1, lysate of pET32a+ transformed bacteria after induction; 2, lysate of pET-MEG990 transformed bacteria without induction; 3, lysate of pET-MEG990 transformed bacteria after induction showing expression of the 57 kDa rMEG990 protein. (B) The rMEG990 protein was purified from bacterial lysate by Ni-NTA Agarose binding and ultrafiltration. Lanes M, standard protein MW marker; 1, Ni-NTA Agarose purified rMEG990 protein; 2, rMEG990 protein after ultrafiltration. (C) Schematic representation of rMEG990 protein depicting C-terminal halves of VP1 of the three serotype (O, A and Asia I) tandemly linked by viral 2A sequences.

3.4. Protective responses in vaccinated cattle against FMDV challenge To determine whether the DNA prime-protein boost vaccination regimen confers protection, we inoculated calves with virulent FMDV serotype ‘O’ intra-dermolingually and monitored for typical FMD signs for 10 days. The protective responses of calves against infection are summarized in Table 2. Both of the unvaccinated control

Neutralizing anbody tres ( Log10)

2.1

1.8

100 μg pSINCMV-Vac-MEG990 + 1 mg MEG proen (n=5) 10 μg pSINCMV-Vac-MEG990 + 1 mg MEG proen (n=5) Inacvated Vaccine (n=5)

1.5

Proen control (n=3) Vector control (n=3)

1.2

0.9

0.6

0.3 0

9

18

28

Days post vaccinaon Fig. 2. Induction of neutralizing antibodies against foot-and-mouth disease virus (FMDV) following DNA prime-protein boost vaccination of bovine calves. Calves were primed with either 10 mg or 100 mg of replicase-based DNA vaccine (pSinCMV-Vac-MEG990) on day 0 and boosted with homologous recombinant protein (rMEG990) on day 18 through intramuscularly route along with appropriate controls. Virus neutralizing activity was analyzed against FMDV serotype‘‘0’’ with BHK21 cell system in micro neutralization assay. Each data point represent mean titer of the group and error bar represent standard error of mean.

animals suffered pyrexia and developed primary lesions on inoculation sites by 2 dpc. Both showed lameness and developed severe vesicular lesions on buccal mucosa and on all four feet by 5 dpc. Only two animals (C8 and C10) in the low dose primed group were protected from challenge and did not develop any secondary lesions. One of the animals (C6) had mild disease with secondary lesion on only one of the hind feet. None of the animals in the high dose prime calves, protein group and vector control group were protected at all rather some among high dose prime group reacted more severely to challenge infection than unvaccinated animals. In inactivated viral vaccine group 4 out of 5 animals were protected and only one animal (C12) developed clinical disease during the observation period. To examine virus replication following challenge, we performed RT-PCR on OPF samples and NSP-serology on post-challenge sera. In unvaccinated animals viral RNA could be detected in OPF on both occasions (i.e. 10 and 21 dpc) and both of them showed sero-conversion to anti3D and anti-3AB antibodies on 21 dpc. Among prime-boost calves viral RNA was detected less frequently in the low dose prime group (3 out of 5) than in high dose prime group (4 out of 5) on 10 dpc and the frequency of detection of viral RNA was found to be decreased in both groups on 21 dpc (2 out of 5 in both groups). The two protected animals in the low dose prime group showed neither the presence of any viral RNA nor antibody response to 3D or 3AB protein. Even unprotected animals of the prime-boost groups showed lower anti-3D and anti-3AB response. Animals in the protein control and the vector control groups were positive for viral RNA and showed seroconversion to anti-NSP antibody. In the inactivated vaccine group the frequency of viral RNA detection, as well as

IFN-gamma release (ng/ml)

P.A. Dar et al. / Veterinary Microbiology 163 (2013) 62–70 4.5

100 μg pSINCMV-Vac-MEG990 + 1 mg MEG proen (n=5)

4

10 μg pSINCMV-Vac-MEG990 + 1 mg MEG proen (n=5)

3.5

67

anti-NSP antibody responses, were lowest and consistent with their protection status after challenge.

Inacvated Vaccine (n=5)

3

Proen control (n=3)

2.5

Vector control (n=3)

3.5. Correlation between IFN-g production and protection against FMDV infection

2

To assess the correlation between post vaccinal IFN-g releases and protection against challenge infection, the animals were categorized into protected and unprotected groups and results were reanalyzed. Comparisons of mean IFN-g releases of protected animals versus non-protected animals are shown in Fig. 4. The post vaccinal IFN-g releases of protected animals were significantly higher than non-protected animals with peak levels achieved on 9 dpv and maintained at higher levels throughout the observation period. Furthermore, the calves which showed post vaccinal IFN-g releases also showed lower frequency of viral RNA detection and seroconversion to anti-NSP antibodies (Table 2). These results indicate that the IFN-g levels correlate to protection against FMDV infection.

1.5 1 0.5 0 0

9

18

28

Days post vaccinaon Fig. 3. Effect of DNA prime-protein boost vaccination on the levels of IFNg released from cultures of whole blood of calves after stimulation with foot-and-mouth disease virus (FMDV) antigen. Calves were primed with either 10 mg or 100 mg of replicase-based DNA vaccine (pSinCMV-VacMEG990) on day 0 and boosted with homologous recombinant protein (rMEG990) on day 18 along with appropriate controls. The post-vaccinal blood from calves were stimulated with FMDV ‘‘O’’ antigen and IFN-g release was measured in supernatant plasma by sandwich ELISA. Each data point represent mean of the group and error bar represent standard error of mean.

4. Discussion Replicase-based DNA vaccines are considered a superior and safe vaccine platform for delivering subunit

Table 2 Summary of protective responses of experimental calves against FMDV infection. Vaccination

Animal ID

Clinical scorea

Protection statusb

Pre-challenge neutralizing Ab titer (log 10)

Peak IFN-g level post-vaccination (ng/ml)

100 mg pSINCMV-Vac-MEG990 + 1 mg MEG990 protein

C1 C2 C3 C4 C5

6 6 3 6 5

NP NP NP NP NP

0.6 0.6 0.9 1.2 1.2

0.17 0.45 0.44 0.36 0.59

10 mg pSINCMV-Vac-MEG990 + 1 mg MEG990 protein

C6 C7 C8 C9 C10

2 5 1 4 1

NP NP P NP P

0.9 1.5 1.5 1.2 0.3

Inactivated vaccine

C11 C12 C13 C14 C15

0 4 0 1 1

P NP P P P

Protein control

C16 C17 C18

4 6 5

Vector control

C19 C20 C21

Unvaccinated control

C22 C23

RT-PCR of OPF samplesc

Sero-conversion to NSPd

10 dpc

21 dpc

3AB

3D

+ +  + +

+ +   

0.23 0.13 0.25 0.37 0.19

0.46 0.35 0.20 0.19 0.24

0.12 0.33 1.61 0.54 3.12

+ +  + 

+ +   

0.30 0.26 0.03 0.12 0.01

0.28 0.15 0.11 0.21 0.00

1.2 0.3 2.1 1.8 1.8

2.11 0.34 4.82 2.72 2.17

 +  + 

 +   

0.18 0.39 0.05 0.11 0.01

0.13 0.55 0.11 0.22 0.05

NP NP NP

0.6 0.3 0.9

0.9 0.82 0.54

+ + +

+ + +

0.54 0.41 0.33

0.63 0.78 0.57

5 6 5

NP NP NP

0.3 0.6 0.6

0.68 0.65 0.11

+ + +

+ + +

0.37 0.29 0.43

0.61 0.77 0.65

5 5

NP NP

– –

– –

+ +

+ +

0.55 0.44

0.61 0.81

a Severity of FMD in calves were scored by summing up the following points: one point for primary vesicle on tongue at the site of inoculation, one point for vesicular lesions on each affected foot and one point for secondary vesicles/erosion on buccal mucosa (on lip, dental pad or gums). The highest score of 6 indicated severe disease with primary lesions on the tongue, secondary lesion on buccal mucosa and all four feet. b Animals with no lesions (Score 0) or primary vesicular lesion at the site of virus inoculation on tongue (Score 1) were considered as protected (P). Secondary lesions were considered as signs of systemic viremia and animals were assigned non-protected (NP). c Results expressed as (+) animal detected positive or () animal detected negative. d Results expressed as OD at 21 dpc – OD at 0 dpc.

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IFN-gamma release (ng/ml)

4 3.5

Vaccinated protected (n=6) Vaccinated unprotected (n=12)

3 2.5 2 1.5 1 0.5 0 0

9

18 Days post vaccinaon

28

Fig. 4. The level of IFN-g release from whole blood of vaccinated animals correlates with protection against the challenge infection. Calves were vaccinated against foot-and-mouth disease and subsequently challenged with virulent FMDV serotype ‘‘O’’ virus on day 28. Development of clinical disease was monitored for 10 days and at the end animals were grouped either protected or unprotected. Comparison of post-vaccinal IFN-g responses between protected versus unprotected calves are made. Data of 18 animals from primeboost groups, inactivated vaccine group and protein control group are included in the analysis. Each data point represent mean of the group and error bar represent standard error of mean.

antigens (Berglund et al., 1998; Ljungberg et al., 2007). At the same, the DNA prime-protein boost approach (heterologous prime-boost) has been proven to be more effective in eliciting antigen specific immune responses than using repeated DNA or protein vaccine alone (homologous prime-boost) (Sabarth et al., 2010; Vaine et al., 2010). Previous study by Yu et al. (2006) showed that a replicasebased DNA vaccine for FMD induces enhanced immunity and virus clearance in mice as compared to conventional DNA vaccine. However, Dory et al. (2009) was unable to demonstrate any improvement of immunity against FMDV in swine’s using a replicase-based DNA vaccine. In this study, we have evaluated a replicase-based DNA vaccine in a DNA prime-protein boost regimen in cattle against FMDV. We demonstrated that using low dose of replicasebased DNA vaccine in a prime-boost protocol could induce significant protective immune responses against FMDV in cattle. However, use of high dose of the vaccine in the prime-boost approach was not able to induce the protective responses. In calves primed with a low dose of pSinCMV-VacMEG990 anti-FMDV immune responses showed marked improvement following the boost, both in the pattern of antibody titers as well as IFN-g responses. Consequently the protection against the virus challenge was higher (Table 2). On the contrary, the calves primed with the high dose of pSinCMV-Vac-MEG990 showed significantly lower immune responses and none of the animals were protected against the viral challenge. The post-boost neutralizing antibody titers were significantly lower as compared to their pre-boost titers (Fig. 2). This decrease in antibody titers following the boost may be due to suppressing activity of the protein at high concentration, which was reflected by the non-responsiveness of blood samples to antigenic or mitogenic stimulation. The post-boost IFN-g releases in whole blood cultures with FMDV antigen were lower than their pre-boost levels (Fig. 3). Reactivity to mitogen was also significantly reduced and occasionally

eliminated (data not shown). This non-responsiveness may be indicative of the compromised immune system. It is possible for vaccines to elicit ineffective responses under certain circumstances. For example, exclusive priming of CD4 T-cell responses can result in the suppression of CD8 T-cell responses on subsequent pathogen challenge (Zhong et al., 2000). Similarly, the inadvertent targeting of antigens that might be poorly expressed at the site of injection might reduce vaccine efficacy (Crowe et al., 2003). This finding also implies that the issue of dose with respect to a given vaccine in a prime-boost regimen may be important for the outcome of immune responses and/or protection against the targeted disease. Our data also shows that the protective responses against FMDV infection were lower in the prime-boost vaccinated calves as compared to the calves vaccinated with inactivated viral vaccine. This difference may be due to the fact that the whole virus contains all the FMDV epitopes that can prime a broader range of immune cells thereby activating more balanced immune responses against FMDV infection. In contrast, our minimal epitope vaccines, pSinCMV-Vac-MEG990 or rMEG990, contained only C-terminal portions of VP1 (residues 127–220 aa) encoding two major epitopes, VP1141–160 and VP1 200–213. These epitopes has been shown to be immuno-dominant and induce major neutralizing antibodies directed against FMDV (Mahapatra et al., 2012). Previous studies with synthetic peptides representing these epitopes showed induction of protection in guinea pigs (Bittle et al., 1982; Doel et al., 1988; Nagarajan et al., 2008), but protection in cattle was limited (DiMarchi et al., 1986; Taboga et al., 1997). Induction of protective immunity against FMDV in cattle may be critically dependent upon specific MHC– peptide–T cell interactions (Baxter et al., 2009; Glass and Millar, 1994). In our study, the protective responses mediated by the pSINCMV-Vac-MEG990 or rMEG990 vaccine were also restricted to a few animals, possibly reflecting the lack of appropriate T helper cell epitopes and/or epitopes presented by broad bovine MHC haplotypes that are able to activate balanced immune responses. To determine the efficacy of vaccination, it is important to be able to diagnostically measure the level of protective immunity. For FMDV vaccines serum neutralizing antibody response show strong correlation to protection against the virus challenge and the titers >1.5 are generally indicative of protective immune status (Goris et al., 2008; Hingley and Pay, 1987; McCullough et al., 1992; Pay and Hingley, 1992). However, this correlation is not absolute as animals with lower titers at times are protected from the disease or animals with higher titers on occasions succumb to the disease (McCullough et al., 1992). Previous studies suggest that IFN-g, a signature cytokine of T-cell activation, may represent an additional correlate for protective immunity against FMDV (Barnett et al., 2004; Parida et al., 2006; Oh et al., 2012). Thus, we measured IFN-g release ex vivo in the blood of vaccinated animals in response to FMDV antigen and correlated the IFN-g levels to the protection status following challenge. We found a positive correlation between IFN-g levels and protection against the FMDV challenge. Six out of total 23 animals used in this experiment were protected and showed higher

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post-vaccinal antibody as well as IFN-g responses except one calf (C11) which had low antibody titers but high IFN-g levels. Three of the protected calves (C10, C11 and C13) which had comparatively higher post-vaccinal IFN-g responses also showed no vesicular lesions on the tongue at virus inoculation sites. This indicates that the high quality or the high payload of FMDV antigen not only exerts inhibitory effect on the systemic virus replication but also inhibits the viral replication at mucosal sites. Similar observation has been reported earlier using high potency vaccines (Barnett et al., 2004; Cox et al., 2006). Further, the calves which had higher post-vaccinal IFN-g levels also showed lower frequency of viral RNA detection and seroconversion to anti-NSP antibodies. This implies that the quantity of IFN-g level may correlate with the capacity of the vaccinated animals to control FMDV infection. In summary, the replicase-based DNA vaccine in proper prime-protein boost combination may offer a safe and effective vaccine strategy against FMD in cattle. The IFN-g may be an additional immunological parameter that correlates with protection against FMDV infection. This latter information is important to further develop IFN-g assay as a adjunct test to predict protection in vaccinated animals which may help to avoid challenge infection during potency testing of FMD vaccines. We are currently investigating the effect of broader range of FMDV epitopes, inparticular T-cell epitopes, on the efficacy of replicasebased DNA vaccines as well as the issues of route and mode of delivery for this novel vaccine strategy. Conflict of interest statement The authors do not have any conflicts of interest to declare. Acknowledgments This project was funded by the Department of Biotechnology (DBT), Government of India. The authors acknowledge the Director, IVRI, Barrielly and Joint Director, IVRI, Bangalore, India, for providing necessary facilities. The first author is the recipient of IVRI Research fellowship during his M.V.Sc. programme. References Albert, M.L., Pearce, S.F., Francisco, L.M., Sauter, B., Roy, P., Silverstein, R.L., Bhardwaj, N., 1998. Immature dendritic cells phagocytose apoptotic cells via alphavbeta5 and CD36, and cross-present antigens to cytotoxic T lymphocytes. J. Exp. Med. 188, 1359–1368. Alexopoulou, L., Holt, A.C., Medzhitov, R., Flavell, R.A., 2001. Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature 413, 732–738. Barnett, P.V., Keel, P., Reid, S., Armstrong, R.M., Statham, R.J., Voyce, C., Aggarwal, N., Cox, S.J., 2004. Evidence that high potency foot-andmouth disease vaccine inhibits local virus replication and prevents the ‘‘carrier’’ state in sheep. Vaccine 22, 1221–1232. Baxter, R., Craigmile, S.C., Haley, C., Douglas, A.J., Williams, J.L., Glass, E.J., 2009. BoLA-DR peptide binding pockets are fundamental for footand-mouth disease virus vaccine design in cattle. Vaccine 28, 28–37. Beard, C., Ward, G., Rieder, E., Chinsangaram, J., Grubman, M.J., Mason, P.W., 1999. Development of DNA vaccines for foot-and-mouth disease, evaluation of vaccines encoding replicating and non-replicating nucleic acids in swine. J. Biotechnol. 73, 243–249.

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