Enhanced protection against FMDV in cattle after prime- boost vaccination based on mucosal and inactivated FMD vaccine

Accepted Manuscript Title: Enhanced protection against FMDV in cattle after prime- boost vaccination based on mucosal and inactivated FMD vaccine Authors: Manar E. Khalifa, Ayman H. El-Deeb, Sayed M. Zeidan, Hussein A. Hussein, Hany I. Abu-El-Naga PII: DOI: Reference:

S0378-1135(17)30624-7 http://dx.doi.org/10.1016/j.vetmic.2017.08.014 VETMIC 7728

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VETMIC

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18-5-2017 20-8-2017 21-8-2017

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Enhanced protection against FMDV in cattle after prime- boost vaccination based on mucosal and inactivated FMD vaccine

Manar E. Khalifaa, Ayman H. El-Deebb, Sayed M. Zeidana, Hussein A. Husseinb,*, Hany I. Abu-El-Nagaa

aDepartment

of Foot and Mouth Disease, Veterinary Serum and Vaccine Research

Institute, Abbassia, Cairo 11381, Egypt. bDepartment

of Virology, Faculty of Veterinary Medicine, Cairo University, Giza

12211, Egypt.

*Corresponding author at: Department of virology, Faculty of Veterinary Medicine, Cairo University, Giza 12211, Egypt. E-mail address: [email protected] Highlights: 

Intranasal immunization of calves with FMD mucosal vaccine elicited lymphocyte proliferation and IgA levels in nasal and salivary secretions.



Intranasal immunization protective levels: 20% and 40% in single dose and double dose, respectively.



Prime boost strategies with parenteral and mucosal vaccines, coupled with mucosal IgA levels, resulted in 100% protection in a challenge test.

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ABSTRACT Improved immunization and control strategies and platforms are greatly needed for foot and mouth disease virus (FMDV) and mucosal vaccines propose an effective strategy for the control FMDV by blocking viral entry. In this study, several immunization strategies, using two FMDV vaccine formulations, including Montanide ISA 206 oil-based FMD inactivated vaccine and Montanide IMS 1313 VG N PR-based concentrated semi-purified FMD mucosal vaccine, were applied. Results of intranasal immunization with the prepared FMD mucosal vaccine, given once or twice, induced IgA levels in both nasal and salivary secretions besides a high response of lymphocyte proliferation with protection levels reaching 20% and 40%, respectively, in a challenge trial in cattle. Immunization with Montanide 206 inactivated FMD vaccine was capable of inducing 80% protection whereas primeboost strategy based on the administration of mucosal vaccine followed by inactivated vaccine appeared to be the most potent strategy by achieving 100% protection against an FMDV challenge. Indeed, the study reports the efficacy of the prepared IMS 1313 FMD mucosal vaccine and the possible use of this vaccine in the context of different vaccination strategies to control FMDV. Abbreviations1 Keywords: FMDV; mucosal vaccine; immunization strategy; prime-boost. 1. Introduction

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Abbreviations: I/N: intranasal; FMDV: foot and mouth disease virus

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Foot and mouth disease virus (FMDV) causes highly infectious and economically important disease in cloven-hoofed animals and is characterized by vesicles in the feet, buccal mucosa, and on udder (OIE, 2012). FMDV is a non-enveloped virus with single-stranded and positive-sense RNA molecules of 8500 nucleotides with icosahedral symmetry within the Family Picornaviridae (Carrillo, 2012). Owing to infectious nature of the virus and high morbidity, vaccination is one of the most successful strategies to control FMD especially in an endemic situation (Rodriguez and Grubman, 2009). However, vaccinations with one or several doses of the parenteral, inactivated FMD vaccine are being applied extensively in routine field vaccinations campaigns against FMDV (Kahn et al., 2002). Such vaccines failed to produce the IgA required in mucosal sites to prevent FMDV entry (Doel, 1996), allowing viral replication in mucosal surfaces causing carrier state in vaccinated animals after live virus challenge (Parida, 2009). From this perspective, mucosal immunity is critical to restrict the entry and spread of FMDV into animals, since secretory IgA is effectively induced by mucosal vaccines and can neutralize the invading FMD virus at the mucosal epithelium and subsequently prevent its entry into host cells (Ogra et al., 2001). Moreover, administration of mucosal vaccines alone did not elicit the required levels of protective serum antibody (Pan et al., 2014). Accordingly, prime-boost immunization strategies combining the advantages of both the mucosal and parenteral vaccines were applied against viral diseases and are showing success in terms of potency and easy in usage (Glynn et al., 2005). The intranasal route administration of FMD vaccine is considered the preferred route due to numerous M (microfold) cells present in nasal-associated lymphoid tissue (NALT) inductive sites that are responsible for antigen uptake and transportation to antigen-presenting cells (APCS). Therefore intranasal route is proposed to counteract 3

a rapid spread of FMDV into the herd especially in emergency situations. On the other hand, the oral route of immunization is accompanied by problems associated with enzymatic degradation of the presented antigen (Sedgmen et al., 2004). In this regard, two FMD vaccine formulations were prepared and were used to immunize cattle. Evaluation of the efficacy of the prime-boost strategy, using both mucosal and inactivated vaccines, was carried out in a challenge trial in calves. The objective of the present study is to design a mucosal vaccine for FMDV to be used in prime-boost strategy with parenteral FMD vaccine for inducing immune responses in both the local and systemic compartments and enhancing protection in cattle against FMDV. 2. Material and methods 2.1 Animals Six-months-old calves (Native breed “Egyptian Baladi cattle”) (n=27), tested negative for FMD antibodies by virus neutralization assay, were used in the challenge trial. This study was carried out in accordance with the recommendations and guidelines of the "European Communities Council Directive 1986 (86/609/EEC)".

All animal

experiments were conducted in isolators of the Veterinary Serum and Vaccine Research Institute. The use of animals and protocols were approved by the Animal Care and Use Committee of Veterinary Serum and Vaccine Research Institute, Egypt. 2.2 FMD viruses O Pan-Asia/2012 virus strain was obtained from Veterinary Serum and Vaccine Research Institute Abbassia, Egypt and used in vaccine preparation and virus neutralization assays. O Pan-Asia seed virus was propagated on the BHK-21 cell line 4

and the harvested virus was inactivated with binary ethylene-imine (BEI) (Bahnemann, 1975). The inactivated virus was concentrated using polyethyleneglycol (PEG)-6000 (Kaaden, et al.,1971) and ultra-filtered using an Amicon 15-10k ultra centrifugal device (Millipore, USA) (Spitteler et al., 2011). For the challenge trial, the reference challenge FMD-virus serotype O Pan-Asia 2012 strain was kindly provided by the Central Laboratory for Evaluation of Veterinary Biologics, Abbassia, Egypt. All experiments involving infectious virus were performed in the isolator facilities of FMD in VSVRI. 2.3 Vaccines Two vaccine formulations were prepared using two types of adjuvants: Montanide ISA 206 (Incomplete Seppic Adjuvant) VG (Vegetative origin) and Montanide IMS (Immuno Stimulating) 1313 VG NPR (Preserved grade with Thimerosal 0.01%) for the preparation of parenteral- inactivated FMD vaccine and mucosal FMD vaccine, respectively. The antigen: adjuvant ratios were prepared following the manufacturer’s recommendations. The antigen content (146S antigenic mass) was quantified by 146S quantitative sucrose density gradient centrifugation (SDG) technique as previously described (Barteling and Meloen, 1974). The 146S dose in inactivated parenteral vaccine was 4µg/dose and 30µg/dose in mucosal vaccine (Pan et al., 2014). Each calf was inoculated with 3 ml of the prepared mucosal vaccine containing 30 µg 146S antigenic mass/dose and for parenteral vaccine, each calf received 2 ml vaccine containing 4 µg 146S antigenic mass/dose. 2.4 Safety and sterility of the prepared FMD vaccines

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Sterility test was performed to confirm the freedom of the prepared vaccines from any contaminations. A safety test was performed (Oie, 2008) to ensure the absence of any abnormal local or systemic adverse reactions post administration. 2.5 Immunization protocols applied in experimental groups of calves A total of 27 animals (calves) were divided into 6 groups. In Group 1, a total of five calves were intranasally vaccinated with FMD IMS 1313 mucosal vaccine, using Sterimatic nasal spray applicator (Sterimatic Worldwide, England) (1x mucosal vaccine). In Group 2, five calves were primed and boosted intranasally with FMD IMS 1313 mucosal vaccine at 21 days (2x mucosal vaccine). In Group 3, Five calves were primed intranasally with FMD IMS 1313 mucosal vaccine and boosted S/C (sub-cutaneous) with inactivated FMD, oil-based vaccine at 21 days (mucosal, parenteral). In Group 4, five calves were primed S/C with inactivated FMD, oil-based vaccine and boosted intranasally with FMD IMS 1313 mucosal vaccine at 21 days (parenteral-mucosal). Finally in Group 5, a total of five calves were vaccinated S/C with inactivated FMD, oil-based vaccine (1x parenteral). Two calves were kept unvaccinated as controls in Group 6. 2.6 Challenge Experiment At 25 days following the last vaccination, the experimental calves were challenged by intradermal lingual inoculation of 0.3ml of 104 ID50 of the FMDV (OIE, 2012). For the challenge trial, the reference challenge FMD-virus O/EGY/2012 strain that belongs to O Pan-Asia strain was kindly provided by the Central Laboratory for Evaluation of Veterinary Biologics, Abbassia, Egypt. Challenged calves were observed daily for 14 days for any rise in temperature, salivation, and appearance of

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vesicles on tongue or foot. Unprotected calves showed vesicular lesions in tongue, hind and fore limbs. 2.7 Virus Neutralization Assay (VN) The titers of the serum neutralizing antibodies were measured using the virus neutralization test. Serum samples were collected from the experimental calves at zero days of vaccination and weekly after vaccine administration; for group 1 and group 5 which received one dose, samples were taken for three weeks and for groups (group 2, group 3and group 4) which received prime boost vaccine, samples were collected for six weeks. The collected serum samples were heat inactivated at 56 0C for 30 min. The tested serum samples were incubated with the prepared FMDV 100 TCID50 for 1 hour then the BHK cells were added to the serum virus mixture and incubated for 48 hours. The cytopathic effects were noticed to determine the titers that were calculated as the log10 of the reciprocal antibody dilution to neutralize 100 TCID50 of the virus (OIE, 2012). 2.8 Evaluation of lymphocyte proliferation response of experimental calves (MTT assay) Lymphocyte proliferation in different groups was evaluated using MTT assay (Nagarajan et al., 2011). Lymphocytes were seeded into 96-well tissue culture plates with 2000 cells /well with adding 100µl of growth media to each well. Triplicate cultures were performed for each sample as follows, the lymphocytes were stimulated by adding inactivated FMDV serotype ‘O’ into the cultured lymphocytes, PHA stimulated lymphocytes were set as a positive control and negative control set of unstimulated cells. The plate was incubated for 24-48 hours in incubator at 37ºC. MTT (1X) was added at 50 µl/well. The plate was incubated for 1-4 hours at 370C.The

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MTT fromazan was dissolved by addition of 150 µl DMSO. The optical density of the samples was read at 570 nm. Results were expressed as SI (Stimulation index)

2.9 IgA Response in experimental calf groups For the detection of FMD VP1 IgA in serum, saliva, and nasal fluids, a recombined virus bovine anti-FMD VP1 IgA serotype O ELISA kit (Alpha Diagnostics Intl.) was used. Serum, nasal swabs and salivary secretions were collected from the experimental calves twice per week post vaccination. For group 1 which received one dose, samples were taken for 24 dpv and for group 2, group 3 and group 4 which received prime boost vaccine; samples were collected for 45 dpv. Nasal swabs were added to PBS in equal volume and salivary secretions then, centrifuged at 7000 rpm for 10 minutes to remove cell debris as previously described (Parida et al., 2006). The test was performed according to the kit manual guide. In antigen coated 8 micro well strip, the positive control was diluted at 2 fold serial dilution in duplicate manner for plotting the standard curve to calculate the O.D readings in relation to the plotted standard curve as corrected O.D readings. The 1x sample diluent was added as blank sample, and the negative control was added at the same volume in duplicate manner. The collected samples were prepared at 1:10 by diluting in 1x sample diluent and added at 100µl /well and incubated for 60 minutes at room temperature (25-280C).The wells were aspirated and washed 3 times with 1x wash buffer. The wells were washed properly to avoid high blank values. The Antibody enzyme conjugate (1x) was added at 100 µl into each well, mixed gently then the plate was incubated for 30 minutes at room temperature. The wells washed 5 times as previously described. The

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TMB substrate was added at 100 µl into each well, mixed gently and then the plate was covered and incubated 15 minutes at room temperature. The reaction was stopped by adding 100 µl of stop solution to all wells and mixed gently. The absorbance was measured at 450 nm using ELISA reader within 15 min. 2.10 Statistical analysis PASW Statistics, version 18.0 software (SPSS Inc., Chicago, IL, USA) was used and graphs were performed using Graph pad prism 7.0 software. The statistical significance of the differences in the means of experimental groups was determined by two-way ANOVA analysis. Data are expressed as the mean ± standard error of the mean. A difference was determined to be statistically significant if p < 0.05. 3. Results 3.1 Complete protection achieved in prime-boost vaccinated calves The unvaccinated calves showed rise in temperature 2 days post-challenge along with lesions in the dorsum of tongue. Characteristic vesicles began to appear 7 days post challenge in all feet without any protection. In contrast, the calves in prime-boost groups (Group 3, mucosal-parenteral) and Group 4 (parenteral-mucosal) showed complete protection from the challenge (Figure 1). On the other hand, calves immunized with the mucosal vaccine once or twice showed 20% and 40% protection, respectively compared to controls. 3.2 FMD mucosal vaccine induced low levels of serum-neutralizing antibodies Calves intranasally primed with mucosal vaccine failed to induce high levels of serum-neutralizing antibodies before booster administration (0.87 ± 0.11), (0.75 ± 0.09) in group 1 and group 2, respectively. Whereas S/C injection with inactivated 9

FMD, oil-based vaccine showed protective antibody titer at 14 days post vaccination (dpv) (1.38 ± 0.056), which increased 21 dpv and reached (1.890 ± 0.089). Calves receiving the mucosal vaccine before and after inactivated vaccines showed increase in neutralizing antibodies at 28 dpv (1.440 ± 0.076) and (1.260 ± 0.076) in group 3 (mucosal-parenteral) and group 4 (parenteral-mucosal), respectively. Neutralizing antibodies continued to increase and reached their peak at 42 dpv, higher in group 3 (2.670 ± 0.073) than in group 4 (1.890 ± 0.059). In contrast, group 2 showed a slight increase following mucosal boost (1.02 ± 0.1). A statistically significant difference was observed between groups of calves as shown in Figure 2. 3.3 Lymphocytic proliferation induced by FMD mucosal vaccine At 3dpv, all mucosally primed groups (group1, group2 and group 3) showed a statistically significant increase in lymphocyte proliferation compared to the unvaccinated calf group. By 10 dpv, the FMD mucosal vaccine was capable of inducing a significant increase in stimulation index (SI) of intranasally vaccinated groups, reaching the peak levels at the 17th day with prominent decline observed at 24 dpv. Following boosting, a second wave of increase appeared in SI levels of all boosted calves, reaching its maximum levels at 38 dpv (0.889±0.011), (0.865±0.035) in group 2 (2xmucosal vaccine) and group 4 (parenteral-mucosal), respectively, indicating the ability of the prepared FMD mucosal vaccine to induce lymphocytic proliferation and to be retained with a booster dose (Figure 3). 3.4 IgA response in mucosal sites IgA in serum

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At 7 dpv, all mucosally primed groups showed positive IgA levels in serum (1.5 ± 0.49), (1.3 ± 0), (1.5 ± 0) in group1, group 2 and group 3, respectively. However, the positive levels were only recalled in group 2 (2x mucosal vaccine) at 31 dpv, which received 2 doses of the mucosal vaccine (1.5 ± 0). No statistically significant difference between time and groups in the dependent variable (Serum IgA); (p-value= 0.299) was detected (Figure 4A). IgA in nasal secretions At 7 dpv, a significant difference from unvaccinated control group was observed in group 2 (2x mucosal vaccine) (2± 0) and group 3 (mucosal-parenteral) (1.4 ± 0.09), reflecting the ability of FMD mucosal vaccine to induce an increase in nasal IgA levels. At 10 dpv, a significant difference from the unvaccinated control group was observed in group 1(1x mucosal vaccine) (1.15± 0.1) and group 3 (mucosalparenteral) (1.1.± 0.07).

At 18 dpv, no significant difference appeared between

vaccinated (group 1, group 2 and group 3) and unvaccinated calves (group 6). Post boosting with the mucosal or parenteral vaccines, calves in group 2, group 3, and group 4 showed high secretory IgA levels, which persisted until 38 dpv (1.25± 0.09), (1.65± 0.3), (1.75± 0.2), respectively. In contrast, at 45 dpv, only group 3 (mucosalparenteral) (1.06± 0.9) and group 4 (parenteral-mucosal) (1.65± 0.9) retained high IgA levels (Figure 4B), p- value = 0.03. IgA in salivary secretions Results at 3 dpv, revealed a significant increase of IgA in salivary secretions in the calves of group 2 (2xmucosal vaccine) (1.25± 0.05) and group 3 (mucosal-parenteral) (0.9± 0.4) in comparison with the control group (0.03± 0); reflecting the capability of FMD mucosal vaccine to induce IgA levels in salivary secretions. At 7 dpv, an 11

increase in salivary IgA levels appeared in all mucosal vaccinated groups (group1, group 2, and group3) (0.9± 0.4), (1.5± 0), (1.25± 0.25), respectively. At 10 dpv, only group 1 (1xmucosal) (0.9± 0.4) showed a statistically significant difference from the control group. At 18 dpv, similar to nasal IgA, no significant difference between vaccinated (group 1, group 2 and group 3) and unvaccinated calves (group 6) were detected in salivary IgA. However, at 24 dpv, group 2 (2xmucosal vaccine) (2 ± 0), and group 4 (parenteral-mucosal) (1.75 ±0.25) showed a significant increase in IgA in salivary secretions, which continued to increase at 28, 31, and 38 dpv in all boosted groups, including group 3(mucosal-parenteral), (1.4± 0.09), (1.75± 0.25), (1.7± 0.29), respectively, pointing out the delay in IgA recall due to parenteral boost. At 45 dpv, there was no statistical difference from the control group in IgA levels in group 2(2x mucosal) (0.38± 0.1); and the control unvaccinated calf group (0.03), whereas in group 3 (mucosal-parenteral) and group 4(parenteral-mucosal), the levels remained high (1.25± 0.75), (1.6± 0.4), respectively (Figure 4C), p- value= 0.004. 4. Discussion FMDV entry and persistence in nasopharyngeal mucosal surfaces and its systemic spread are major challenges in its control (Pacheco et al., 2015). FMD commercial vaccines are not capable of producing sufficient mucosal immunity required to block FMD viral entry through mucosal sites (DOEL, 1996; Pan et al., 2014). Recently, studies were greatly concerned with producing FMD mucosal vaccine capable of inducing sufficient IgA levels to prevent viral invasion and the establishment of infection (Fischer et al., 2003; Song et al., 2005; Pan et al., 2014; Wang et al., 2015). However, no studies have focused on combining both parenteral and mucosal vaccines in a prime-boost strategy for protection against FMD in cattle. In the current study, a trial for designing an appropriate FMD mucosal vaccine was carried out 12

considering the design properties mentioned by Holmgren (Holmgren and Czerkinsky, 2005), who recommended the criteria for targeting the mucosal inductive sites, stimulating the innate immune response that would consequently evoke the adaptive immune response with special concern to combined parenteral and mucosal approaches. We have assessed the protection levels following intra-lingual FMD challenge in experimental calves since it is the gold standard for evaluation of the prepared FMD vaccines in vivo (Feng et al., 2016). Collective analysis of the results of the mucosally vaccinated groups has revealed that the protection levels of 20% in group1 (1x mucosal vaccine) and 40% in group 2 (2x mucosal vaccine) (Figure 1), while a prime-boost strategy using mucosal administration, followed by parenteral vaccine or vice versa, revealed satisfactory results with complete protection (100%) in both groups (group 3 and group 4), indicating successful interaction between the virus and virus-specific antibody forming a complex which is phagocytosed by the macrophages (Mccullough et al., 1992). The mucosally vaccinated groups (group1 and group 2) which received mucosal vaccine only, showed low antibody titers in serum, despite the mucosal boost administered in group 2 (Figure 2) since the nature of antigen presentation affects the outcome of the prime-boost vaccination strategies (Barefoot et al., 2009). Alternatively, high antibody titers were produced after administration of FMD inactivated vaccine in group 5 (1x parenteral vaccine). While, group 3 (mucosalparenteral) and group 4 (parenteral-mucosal) showed remarkably high levels of serum neutralizing antibodies following booster administration that lend further support to the combination of parenteral and mucosal delivery in prime-boost for effective mucosal vaccine strategies (Neutra and Kozlowski, 2006). As the booster dose is responsible for directing the final location of effector lymphocytes which are 13

produced after primary exposure to the antigen (Mccluskie et al., 2002), this can explain the high neutralizing antibody levels in group 3 which received parenteral boost in comparison to group 4 which received mucosal boost (Figure 2). It is likely that the significant increase in stimulation indices of the lymphocytes from the unvaccinated control group at 3 dpv in mucosally primed groups (Figure 3), is linked to the micro emulsion formulation of the designed mucosal vaccine with Montanide IMS 1313 VG adjuvant that allowed antigen up-take and adherence to M cells (microfold cells) (Neutra and Kozlowski, 2006; Quattrocchi et al., 2014). Moreover, the high antigenic payload of 146S in the prepared mucosal vaccine, allowed the retention in mucosal tissues (DOEL & CHONG, 1982; Neutra and Kozlowski, 2006;Maroof et al., 2014), thereby activating

the adaptive immune

response (Quattrocchi et al., 2014). On the other hand, lymphocytic proliferation levels induced by the FMD conventional vaccine were demonstrated by (Dar et al., 2013) who showed a significant increase at 14 and 21dpv, indicating the rapid stimulation of lymphocytes induced by the designed mucosal vaccine compared to the conventional FMD vaccines. It has been well established that, IgA is critically required in FMD vaccines for either immune exclusion, preventing FMDV entry into epithelial cells, or immune elimination, which is the transportation of the FMD that invaded the epithelial barrier back into the lumen (Bouvet et al., 2002; Neutra and Kozlowski, 2006; Macpherson et al., 2008). In our study, a significant increase of IgA levels from the unvaccinated control group from 3-10 dpv in salivary secretions and 7-10 dpv in nasal swabs of mucosally primed groups was observed (Figures 4B, 4C) as a result of the evoked adaptive immune response (Neutra and Kozlowski, 2006) and indicates the presence of effector plasma cells in nasal and salivary secretions (Fiorino et al., 2013). 14

Because intranasal immunization leads to induction of an immune response in NALT, performing its secretory effector function in both nasal and salivary secretions explains the appearance of IgA in salivary secretions despite nasally administered antigen (Davis, 2001), rendering the intranasal route the most appropriate route for FMD vaccine delivery. The IgA results are quite similar to those obtained previously (Pan et al., 2014), who demonstrated IgA levels 4dpv, following mucosal immunization with Chi-PLGA-DNA nanoparticles. Interestingly, the high IgA levels in group 3 (mucosal-parenteral), followed by parenteral boost, may be attributed to the migration of the antigen to the draining lymph node and uptake by antigenpresenting cells (APCs) reaching NALT where B-and T-cells activation or direct transportation of the antigen to NALT occurs (Bouvet et al., 2002). It is observed that, groups (group 3 and group 4) which received both parenteral and mucosal immunizations showed prolonged IgA levels in nasal and salivary secretions that persisted till 45 dpv than in group 2 (2x mucosal vaccine), which received the antigen with the same route of immunization. This can be correlated to the strong immune response elicited following the combination of both mucosal and parenteral vaccines, than that produced from administration of either mucosal or parenteral vaccination alone, demonstrating the interaction between mucosal and systemic immune systems (Mccluskie et al., 2002; Glynn et al., 2005). Giving intranasal immunization twice, stimulated a direct mucosal immune response in both nasal and salivary secretions and poor systemic neutralizing antibodies. By combining both mucosal and parenteral FMD vaccines in a prime-boost strategy, the magnitude and localization of the evoked immune response was affected, and complete protection with high-neutralizing antibodies and increased mucosal IgA response lasted until 45 dpv was achieved (Fiorino et al., 2013). 15

However the conducted study has met its objectives to a great extent, there were some unavoidable limitations. The antigenic dose of the mucosal vaccine was high, so further studies need to be carried out in order to optimize the 146S dose in FMD mucosal vaccine. Moreover, the duration of immunity for prime-boost strategies was not pursued that will be carried out in upcoming studies. In conclusion, in the present study we have achieved 100% protection against FMD challenge when prime-boost strategy was applied in which mucosal and inactivated parenteral FMD vaccines were used.

Application of mucosal vaccine can be

effectively applied to restrict the spread of FMD outbreaks in context of different vaccination regimens. Conflict of interest: None. Funding: This work was supported by grants from the veterinary serum and vaccine research institute.

AUTHOR CONTRIBUTIONS: Study conception and design: Hussein

Acquisition of data: Hussein, Khalifa, Abu El-naga

Analysis and interpretation of data: Hussein, Khalifa, Abu El-naga, El Deeb, Zeidan

Drafting of manuscript: Hussein, Khalifa, El-Deeb, Abu El-naga

Critical revision: Hussein, El-Deeb, Zeidan, Khalifa

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Approval of the version of the manuscript to be published: Hussein, Khalifa, ElDeeb, Zeidan, Abu El-naga

Acknowledgement: We would like to acknowledge Dr. Muhammad Munir at avian innate immunity & host genetic diversity at Pirbright institute, for reviewing the manuscript.

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doi:10.1128/CMR.14.2.430 OIE, 2008. Principles of veterinary vaccine production. OIE Terr. Man. 90–104. OIE, 2012. Foot and mouth disease. OIE Terr. Man. Pacheco, J.M., Smoliga, G.R., O’Donnell, V., Brito, B.P., Stenfeldt, C., Rodriguez, L.L., Arzt, J., 2015. Persistent foot-and-mouth disease virus infection in the nasopharynx of cattle; tissue-specific distribution and local cytokine expression. PLoS One 10, 1–20. doi:10.1371/journal.pone.0125698 Pan, L., Zhang, Z., Lv, J., Zhou, P., Hu, W., Fang, Y., Chen, H., Liu, X., Shao, J., Zhao, F., Ding, Y., Lin, T., Chang, H., Zhang, J., Zhang, Y., Wang, Y., 2014. Induction of mucosal immune responses and protection of cattle against directcontact challenge by intranasal delivery with foot-and-mouth disease virus antigen mediated by nanoparticles. Int. J. Nanomedicine 9, 5603–5618. doi:10.2147/IJN.S72318

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Parida, S., 2009. Vaccination against foot ‑ and ‑ mouth disease virus : strategies and effectiveness. Expert Rev. Vaccines 8, 347–365. Parida, S., Anderson, J., Cox, S.J., Barnett, P. V., Paton, D.J., 2006. Secretory IgA as an indicator of oro-pharyngeal foot-and-mouth disease virus replication and as a tool

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doi:10.1016/j.vaccine.2005.09.006 Quattrocchi, V., Pappalardo, J.S., Langellotti, C., Smitsaart, E., Fondevila, N., Zamorano, P., 2014. Early protection against foot-and-mouth disease virus in cattle using an inactivated vaccine formulated with Montanide ESSAI IMS D 12802

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doi:10.1016/j.vaccine.2014.02.061 Rodriguez, L.L., Grubman, M.J., 2009. Foot and mouth disease virus vaccines. Vaccine 27, 90–94. doi:10.1016/j.vaccine.2009.08.039 Sedgmen, B.J., Meeusen, E.N.T., Lofthouse, S.A., 2004. Alternative routes of mucosal immunization in large animals. Immunol. Cell Biol. 82, 10–16. doi:10.1111/j.1440-1711.2004.01214.x Song, H., Wang, Z., Zheng, D., Fang, W., Li, Y., Liu, Y., Niu, Z., Qiu, B., 2005. A novel mucosal vaccine against foot-and-mouth disease virus induces protection in mice and swine. Biotechnol. Lett. 27, 1669–1674. doi:10.1007/s10529-0052727-4 Spitteler, M.A., Fernández, I., Schabes, E., Krimer, A., Régulier, E.G., Guinzburg, M., Smitsaart, E., Levy, M.S., 2011. Foot and mouth disease (FMD) virus: Quantification of whole virus particles during the vaccine manufacturing process 21

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doi:10.1016/j.vaccine.2011.05.078 Wang, M., Pan, L., Zhou, P., Lv, J., Zhang, Z., Wang, Y., Zhang, Y., 2015. Protection against foot-and-mouth disease virus in Guinea pigs via oral administration of recombinant Lactobacillus plantarum expressing VP1. PLoS One 10, 1–18. doi:10.1371/journal.pone.0143750

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Figures & captions: 100%

100

100%

G p .1 1 x m u c o s a l

80%

PPG%

G p .2 2 x m u c o s a l G p .3 M u c o s a l-p a re n te ra l G p .4 P a re n te ra l- m u c o s a l

40%

50

G p .5 1 x P a re n te ra l G p .6 C o n tro l

20%

0% l o tr

ra

n

te c

o

n re a P x

n

1

ra te

l

l s o c u

m l-

p la s o P

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l ra te n

re a

m x 2

1

x

m

u

u

c

c

o

o

s

s

a

a

l

l

0

Figure1. The percentage of protection against pedal generalization (PPG %) against intradermal-lingual FMDV challenge at 25 days post last vaccination. Group1: Vaccinated once with FMD mucosal vaccine, Group 2: Vaccinated twice with FMD mucosal vaccine, Group 3: Calves primed with FMD mucosal vaccine and boosted with FMD, oil-based vaccine (mucosal, parenteral), Group 4: Calves primed with FMD, oil-based vaccine and boosted with FMD mucosal vaccine (parenteralmucosal), Group 5: Calves vaccinated with FMD, oil-based vaccine (1x parenteral), Group 6: Unvaccinated calves (control group).

23

o

f

D a y s p o s t v a c c in a t io n

f

v

24

4

3

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8

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d

d

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v

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3

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2

1

2

5

8

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7

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D a y s p o s t v a c c in a t io n d

d

d

d

d

p

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p

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p

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v

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v

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v

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v

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S e r u m n e u tr a liz in g a n t ib o d y t it e r s ( L o g 1 0 )

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0 .5

2

f

S e r u m n e u tr a liz in g a n tib o d y t it e r s ( lo g 1 0 ) 0 .6

4

y

o

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d

a

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n

S e r u m n e u t r a l iz in g

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a n t ib o d y t it e r s ( L o g 1 0 )

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7

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1 .0

0 .5

0 .0

D a y s p o s t v a c c in a t io n

P a r e n t e r a l- M u c o s a l

2 .0

1 .5

1 .0

0 .5

0 .0

D a y s p o s t v a c c in a tio n

0 .4 6

C o n tro l

0 .4 4

0 .4 2

0 .4 0

0 .3 8

0 .3 6

0 .3 4

Figure2. Mean neutralizing antibody titers (log10) induced by the prepared FMD vaccines in serum over time assessed by (VNT) in experimental groups (group1, group2, group3, group4 and group 6), weekly from 0 day of vaccination (n=5). For group1 and group 5 which received one dose, samples were taken for three weeks (21 dpv). For groups (group 2, group 3, and group 4) which received prime boost vaccine, samples were collected for six weeks (42 dpv). Titers were expressed as mean log10 of antibody titers ± SEm. Error bars represent the SEm. pvalue = 0.0000001. Group1: Calves vaccinated once with FMD mucosal vaccine, Group 2: Calves vaccinated twice with FMD mucosal vaccine, Group 3: Calves primed with FMD mucosal vaccine and boosted with FMD, oil-based vaccine (mucosal, parenteral), Group 4: Calves primed with FMD, oil-based vaccine and boosted with FMD mucosal vaccine (parenteral-mucosal), Group 5: Calves vaccinated with FMD, oil-based vaccine (1x parenteral), Group 6: Unvaccinated calves (control group). Abbreviations: I/N: Intra nasally, S/C: Subcutaneously, FMDV: Foot and mouth disease virus, SEm: Standard error of mean.

25

*

1 .0

*

*

* P I (P r o life r a tiv e In d e x )

*

G ro u p 1 1 x M u c o s a l G ro u p 2 2 x M u c o s a l

0 .8

* *

G ro u p 3 M u c o s a l-P a re n te ra l

0 .6

G ro u p 4 P a re n te ra l-M u c o s a l G ro u p 6 C o n tro l

0 .4

0 .2

V

V

P s

s

y 4

5

d

a

a 3

8

d 1 3

d

a

y

s y

s y a d 4 2

P

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V P

V P s y a d

1

7

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3

d

a

a

y

y

s

s

P

P

V

V

0 .0

D a y s p o s t v a c c ia n t o n

Figure3. Lymphocyte proliferation assay of PBMCS in experimental calvs following immunization with the prepared FMD vaccines (MTT assay) from 3 days post vaccination to 45 days post vaccination. Data expressed as Mean of (SI) (570 nm) ± SE. (n=2). Error bars represent the SEm. Group1: Calves vaccinated once with FMD mucosal vaccine, Group 2: Calves vaccinated twice with FMD mucosal vaccine, Group 3: Calves primed with FMD mucosal vaccine and boosted with FMD, oil-based vaccine (mucosal, parenteral), Group 4: Calves primed with FMD, oilbased vaccine and boosted with FMD mucosal vaccine (parenteral-mucosal) sample NT form 3dpv-18dpv, Group 5: Calves vaccinated with FMD, oil-based vaccine (1x parenteral) sample NT, Group 6: Unvaccinated calves (control group). Abbreviations: I/N: Intra nasally, S/C: Subcutaneously, FMDV: Foot and mouth disease virus, SEm: Standard error of mean. (*): refer to statistical significant difference from control un vaccinated group.

26

2 .5

S e r u m Ig A

( c o r r e c t e d O .D 4 5 0 n m )

G p .1 1 x m u c o s a l G p .2 2 x m u c o s a l

2 .0

G p .3 m u c o s a l-p a r e n te r a l 1 .5

G p .4 p a r e n te ra l- m u c o s a l G p .6 c o n tr o l g r o u p

1 .0

0 .5

v 4

5

d

p

v p d 8 3

3

1

d

d

p

p

v

v

v 2

4 2

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d

p d 1

8

d 0 1

p

v

v p

v p d 7

3

d

p

v

0 .0

D a y s p o s t v a c c ia n to n

Figure 4A. IgA levels in serum induced by the prepared FMD vaccines in experimental cattle groups performed by ELISA from 3 days post vaccination to 45 days post vaccination. Data expressed as corrected O.D readings (n=2). Note: Negative control values less than the cut off control 0.500. Positive control higher than the cut off and at least 0.800 or higher. Blank values less than 0.400. Error bars represent the SEm. p-value= 0.299. Group1: Calves vaccinated once with FMD mucosal vaccine, Group 2: Calves vaccinated twice with FMD mucosal vaccine, Group 3: Calves primed with FMD mucosal vaccine and boosted with FMD, oilbased vaccine (mucosal, parenteral), Group 4: Calves primed with FMD, oil-based vaccine and boosted with FMD mucosal vaccine (parenteral-mucosal) sample NT form 3dpv-18dpv, Group 5: Calves vaccinated with FMD, oil-based vaccine (1x parenteral) sample NT, Group 6: Unvaccinated calves (control group). Abbreviations: I/N: Intra nasally, S/C: Subcutaneously, FMDV: Foot and mouth disease virus, SEm: Standard error of mean.

27

*

2 .5

* *

*

N a s a l Ig A c o r r e c t e d

O .D r e a d in g s 4 5 0 n m

*

* *

2 .0

G p .1 1 x m u c o s a l *

G p .2 2 x m u c o s a l

1 .5

*

* G p .3 m u c o s a l-p a r e n te r a l

1 .0

G p .4 p a r e n te ra l- m u c o s a l 0 .5

G p .6 c o n tr o l

v 5 4

8

d

p

v p d

p 3

3

1

d

d 2

8

d

p

v

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v p

v 4 2

8 1

0 1

d

d

p

v p

v p d 7

3

d

p

v

0 .0

D a y s p o s t v a c c ia n to n

Figure 4B. IgA levels in nasal swabs induced by the prepared FMD vaccines in experimental cattle groups performed by ELISA from 3 days post vaccination to 45 days post vaccination. Data expressed as corrected O.D readings (n=2). Note: Negative control values less than the cut off control 0.500. Positive control higher than the cut off and at least 0.800 or higher. Blank values less than 0.400. Error bars represent the SEm. p-value= 0.03. Group1: Calves vaccinated once with FMD mucosal vaccine, Group 2: Calves vaccinated twice with FMD mucosal vaccine, Group 3: Calves primed with FMD mucosal vaccine and boosted with FMD, oilbased vaccine (mucosal, parenteral), Group 4: Calves primed with FMD, oil-based vaccine and boosted with FMD mucosal vaccine (parenteral-mucosal) sample NT form 3dpv-18dpv, Group 5: Calves vaccinated with FMD, oil-based vaccine (1x parenteral) sample not taken, Group 6: Unvaccinated calves (control group). Abbreviations: I/N: Intra nasally, S/C: Subcutaneously, FMDV: Foot and mouth disease virus, SEm: Standard error of mean. (*): refer to statistical significant difference from control un vaccinated group.

28

S a liv a ry Ig A

c o r r e c te d O .D r e a d in g s (4 5 0 n m )

2 .5

* *

*

*

*

*

G p .1 1 x m u c o s a l

2 .0 *

G p .2 2 x m u c o s a l 1 .5

* *

*

G p .3 m u c o s a l-p a re n te ra l

1 .0

G p .4 p a r e n te r a l- m u c o s a l

0 .5

G p .6 c o n tr o l g r o u p v 4

5

d

p

v d 8 3

3

1

d

d

p

p

v

v p

v 8 2

4

d

p

v 2

1

8

d 0 1

d

p

p

v

v p d 7

3

d

p

v

0 .0

D a y s p o s t v a c c ia n to n

Figure 4C. IgA levels in salivary secretions induced by the prepared FMD vaccines in experimental cattle groups performed by ELISA from 3 days post vaccination to 45 days post vaccination. Data expressed as corrected O.D readings (n=2). Note: Negative control values less than the cut off control 0.500. Positive control higher than the cut off and at least 0.800 or higher. Blank values less than 0.400.Error bars represent the SEm. p-value= 0.004. Group1: Calves vaccinated once with FMD mucosal vaccine, Group 2: Calves vaccinated twice with FMD mucosal vaccine, Group 3: Calves primed with FMD mucosal vaccine and boosted with FMD, oil-based vaccine (mucosal, parenteral), Group 4: Calves primed with FMD, oil-based vaccine and boosted with FMD mucosal vaccine (parenteral-mucosal) sample NT form 3dpv-18dpv, Group 5: Calves vaccinated with FMD, oil-based vaccine (1x parenteral) sample not taken, Group 6: Unvaccinated calves (control group). Abbreviations: I/N: Intra nasally, S/C: Subcutaneously, FMDV: Foot and mouth disease virus, SEm: Standard error of mean. (*): refer to statistical significant difference from control un vaccinated group.

29

30