We are reporting you an effective DNA vaccine for FMD virus, and it was prepared using mannosylated chitosan (MC) nanoparticles to form the FMDV-pVAC-VP1-OmpA complex particles. These particles were characterized for their physical properties like morphology, size and charge prior evaluating the actual vaccine delivery effect of MC-nanoparticles. The immunological evaluation indicated that the 20 μg of DNA vaccine complexed with MC nano-particles was found optimum in inducing the immune response in G pigs as measured by FMDV specific neutralising antibodies and Th1/Th2 responses using micro SNT and ELISA, respectively.

Foot and mouth disease (FMD) is a highly contagious, economically important, office international des epizooties notifiable transboundary viral disease of cloven-hoofed animals caused by FMD virus, an Aphthovirus. The disease is associated with high morbidity and less mortality in endemic countries with international trade restrictions. In a country where test and slaughter policy is difficult to follow, vaccination is a viable strategy for controlling the disease ^[5]. The drawbacks of current inactivated virus vaccines are live virus handling, short duration of immunity, improper inactivation of virus with possible vaccine related outbreaks and poor elicitation of cell mediated immune response (CMI) ^[12]. Among the available alternative strategies, the DNA vaccine is gaining importance ^[1]. DNA immunization elicits CMI, humoral immune responses (HIR), antigen-specific cytotoxic T lymphocytes (CTL) and neutralizing antibodies ^[5]. However in the case of FMD, DNA vaccines have been reported to provide partial protection, warranting improvement in the current DNA vaccine strategies ^[4]. In particular, the immune response induced by DNA vaccines can be improved using suitable delivery systems ^[8]. Recently, the nanoparticle based techniques have emerged as potential DNA delivery systems as they stabilize DNA and also provide adjuvant effect ^{[1, 3]}. In this regard, chitosan (C) nanoparticles are beneficial compared to non viral vectors as they are inert, less toxic, rapidly biodegradable, and biocompatible ^{[2, 3, 6]}. In view of these, the present study was conceptualized to prepare and physically characterize the mannosylated chitosan (MC) ^{[7, 10]} nanoparticles complexed with an FMDV DNA vaccine construct, FMDV-pVAC-VP1-OmpA and optimise dose of FMDV construct for immunization in G pigs.

Initially, the water soluble chitosan was prepared by the N-acetylation method ^[7], dried at 50℃ and stored at room temperature. Chitosan was mannosylated and the MC-DNA vaccine complex was prepared ^[10]. FMD seronegative G pigs of either sex, each weighing 400-500 g were used. Recombinant DNA construct pVAC-VP1-OmpA, carrying the VP1 gene of FMDV type 'O' upstream of the Omp A gene of S. typhimurium in pVac1 vector, was harvested using E. coli DH5α bacterial cultures. The plasmid DNA was confirmed by digesting with Eco R Ⅴ and Bgl Ⅱ and agarose gel electrophoresis for the presence of VP1-OmpA ~1.7 kb gene in the DNA vaccine construct. The endotoxin free vaccine construct was bulk prepared for in vivo immunization using a PerfectPrepTM Endofree Maxi kit (5PRIME, Germany). The concentration and purity of the plasmid DNA was determined by agarose gel electrophoresis and spectrophotometry (nanodrop). Later, the particle size and zeta potential of the MC nanoparticles and DNA loaded MC nanoparticles was measured in a Malvern Master sizer 2000 (Malvern Instruments, UK), and the mannosylation of chitosan was analysed by high resolution 1^H NMR spectra in an NMR spectroscope (400 MHz Bruker, Germany, Ultrashield TM plus). Likewise, the DNA binding efficiency of MC was calculated as the ratio between the bound DNA and the total DNA used in the preparation of nanoparticles. The MC-DNA complex formation was confirmed by 1.0% agarose gel electrophoresis.

FMDV serotype 'O' at passage 5 in BHK₂₁ cells was titrated ^[21] and whole virus (146S) inactivated antigen was prepared ^[14]. Further, the optimal dose of MC-DNA vaccine was assessed in five groups (Group I -V) of G pigs with a group size of 8 animals. These groups were respectively immunized intramuscularly with 2, 5, 10, 20 and 50 μg of DNA vaccine complexed with MC nano-particles and observed over a period of 30 days. The animals were bled on 0, 4, 10, 16, 22 and 30 day post immunization (dpi) and serum was collected and heat inactivated. These sera were monitored for the presence of FMDV O serotype specific virus neutralizing antibody by SNT ^[14] and whole IgG, IgG1 and IgG2 response by respective indirect ELISAs ^[4]. The working dilutions of antigen, positive serum, negative serum and conjugate were determined by chequerboard titration. The peroxidase conjugated goat anti-guinea pig IgG1 and IgG2 were used at 1:5000, respectively.

The ~5.4 kb plasmid DNA (pVac-VP1-OmpA) extracted from positive E.coli and the recombinant plasmid DNA were intact upon electrophoresis. Further, the digestion of pVac-VP1-OmpA with Eco R Ⅴ and Bgl Ⅱ released a 1.7 kb VP1-OmpA fragment and a 3.7 kb vector fragment. However, pVac-VP1-OmpA linearized with Eco R Ⅴ showed a single band of 5.4 kb. The chitosan used was soluble in water. The morphology of MC nanoparticles and MC DNA under scanning electron microscope appeared spherical with some irregularities. The NMR spectroscopy also revealed distinctness in the MC as compared to the chitosan nanoparticles. The particle size of the MC and MCDNA nanoparticles were 60-300 and 80-500 nm, respectively. The zeta potential of MC and MC-DNA nanoparticles were +32 to +60 mV, and +22 and +50 mV, respectively. Further agarose gel electrophoresis was carried out to determine the DNA loading efficiency and formation MC-DNA complex. Accordingly, MC-DNA complex remained inside the gel, whereas, un-bound plasmid DNA migrated down the gel lane indicating that the DNA bound to MC-DNA complex. In case of pVac-VP1-OmpA complexed with un-mannosylated chitosan, an amount of DNA was migrating into the gel probably due to separation of weakly bound DNA. The results demonstrated that MC complexed better with DNA than unmannosylated chitosan. The DNA loading efficiency (expressed as the ratio between the bound DNA and the total DNA used to prepare nanoparticles) was 0.81 (81%).

The dose optimization studies in G pigs indicated that the virus neutralizing antibodies were induced in all immunized groups. But the group immunized with the MC-DNA complex containing 20 μg of DNA, showed a consistent rise in SN titres ranging from 0.6 to 1.8, respectively from pre-vaccination to 30 day post immunization (dpi) and sustained throughout the study period. Although the concentration greater than 20 μg (i.e. 50 μg) also induced SN antibody, the titres were not sustained beyond 16 dpi and it receded to 1.5 on 16 dpi onwards (Table 1). In order to monitor the Th2/Th1 responses elicited by different doses of MC-DNA vaccine complex, IgG1 and IgG2 responses were monitored in all the sera collected from G pigs by respective indirect ELISAs. The immune responses were represented in terms of OD_492nm. There was a graded increase in total IgG1 response from 0 to 30 dpi. The IgG1 response increased with an increase in dose of MC-DNA up to 20 μg, attaining a peak OD of 0.46±0.006 as observed and this was maintained up to 30dpi; whereas, G pigs immunized with a 50 μg dose showed an average OD of 0.38±0.006 on 30 dpi. Similarly, total IgG2 response showed an increasing trend from day 0 to 22 dpi with gradual decline thereafter. However, the peak titre was observed in the group immunized with 20μg dose both on 22 and 30 dpi (Table 2).

Table 1.  Serum neutralizing antibody titre against FMDV serotype 'O' in different immunized groups of Guinea pigs at different time intervals (values indicate the mean ± SD)

Table 2.  FMDV Type O specific IgG1 and IgG2 antibody response of various immunized groups of guinea pigs at different time intervals in the respective indirect ELISAs. The values indicate the mean ± SD at OD₄₉₂nm.

In the present study, chitosan and mannosylated chitosan were tested for effective delivery of a DNA vaccine cassette containing VP1 (immunodominant) region of FMDV along with full length outer membrane protein A (OmpA) gene of S. typhimurium cloned into pVAC plasmid DNA vector. Initially the morphology, size and charge of the MC nanoparticles prepared were conformed to the observations of other researchers ^{[8, 13]}. Incorporation of the FMDV-DNA construct into the nanoparticles reduced their positive charge, which is in accordance with Bordetella bronchoseptica dermonecrotoxin ^[8] and Treponema pallidum Tp92 DNA vaccine vectored by MC/chitosan naonparticles, respectively ^[17]. Also, mannose receptor mediated uptake enhances the HLA class Ⅱ restricted antigen presentation by cultured DCs ^[16]. In general, a higher dose of DNA vaccine is required to induce a good immune response, but in the present experiment 20 μg was found optimum. This is true to the statement that the delivery with cationic polymers such as MC nanoparticles reduces the quantity of DNA antigen due to the effective targeting and presentation through mannose receptors on the APCs for better immunity. The presence of positive charge on these particles facilitates the formation of polyelectrolyte complexes with negatively charged nucleic acids ^{[6, 11]}. Similarly after complexing chitosan/MC with DNA construct, the zeta potential of these complexes remained positive that might had provided a more stable colloidal dispersion ^[13] as chitosan derivatives have already been proved to enhance the immunity of DNA vaccine encoding hepatitis B virus core ^[9]. Furthermore, it is also possible to select the type of immune response essential to help the host to engender the type of protective immune response with the minimum possible antigen dose. It is warranted that the results in G pigs (an FMD animal model) needs to be evaluated in the natural host, bovine for its ultimate use. MC as a potent adjuvant can be assessed with other DNA vaccines and bacterial/viral antigens.