Foot-and-mouth disease virus (FMDV) is a member of genus Aphthovirus of the family Picornaviridae and causes the highly contagious vesicular disease foot-and-mouth (FMD) of cloven-hoofed animals [10, 21]. Unlike cattle and pigs, FMD in sheep is frequently mild or non apparent, so that subsequent transmission to other susceptible species can produce devastating consequences [13, 9]. Thus sheep can be considered an important target species for FMDV vaccines. Although traditionally inactivated vaccines play a key role for control and eradication of the disease in some parts of the world, these chemically inactivated vaccines have several disadvantages including the need to store under refrigeration  and difficult to discrimination between infected and vaccinated animals [14, 16]. Additionally, there is a potential risk of live virus escaping from vaccine plants [1, 2, 13].
Epitope vaccines againt FMDV are a novel vaccine and represented one of the safest methods for eliciting neutralizing antibodies to FMDV and confering complete protection in small animals [20, 23]. However, this vaccine only could offer limited protection in the natural hosts [17, 22, 23]. This may be due to the poor immunogenicity of antigenic epitopes and the possible lack of the appreciate T-helper cell epitopes [3, 8]. Previous reports have indicated that the host-self immunoglobulin G heavy constant region as a potential protein carrier could not only significantly improve the immunogenicity of antigenic epitopes, but could also reduce side-effects compared with that of other foreign protein carriers [7, 24, 25].
In this study, to develop a promising multiple-epitope vaccine against FMDV type Asia 1 in sheep, a tandem repeated multiple-epitope vaccine coupled with the ovine IgG heavy constant region gene was developed. The potential of the multiple-epitope recombinant vaccine was evaluated in guinea pigs and sheep. The results showed that the multiple-epitope recombinant vaccine could elicit high titers of neutralizing antibodies to FMDV in guinea pigs, and conferred full protection in guinea pigs against virus challenge with 103 GPID50 of FMDV. Particularly, the recombinant protein RE-oIgG was able to elicit protective levels of neutralizing antibodies in sheep. We speculated that this multiple-epitope recombinant vaccine is a promising vaccine regime which may be used for control and eradication of FMDV in the future.
Characterization of the recombinant antigens
The sequencing results showed that all recom-binant expression plasmids were constructed succes-sfully and the chimeric genes were inserted correctly in the plasmid open reading frame. The recombinant proteins, RE and RE-oIgG, were specifically expressed as a formation of inclusion bodies in E. coli, and the specific band could be clearly visualized on an 12% SDS-PAGE gel (Fig. 2). Western blotting showed that the recombinant proteins, RE and RE-ocIgG, could react specifically with the anti-FMDV positive sera (type Asia 1) (Fig. 3). Although the recombinant protein was expressed as a formation of inclusion bodies, our experiments showed that the denatured protein did not affect the immunogenicity and the function of IgG. This result was confirmed in other experiments in our laboratory .
Figure 2. The expression analysis of the recombinant proteins in E.coli cells. A: Expression of the recombinant protein RE-oIgG. B: Expression of the recombinant protein RE. Lane 1-3. standard protein marker, before and after induction with IPTG respectively
Figure 3. Western blot of recombinant proteins RE-oIgG and RE. Lane 1-4, Standard protein marker, negative control, recom-binant protein RE-oIgG and RE, respectively
Potency of the recombinant vaccine in guinea pigs
As shown in Fig. 4, the recombinant protein RE-oIgG elicited significantly higher titers of neutralizing antibodies in vaccinated guinea pigs (t-test, p < 0.01) compared to the RE recombinant. Furthermore, RE-oIgG was able to elicit a level of neutralizing antibodies as high as that induced by the traditional inactivated vaccine (t-test, p > 0.05). Neutralizing antibodies to FMDV still remained negative in a control group.
Figure 4. Profile of neutralizing antibodies to FMDV type Asia 1 in guinea pigs prior to or after vaccination. PBS, The negative control; RE, The recombinant protein RE; RE-oIgG, The fusion protein of RE and oIgG. Ⅳ, The traditionally inactivated vaccine
Inspiringly, as shown in Table 1, all guinea pigs vaccinated with either a commercial vaccine or the recombinant protein RE-oIgG were completely protected against the challenge. Although RE could not offer protection in guinea pigs, it could delay the appearance of clinical signs and reduce the severity of disease. The negative control group was completely susceptible and developed the typical vesicles at hours 24 after challenge.
Table 1. The results of the vaccinated guinea pigs challenged with FMDV type Asia Ⅰ
Lymphocytes proliferation assay
As shown in Fig. 5, higher percentages of lymphocytes proliferation were obtained from RE-oIgG, which was significantly higher than that observed for recombinant protein RE (p < 0.01, t-test). Inspiringly, the recombinant protein RE-oIgG was able to elicit the same level of lymphocytes proliferation as induced by the traditionally inactivated vaccine (p > 0.05, t-test). In addition, the negative control and non-correlated antigen BSA did not induce significant proliferation responses.
Figure 5. Profile of lymphocytes proliferation in PBMCs selected from the vaccinated guinea pigs on day 42 postvaccination. N, negative control; BSA, A non-correlated antigen control; RA, recombinant anitigen. Ⅳ, inactivated whole virus antigen; ConA, nonnspecific stimulator
Immune response in sheep
As shown in Fig. 6, the higher titers of neutralizing antibodies were elicited in sheep vaccinated with RE-oIgG. A significant difference in the titers of neutralizing antibodies was found between RE and RE-oIgG. However, the negative control PBS maintained negligible antibody responses in the trial period.