Vaccines based on synthetic peptides offer several advantages when compared with conventional vaccines based on attenuated or inactivated microorganisms, particularly regarding safety, thermal stability and ease of production. Despite the potential advantages of this approach, the development of successful peptide vaccines has been limited mainly by difficulties associated with in vivo stability, poor immunogenicity of linear peptides, and by the MHC polymorphism of the host species [19, 20]. However, recent advances on the requirements for induction and maintenance of immune responses, as well as on the pharmacokinetics of peptides, have provided new strategies to enhance both, peptide immunogenicity and stability, which are leading to the return of peptide based technologies to the forefront of vaccine design [21, 22].
The use of one of such approach, the multimerization of peptides by dendrimeric constructs, has recently allowed us to report solid protection against FMDV challenge . The peptide dendrimer used B4T, contained four copies of an immunodominant B-cell epitope, named site-A, branched out a selected Th epitope of FMDV broadly recognized by T-cells from outbred pigs . This dendrimeric construction specifically induced high titers of FMDV-neutralizing antibodies, activated T-cells, release of IFNγ and a potent anti-FMDV IgA response . However, whether the immunogenicity and protection elicited by this dendrimeric peptide was due to incorporation of a relevant T-cell epitope and/or to the presence of a dendrimeric structure containing repeated B-cell epitopes remained to be addressed.
As part of the understanding of the determinants of the protection afforded by peptide B4T, we have analyzed the immune response and the protection conferred in pigs by linear peptides containing the B- and the T-cell epitopes displayed in the dendrimeric peptide.
As shown in Figure 1, immunization of pigs with linear peptides (B or TB) was not able to confer full protection as that reached using peptide B4T. However, significant differences on the maximum clinical scores were observed between the two groups of immunized animals (Figure 1B), being the values lower for animals immunized with peptide TB (group 2) (Figure 1B). These results correlate with the significant reduction of FMDV RNA detection after viral challenge in blood, nasal and pharingeal swabs in animals immunized with peptide TB (Table 2). Therefore, vaccination with a linear peptide displaying a specific T-cell epitope, can reduce virus excretion in pigs, which might contribute to reduced transmission of FMDV in the field, even if the pigs are not fully protected. Similar improved control of FMDV replication in mice has been achieved by DNA vaccines encoding one B and two T cell epitopes . However, to reach the same protection results in pigs, the B and T epitopes encoded by the DNA vaccine requires to be tagerted to Class-II swine leukocyte antigens .
The protection against FMDV conferred by conventional whole virus vaccine broadly correlates with the virus neutralizing antibody levels present in serum in cattle and, to a lesser extent, in pigs [7, 25, 26]. Our results show a lack of correlation between neutralizing antibody levels and protection, as the levels of virus neutralizing antibodies induced by both peptides B and TB were similar (around 2 log10), and even similar to those previously reported for peptide B4T . A similar lack of correlation has been reported in animals immunized with vaccines different from those based on inactivated FMDV .
Antibodies may neutralize FMDV by different mechanisms . Thus, the lower protective effect of the antibodies induced by linear peptides (B or TB) could be due to differences in either the mechanisms of neutralization they exert (i.e. direct neutralization of receptor binding, Fc-γ-receptor-dependent viral clearance, complement-mediated lysis, antibody-dependent cytotoxicity) or to affinity differences. To assess other humoral factors potentially contributing to limited protection conferred by the linear peptides, the spectrum of immunoglobulin subclasses induced by peptides T and TB was analysed. Our results show that linear peptides were able to induce significant titres of specific IgG1, IgG2 and IgA at day 39 (18 days after boost). The magnitude of these isotype responses was similar after B and TB immunization, suggesting that the inclusion of the T-cell epitope has no influence on the antibody isotype switch. However, the IgA titres induced by both linear peptides are between 1 and 2 orders of magnitude lower than those reported previously for dendrimer vaccination . Therefore, IgA induction seems to be dependent on the branched structure and not only on the presence of the T-cell epitope. Induction of IgA responses has been recently correlated with complete protection against FMDV challenge of pigs immunized with a highly concentrated, inactivated vaccine . All together, this result indicates that the lack of full protection conferred by linear peptides is, a large extend due to the inefficient induction of specific IgA.
The highly repetitive array of epitopes on viral surfaces allows efficient crosslinking of antigen-specific immunoglobulins on B cells, constituting a strong activation signal that may even overcome B cell tolerance . In contrast to linear monomeric peptides, dendrimers display a repetitive configuration, which could allow direct activation of B cells, leading to a rapid B cell proliferation and production of antibodies. Likewise the triggered B cells are able to activate T helper cells, leading to cytokine secretion that can result in a suitable environment for generation of B cell memory. Therefore the activation of these early mechanisms by dendrimeric peptides, but not by monomeric peptides, can be relevant for the induction of protective immune responses against FMDV. Further studies are required to test this hypothesis, by analyzing the interaction between dendrimeric versus monomeric peptides with different antigen presenting cells essential in the early specific responses (B cells, Dendritic cells, γδ T lymphocytes, etc.).
Besides the direct neutralization of viral infectivity, other mechanisms of viral clearance may operate in vivo, as T-cell activation and the balance of cytokines they release. Immunization with peptides B or TB elicited T cells that consistently proliferate when stimulated with the peptides (Figure 2). However, the lymphoproliferative response to FMDV was significantly higher for TB-immunized pigs (Figure 2). These results show that the inclusion of the specific T-cell epitope in the peptide formulation allows priming of T cells that can recognize more efficiently the viral epitopes presented in the context of a subsequent virus encounter.
Upon in vitro stimulation, primed T cells from two pigs immunized with the linear peptide TB released IFNγ, which was not detected in any of the animals immunized with peptide B. These results indicate that the inclusion of the FMDV T-cell epitope enhances the IFNγ release. However, since the IFNγ release is higher after dendrimer vaccination , we can assume that optimal IFNγ production requires the inclusion of an efficient T-cell epitope, as well as a suitable configuration. IFNγ is a major activator of macrophages, enhancing their antimicrobial activity and their capacity for processing and presenting antigens to T lymphocytes . It has been reported that IFNγ stimulates MHC expression in antigen-presenting cells and that efficiently inhibits FMDV replication . Therefore, these results suggest that the better clinical protection conferred by the TB peptide compared to B peptide, is mostly due to the induction of a more efficient lymphoproliferative response and IFNγ release.
Taken together, our results show that the incorporation of a specific T-cell epitope in the peptide formulation seems to be necessary but not sufficient to enhance the protective cellular response to the protective levels achieved by a dendrimeric peptide. Thus, even though further work is required to understand the details of mechanisms leading to the solid protection conferred by dendrimeric peptides, our results suggest that branched configuration along with the inclusion of a T-cell epitope are essential to induce protective immune responses characterized mainly by high specific IgA responses and production of IFNγ.