Gene-gun based DNA vaccination has been very successful in different animal models[13
]. Because the delivery route plays such a critical role in outcome of vaccination [37
], alternative delivery methods have been pursued and tested [22
]. We have used gene-gun delivery to achieve strong and specific protective and therapeutic immunity in our rabbit papillomavirus model [10
]. Our recently established HLA-A2.1 transgenic model has allowed us to target a single epitope that promotes complete protective immunity in animals following DNA vaccination [20
]. However, the gene-gun delivery system requires pure gold particles which are expensive. A low-cost vaccine delivery method is desired. Most vaccines are delivered by injection, which increases the risk of infection from HIV, hepatitis, and other serious diseases in areas where sterilization is problematic. Moreover, injecting vaccines can be a complex process, and used syringes and needles create a major waste disposal problem. Therefore, a noninvasive and effective vaccine delivery method is highly desirable for clinical use [44
]. Peptide immunization in mice has shown promising results [24
]. Mucosal delivery of peptide has been shown to be effective in the ocular HSV/rabbit model [28
]. To achieve an optimal result, we tested different adjuvants to augment the protection. Our data suggest that delivery route plays a critical role in the vaccination outcome. Mucosal peptide delivery in combination with DNA vaccination achieved the best result.
Adjuvants are added to many vaccines to increase their immunogenicity and efficacy [22
]. Many adjuvants help to trigger innate immunity or to direct Th-1 immunity in the host. While some of the adjuvants show universal effects in all species, others work in a more species-restricted manner [47
]. In this study, adjuvant (HBV core) that worked well in HHD mice was found not to be effective in rabbits. On the other hand, TT helper and PADRE adjuvants worked much better in rabbits with TT helper showing more potent response in peptide immunization. Previous studies demonstrated that strong Th1 response was generated by mucosally delivery of HSV D-1 multi epitope peptides together with CpG2007 in an ocular HSV/ rabbit model [29
]. In our study, we did not test CpG2007 with peptide alone. However, we found that stronger immunity was generated in rabbits when CpG2007 was used together with TT helper motif than with PADRE motif. Therefore, knowledge as to how to formulate more than two adjuvants for a vaccine could impact the outcome significantly. We have also shown that the delivery route used for peptide immunization is important for the outcome. The ocular HSV/rabbit model has demonstrated that mucosal delivery of lipopeptide without adjuvant stimulated strong immunity [28
] indicating the mucosal routes are optimal delivery routes for this animal. Our study with the rabbit papillomavirus model further confirmed this observation. In this study, we combined intranasal and ocular routes together for delivery. It would be interesting to find if these two mucosal delivery routes are equivalent in stimulating immune responses for peptide immunization in our animals. We demonstrated that intradermal injection of peptide emulsion did not initiate an immune response in rabbits although it did in mice. However, when we delivered CRPVE1/303–311 peptide together with TT helper peptide mucosally, significant and specific protection was found. Therefore, both adjuvant and delivery route play important roles in the outcome of immune response in vivo.
Long peptides that include CD4 and CD8 epitopes have been demonstrated to be powerful for therapeutic vaccination [50
]. Other adjuvants such as CpG-ODN and Th-fusion have also been reported to enhance the immunotherapy effect [24
]. In this study, we fused our epitope with TT helper motif or PADRE and tested whether the chimeric products could provide improved immunity in animals. Although these chimeric peptides failed to stimulate a T cell response in HHD mice and rabbits after in vitro stimulation, they did prime strong protective immunity in A2 rabbits when combined with CpG2007. The difference on the immunogenicity of these chimeric peptides in HHD mice and HLA-A2.1 transgenic rabbits might result from the constitution of these two transgenic animals. HHD mice express a chimeric form of HLA-A2.1 with alpha 1 and alpha 2 domains derived from the human HLA-A2 and alpha 3 derived from mouse H-2Db and human beta-2 microglobulin covalently attached [6
]. The transgenic rabbits, on the other hand, contain the whole human HLA-A2.1 heavy chain together with rabbit beta-2 microglobulin [19
]. The compatibility of the peptide/MHCI complex from these two transgenic animals might influence outcome in terms of immunogenicity. In other words, the A2 rabbit has a better chance to recognize these epitopes when compared with A2 mice. We have demonstrated that some epitopes that are missed by A2 mice can be detected by A2 rabbits. This further confirms the advantage of this novel transgenic rabbit model. In addition, rabbits show higher genetic homology to humans and thus, results generated from this humanized rabbits could be more relevant to the human situation. In summary, chimeric peptide is an additional avenue for peptide based vaccine in rabbits.
Gene gun-delivered DNA vaccination has been shown to provide superior immunity when compared to biojector and syringe injection in mice [18
]. Our studies have demonstrated that gene gun- delivered DNA vaccination is effective in protecting rabbits from subsequent viral infection [13
]. Three immunizations were usually administered to achieve the best protection in our previous studies [45
]. In one of our most recent studies, we showed that a single DNA immunization failed to provide any protection [10
]. In the study presented here, peptide priming followed by a single half-dose DNA booster immunization provided strong and specific protective immunity in rabbits. This suggests that the peptide immunization primed a strong immunity for DNA vaccination because no such strong protection was seen with peptide immunization alone. Because DNA vaccination by gene-gun is more expensive and time consuming than peptide immunization, the combination of peptide priming and low dose gene gun boosting could provide a satisfactory strategy. By combining both delivery methods, we achieved strong protective immunity comparable to that of DNA vaccination alone. Different laboratories have tested numerous prime/boost strategies in attempts to find the best combinations. DNA prime-protein booster has been shown to induce high titers of neutralizing antibody [56
]; A DNA priming/ vaccinia booster regime stimulated strong T cell response in vivo [57
]. In our study, we found priming with peptide and boosting with DNA could stimulate strong protective immunity in animals. Because both peptide and DNA delivery methods are noninvasive, this combined immunization strategy can be potentially adapted for future clinical trials.
In this study, we also compared two HLA-A2.1 restricted epitopes located on the mutant genome (CRPV containing HPV16E7/82–90) to see how the host immune system responded to each of them. CRPVE1/303–311 has been shown to be a very strong target for host immunity in an earlier study while HPV16E7/82–90 was found to be relatively weaker [19
]. We immunized HLA-A2.1 rabbits with either CRPVE1/303–311 or HPV16E7/82–90 and challenged them with CRPV DNA mutant containing HPV16E7/82–90. Our data further confirmed that CRPVE1/303–311 showed significantly stronger protection when compared with HPV16E7/82–90. Therefore, our HLA-A2.1 transgenic rabbit model can not only be used to screen immunogenicity of a certain epitope but can also be used to compare the strength of different epitopes.
In summary, peptides delivered mucosally can prime an immune response in rabbits that, when boosted by a low dose of gene-gun delivered DNA, can provoke strong and specific protective immunity in our HLA-A2.1 transgenic rabbit model. This noninvasive vaccination regime will be attractive for clinical application.