Our previous studies showed that the norovirus P particle is easily produced, extremely stable, and highly immunogenic and can be used as a subunit vaccine against noroviruses (30
). In this study, we further demonstrated that the P particle can also be used as a novel vaccine platform for immune enhancement of a foreign antigen. We have shown (i) that the surface loops of the P particle are excellent sites for foreign antigen insertion without affecting the formation and production of the P particle, (ii) that the P particle tolerates a foreign antigen in a size up to at least 159 amino acids, and (iii) enhanced immune responses to inserted antigens demonstrated by both in vitro
neutralization and in vivo
protection experiments. The P particle-VP8 chimera served as a promising dual vaccine against both rotavirus and norovirus. Thus, the simple procedure to generate chimeric particles and the multiple surface loops with potential for multipolyvalent foreign insertion make the P particle an attractive vaccine platform for antigen presentation for infectious diseases or other medical conditions that would benefit from an efficient vaccine.
A primary goal of this study was to examine whether the P particle can enhance the immunogenicity of a small polypeptide antigen that generally has a low level of immunogenicity. We first studied this issue by using the His tag as a model and obtained excellent results. Two major factors may be responsible for the observed immune enhancement, namely, multicopy number and surface exposure of the inserted antigens. The P particle is composed of 24 copies of P monomers, which may explain its enhanced immune responses compared with a free His tag peptide and the His tag fused to a P dimer (Fig. ). Thus, the P particle may act as an adjuvant by its large size (830 kDa) and proper presentation of a foreign antigen that otherwise has low immunogenicity. The increased multicopy number of an antigen per particle is another feature that may explain the increased immune responses.
In addition to inserting the His tag peptide, we have successfully inserted a number of other small peptides into the P particles, including the T cell epitope of murine cytomegalovirus (9 aa), the Epi8 epitope of Pseudomonas aeruginosa
(14 aa), the T cell epitope of murine rotavirus VP6 (14 aa), and the M2 extracellular epitope of influenza virus (M2e, 23 aa) (M. Tan and X. Jiang, unpublished). Significantly increased immune response and protection via the M2e antigen through the P particle platform has also been demonstrated in a mouse model (M. Xia, M. Tan, and X. Jiang, unpublished). These data suggested that the P particle may be readily useful for immune enhancement with a wide variety of small polypeptide antigens. The successful insertion of different rotavirus VP8s (159 aa) and the green fluorescent protein (GFP; 238 aa) into P particles (this report; M. Tan and X. Jiang, unpublished) has greatly extended the application of the P particle platform for a wider range of larger foreign antigens. The rotavirus VP8 is a spike protein on the viral capsid and is believed to be important for rotavirus infectivity. It is also one of two rotavirus antigens that induce neutralizing antibodies. A number of neutralizing epitopes have been identified on the VP8 protein (11
). The success of the P particle-VP8 chimera in inducing a neutralizing antibody response and providing protection against rotavirus shedding in mice suggested that these epitopes have been preserved on the P particle carrier.
Future study to develop the P particle-VP8 chimera into a useful vaccine against rotaviruses is warranted. While the two recently introduced rotavirus vaccines are highly effective, new-generation vaccines may be needed for potentially new emerging viruses. Noninfectious subunit vaccines do not have a risk of reversion to virulent strains. The development of a VLP vaccine for rotaviruses has been proposed for years. However, a rotavirus VLP vaccine faces the challenges of low-efficiency expression and high cost of manufacturing because of the requirement for cotransfection of several capsid genes to the baculovirus host. In contrast, generation of the P particle-VP8 chimera requires only a routine E. coli-based cloning and expression procedure, which is highly efficient and low in cost. In addition, cross-neutralization epitopes on VP8 have been described. We have shown in this study that immunization of mice with a chimeric P particle containing VP8 from a P virus conferred cross-neutralization of a P rotavirus (Fig. ). A cocktail vaccine containing a minimal number of P types may also be cost-effective.
The ability of antibodies induced by the P particle-VP8 chimera to block the binding of norovirus VLPs to HBGAs is unexpected. As shown in Fig. , the distal surface of the P dimers, including the HBGA binding interfaces (3
) of the P particle, is most likely to be covered by the inserted VP8s. This leads to the loss of the capability of the chimera to bind to HBGA receptors (data not shown). One possibility for the continued blocking ability seen is that the epitopes of the HBGA binding interfaces of the P particle-VP8 chimera are still accessible for host immune response even though they are covered by the inserted VP8 antigens. Alternatively, the observed carbohydrate blockage may be due to an antibody binding in the vicinity of the carbohydrate binding site. No matter which mechanism is involved, the ability of the chimera-induced antibody to block binding of norovirus VLPs to HBGAs adds additional value to the P particle platform. The concept of the P particle-VP8 chimera as a dual vaccine against both norovirus and rotavirus may be particularly valuable for specific populations at risk for both infections.
Although only data on loop 2 of the P particle (Fig. ) are reported here, success with various antigen insertions in the other two loops has been demonstrated (L. Y. Wang, M. Tan, and X. Jiang, unpublished). The availability of three surface loops per P monomer provides opportunities for versatile vaccine designs. For example, to increase immune responses, the same epitope or antigen can be inserted in all three loops to reach 72 copies of the antigen per particle. An even higher copy number can also be generated by insertion of tandem repeats of individual antigens. Alternatively, different antigens can be inserted into each of the three loops, resulting in a multivalent vaccine against different pathogens. Additional vaccine templates may also be generated by insertion of functional tags for different purposes. For example, insertion of a His tag would further simplify the purification procedure. Special ligands or signal molecules may also be used to stimulate immune responses by targeting the vaccine to special organs, tissues, or cells of the host. A potential drawback of using a norovirus particle vaccine platform could be the preexisting antibodies to noroviruses in humans. Thus, further studies are required to clarify this issue.
A further application of the P particle platform is for production of antibodies against small peptides for research and diagnostic uses. A variety of disease biomarkers (mainly peptide epitopes) has been identified, and antibodies against these biomarkers are important for diagnostic purposes. Small peptides can be easily inserted into a loop of the P particle by a simple DNA cloning procedure. We have developed convenient P particle vectors containing cloning cassettes that would further facilitate the process. Following expression of the recombinant chimeric P particle in bacteria, high-titer antibodies specific to the inserted peptide antigens can be made by immunization of laboratory animals with the chimeric P particle according to the established procedure in this study. Antibody production in this way will avoid the costly steps of peptide synthesis and conjugation of the peptide to a macromolecule such as keyhole limpet hemocyanin (KLH) for immune enhancement. Since the norovirus P domain has a unique sequence that shares no homology with any other proteins, cross-reactivity with other proteins should not be a concern. Thus, the P particle vaccine platform can be used as a convenient tool for antibody production in many areas of biomedical research.