Capsomeres are considered to be an alternative to viruslike particle (VLP)-based vaccines as they can be produced in prokaryotic expression systems. So far, no detailed side-by-side comparison of VLPs and capsomeres has been performed. In the present study, we immunized mice with insect cell-derived human papillomavirus type 16 VLPs and capsomeres. VLPs induced consistently higher antibody titers than capsomeres but the two forms induced similar CD8 T-cell responses after subcutaneous, intranasal, and oral immunization, and at least 20 to 40 times more L1 in the form of capsomeres than in the form of VLPs was needed to achieve comparable antibody responses. These results were confirmed by DNA immunization. The lower immunogenicity of capsomeres was independent of the isotype switch, as it was also observed for the early immunoglobulin M responses. Although there were differences in the display of surface epitopes between the L1 particles, these did not contribute significantly to the differences in the immune responses. capsomeres were less immunogenic than VLPs in Toll-like receptor 4 (TLR4)-deficient mice, suggesting that the lower immunogenicity is not due to a failure of capsomeres to trigger TLR4. We observed better correlation between antibody results from enzyme-linked immunosorbent assays and neutralization assays for sera from VLPimmunized mice than for sera from capsomere-immunized mice, suggesting qualitative differences between VLPs and capsomeres. We also showed that the lower immunogenicity of capsomeres could be compensated by the use of an adjuvant system containing MPL. Taken together, these results suggest that, presumably because of the lower degree of complexity of the antigen organization, capsomeres are significantly less immunogenic than VLPs with respect to the humoral immune response and that this characteristic should be considered in the design of putative capsomere-based prophylactic vaccines.

Certain types of human papillomaviruses (HPV) are causatively associated with cervical carcinoma, the second most common cancer in women worldwide. Due to limitations in the availability of currently used virus-like particle (VLP)-based vaccines against HPV to women of developing countries, where most cases of cervical cancer occur, the development of a cost-effective second-generation vaccine is a necessity. Capsomeres have recently been demonstrated to be highly immunogenic and to have a number of advantages as a potential cost-effective alternative to VLP-based HPV vaccines. We have expressed a mutated HPV-16 L1 (L1_2xCysM) gene that retained the ability to assemble L1 protein to capsomeres in tobacco chloroplasts. The recombinant protein yielded up to 1.5% of total soluble protein. The assembly of capsomeres was examined and verified by cesium chloride density gradient centrifugation and sucrose sedimentation analysis. An antigen capture enzyme-linked immunosorbent assay confirmed the formation of capsomeres by using a conformation-specific monoclonal antibody which recognized the conformational epitopes. Transplastomic tobacco plants exhibited normal growth and morphology, but all such lines showed male sterility in the T₀, T₁ and T₂ generations. Taken together, these results indicate the possibility of producing a low-cost capsomere-based vaccine by plastids.

Human papillomaviruses have been recognized as the causative agent of anogenital cancer. In 2006, a commercial vaccine based on virus-like particles composed of the L1 major capsid protein of the papillomaviruses has been available. This vaccine induces virus-neutralizing antibody responses upon parenteral injection. Here we investigated the oral immunogenicity of different assembly forms of HPV 16 L1, that is: T7-VLPs, T1 particles and capsomeres. Our results show that all three assembly forms induce humoral and cellular immune responses after oral vaccination of mice. The anti-L1 antibodies were conformation-specific and showed neutralizing activity in a pseudovirion-based assay. We also investigated if adjuvants have an influence on the oral immunogenicity of the different L1 forms. For saponins we observed a significant toxicity if applied orally. Co-administration of either CpG DNA or Escherichia coli heat-labile enterotoxin LT(R192G) had no apparent enhancing effect on the production of anti-L1 antibodies. More pronounced was the effect of CpG administration on the long-term immunity as we observed a significantly stronger recall response 244 days after the first vaccination. Compared to capsomeres, VLPs induced stronger humoral immune responses while the CTL responses were induced at comparable levels. Finally, we were also able to induce neutralizing antibodies and L1-specific cytotoxic T-lymphocytes after oral administration of crude extracts of L1-expressing insect cells. In conclusion, all three assembly forms of the L1 protein are immunogenic when administered orally.