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.

L1 capsomeres purified from Escherichia coli represent an economic alternative to the recently launched virus-like particle (VLP)-based prophylactic vaccines against infection with human papillomavirus types 16 and 18 (HPV-16and HPV-18), which are causative agents of cervical cancer. It was recently reported that capsomeres are much less immunogenic than VLPs. Numerous modifications of the L1 protein leading to the formation of capsomeres but preventing capsid assembly have been described, such as the replacement of the cysteine residues that form capsid-stabilizing disulfide bonds or the deletion of helix 4. So far, the influence of these modifications on immunogenicity has not been thoroughly investigated. Here, we describe the purification of eight different HPV-16 L1 proteins as capsomeres from Escherichia coli. We compared them for yield, structure, and immunogenicity in mice. All L1 proteins formed almost identical pentameric structures yet differed strongly in their immunogenicity, especially regarding the humoral immune responses. Immunization of TLR4 ؊/؊ mice and DNA immunization by the same constructs confirmed that immunogenicity was independent of different degrees of contamination with copurifying immune-stimulatory molecules from E. coli. We hypothesize that immunogenicity correlates with the intrinsic ability of the capsomeres to assemble into larger particles, as only assembly-competent L1 proteins induced high antibody responses. One of the proteins (L1⌬N10) proved to be the most immunogenic, inducing antibody titers equivalent to those generated in response to VLPs. However, preassembly prior to injection did not increase immunogenicity. Our data suggest that certain L1 constructs can be used to produce highly immunogenic capsomeres in bacteria as economic alternatives to VLP-based formulations.Certain types of human papillomavirus (HPV) are the cause of cervical cancer, most frequently HPV types 16 and 18 (HPV-16 and HPV-18), which are responsible for about 50% and 20% of cases, respectively (8,15,16). Recently, two vaccines that prevent infection with HPV-16 and HPV-18 have been introduced to the market. These vaccines are based on the viral major structural protein L1, which can spontaneously self-assemble in vitro into empty virus-like particles (VLPs) that resemble the native virions in size and shape. VLPs have been shown to be highly immunogenic, as they can induce high titers of neutralizing antibodies (29,30). HPV virions and VLPs consist of 72 L1 pentamers, also called capsomeres, which are arranged in an icosahedral Tϭ7 particle lattice with a diameter of 55 nm. Cryo-electron microscopic analysis has revealed the presence of 60 hexavalent and 12 pentavalent capsomeres (4).Capsid assembly has been reported to be optimal at low pH (pH 5.4) and high ionic strength, whereas both high pH (pH 8.2) and the presence of reducing agents favor disassembly into capsomeres, the latter because the viral particles are stabilized by intercapsomeric disulfide bonds between two conserved cysteine resi...

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