Adeno-associated virus type 2 (AAV2) capsid assembly requires the expression of a virally encoded assemblyactivating protein (AAP). By providing AAP together with the capsid protein VP3, capsids are formed that are composed of VP3 only. Electron cryomicroscopy analysis of assembled VP3-only capsids revealed all characteristics of the wild-type AAV2 capsids. However, in contrast to capsids assembled from VP1, VP2, and VP3, the pores of VP3-only capsids were more restricted at the inside of the 5-fold symmetry axes, and globules could not be detected below the 2-fold symmetry axes. By comparing the capsid assembly of several AAV serotypes with AAP protein from AAV2 (AAP-2), we show that AAP-2 is able to efficiently stimulate capsid formation of VP3 derived from several serotypes, as demonstrated for AAV1, AAV2, AAV8, and AAV9. Capsid formation, by coexpressing AAV1-, AAV2-, or AAV5-VP3 with AAP-1, AAP-2, or AAP-5 revealed the ability of AAP-1 and AAP-2 to complement each other in AAV1 and AAV2 assembly, whereas for AAV5 assembly more specific conditions are required. Sequence alignment of predicted AAP proteins from the known AAV serotypes indicates a high degree of homology of all serotypes to AAP-2 with some divergence for AAP-4, AAP-5, AAP-11, and AAP-12. Immunolocalization of assembled capsids from different serotypes confirmed the preferred nucleolar localization of capsids, as observed for AAV2; however, AAV8 and AAV9 capsids could also be detected throughout the nucleus. Taken together, the data show that AAV capsid assembly of different AAV serotypes also requires the assistance of AAP proteins.

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...