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.

Toll-like receptor (TLR) ligands are being considered as adjuvants for the induction of antigen-specific immune responses, as in the design of vaccines. Polyriboinosinic-polyribocytoidylic acid (poly I:C), a synthetic double-stranded RNA (dsRNA), is recognized by TLR3 and other intracellular receptors. Poly ICLC is a poly I:C analogue, which has been stabilized against the serum nucleases that are present in the plasma of primates. Poly I:C12U, another analogue, is less toxic but also less stable in vivo than poly I:C, and TLR3 is essential for its recognition. To study the effects of these compounds on the induction of protein-specific immune responses in an animal model relevant to humans, rhesus macaques were immunized subcutaneously (s.c.) with keyhole limpet hemocyanin (KLH) or human papillomavirus (HPV)16 capsomeres with or without dsRNA or a control adjuvant, the TLR9 ligand CpG-C. All dsRNA compounds served as adjuvants for KLH-specific cellular immune responses, with the highest proliferative responses being observed with 2 mg/animal poly ICLC (p = 0.002) or 6 mg/animal poly I:C12U (p = 0.001) when compared with immunization with KLH alone. Notably, poly ICLC—but not CpG-C given at the same dose—also helped to induce HPV16-specific Th1 immune responses while both adjuvants supported the induction of strong anti-HPV16 L1 antibody responses as determined by ELISA and neutralization assay. In contrast, control animals injected with HPV16 capsomeres alone did not develop substantial HPV16-specific immune responses. Injection of dsRNA led to increased numbers of cells producing the T cell–activating chemokines CXCL9 and CXCL10 as detected by in situ hybridization in draining lymph nodes 18 hours after injections, and to increased serum levels of CXCL10 (p = 0.01). This was paralleled by the reduced production of the homeostatic T cell–attracting chemokine CCL21. Thus, synthetic dsRNAs induce an innate chemokine response and act as adjuvants for virus-specific Th1 and humoral immune responses in nonhuman primates.

Since their discovery as a tool for gene transfer, vectors derived from the adeno-associated virus (AAV) have been used for gene therapy applications and attracted scientist to this field for their exceptional properties of efficiency of in vivo gene transfer and the level and duration of transgene expression. For many years, AAVs have been considered as low immunogenic vectors due to their ability to induce long-term expression of non-self-proteins in contrast to what has been observed with other viral vectors, such as adenovirus, for which strong immune responses against the same transgene products were documented. The perceived low immunogenicity likely explains why the use of AAV vectors for vaccination was not seriously considered before the early 2000s. Indeed, while analyses conducted using a variety of transgenes and animal species slowly changed the vision of immunological properties of AAVs, an increasing number of studies were also performed in the field of vaccination. Even if the comparison with other modes of vaccination was not systemically performed, the analyses conducted so far in the field of active immunotherapy strongly suggest that AAVs possess some interesting features to be used as tools to produce an efficient and sustained antibody response. In addition, recent studies also highlighted the potential of AAVs for passive immunotherapy. This review summarizes the main studies conducted to evaluate the potential of AAV vectors for vaccination against infectious agents and discusses their advantages and drawbacks. Altogether, the variety of studies conducted in this field contributes to the understanding of the immunological properties of this versatile virus and to the definition of its possible future applications.

The human papillomavirus (HPV) minor capsid protein L2 is a promising candidate for a broadly protective HPV vaccine yet the titers obtained in most experimental systems are rather low. Here we examine the potential of empty AAV2 particles (AAVLPs), assembled from VP3 alone, for display of L2 epitopes to enhance their immunogenicity. Insertion of a neutralizing epitope (amino acids 17–36) from L2 of HPV16 and HPV31 into VP3 at positions 587 and 453, respectively, permitted assembly into empty AAV particles (AAVLP(HPV16/31L2)). Intramuscularly vaccination of mice and rabbits with AAVLP(HPV16/31L2)s in montanide adjuvant, induced high titers of HPV16 L2 antibodies as measured by ELISA. Sera obtained from animals vaccinated with the AAVLP(HPV16/31L2)s neutralized infections with several HPV types in a pseudovirion infection assay. Lyophilized AAVLP(HPV16/31L2) particles retained their immunogenicity upon reconstitution. Interestingly, vaccination of animals that were pre-immunized with AAV2 - simulating the high prevalence of AAV2 antibodies in the population - even increased cross neutralization against HPV31, 45 and 58 types. Finally, passive transfer of rabbit antisera directed against AAVLP(HPV16/31L2)s protected naïve mice from vaginal challenge with HPV16 pseudovirions. In conclusion, AAVLP(HPV16/31L2) particles have the potential as a broadly protective vaccine candidate regardless of prior exposure to AAV.

b Adeno-associated virus (AAV) capsid assembly requires expression of the assembly-activating protein (AAP) together with capsid proteins VP1, VP2, and VP3. AAP is encoded by an alternative open reading frame of the cap gene. Sequence analysis and site-directed mutagenesis revealed that AAP contains two hydrophobic domains in the N-terminal part of the molecule that are essential for its assembly-promoting activity. Mutation of these sequences reduced the interaction of AAP with the capsid proteins. Deletions and a point mutation in the capsid protein C terminus also abolished capsid assembly and strongly reduced the interaction with AAP. Interpretation of these observations on a structural basis suggests an interaction of AAP with the VP C terminus, which forms the capsid protein interface at the 2-fold symmetry axis. This interpretation is supported by a decrease in the interaction of monoclonal antibody B1 with VP3 under nondenaturing conditions in the presence of AAP, indicative of steric hindrance of B1 binding to its C-terminal epitope by AAP. In addition, AAP forms high-molecular-weight oligomers and changes the conformation of nonassembled VP molecules as detected by conformation-sensitive monoclonal antibodies A20 and C37. Combined, these observations suggest a possible scaffolding activity of AAP in the AAV capsid assembly reaction.A deno-associated virus (AAV) is a nonenveloped singlestranded DNA virus of the Parvoviridae family (15). To date, 13 distinct human or nonhuman primate AAV serotypes have been described and numerous recombinant species have been isolated (10). The AAV assembly pathway proposed by Myers and Carter suggests the rapid formation of empty capsids into which the single-stranded genome is inserted in a slow reaction (16). While the process of genome replication has been elucidated in great detail (15,22), molecular events underlying capsid formation and genome encapsidation are less well understood (12).Capsid assembly occurs in the nuclei of infected cells, where capsids are first detectable in the nucleoli but are spread throughout the nucleus at later stages of infection (23). Expression of the cap gene is sufficient for capsid formation. Besides the three capsid proteins, VP1, VP2, and VP3, known to be expressed from open reading frame 1 (ORF1), the cap gene encodes an assembly factor, the assembly-activating protein (AAP), from a second ORF, ORF2 (21). AAP is essential for capsid assembly. It targets newly synthesized capsid proteins to the nucleolus and promotes capsid formation in a still unknown way. AAPs of some, but not all, AAV serotypes can cross-complement each other in the assembly reaction (20). AAP is a rather unstable protein but becomes stabilized upon the coexpression of capsid protein VP3. However, this stabilizing effect depends very much on the serotype of the coexpressed capsid protein, indicating specific AAP-VP protein interactions (20). AAP amino acid sequence alignment of serotypes 1 to 13 shows a high degree of homology. Only AAPs from serotypes 4, 5, 1...

Cervical cancer is the second most common cancer in women worldwide. Persistent high-risk human papillomavirus (HPV) infection has been identified as the causative event for the development of this type of cancer. Recombinant adeno-associated viruses (rAAVs) are currently being developed and evaluated as vaccine vector. In previous work, we demonstrated that rAAVs administered intranasally in mice induced high titers and long-lasting neutralizing antibodies against HPV type 16 (HPV16). To extend this approach to a more human-related species, we immunized rhesus macaques (Macaca mulatta) with AAVs expressing an HPV16 L1 protein using rAAV5 and 9 vectors in an intranasal prophylactic setting. An rAAV5-L1 vector followed by a boost with rAAV9-L1 induced higher titers of L1-specific serum antibodies than a single rAAV5-L1 immunization. L1-specific antibodies elicited by AAV9 vector neutralized HPV16 pseudovirions and persisted for at least 7 months post immunization. Interestingly, nasal application of rAAV9 was immunogenic even in the presence of high AAV9 antibody titers, allowing reimmunization with the same serotype without prevention of the transgene expression. Two of six animals did not respond to AAV-mediated intranasal vaccination, although they were not tolerant, as both developed antibodies after intramuscular vaccination with HPV16 virus-like particles. These data clearly show the efficacy of an intranasal immunization using rAAV9-L1 vectors without the need of an adjuvant. We conclude from our results that rAAV9 vector is a promising candidate for a noninvasive nasal vaccination strategy.

True hermaphroditism (TH) is an unusual form of sex reversal, characterized by the development of testicular and ovarian tissue in the same subject. Approximately 60% of the patients have a 46,XX karyotype, 33% are mosaics with a second cell line containing a Y chromosome, while the remaining 7% are 46,XY. Molecular analyses have demonstrated that SRY is present in only 10% of TH with a 46,XX karyotype; therefore, in the remaining 90%, mutations at unknown X-linked or autosomal sex determining loci have been proposed as factors responsible for testicular development. True hermaphroditism presents considerable genetic heterogeneity with several molecular anomalies leading to the dual gonadal development as SRY point mutations or SRY hidden gonadal mosaicism. In order to identify genetic defects associated with subjects with the disease, we performed molecular analyses of the SRY gene in DNA from blood leukocytes and gonadal tissue in 12 true hermaphrodites with different karyotypes. Our results using PCR and FISH analyses reveal the presence of hidden mosaicism for SRY or other Y sequences in some patients with XX true hermaphroditism and confirms that mosaicism for SRY limited to the gonads is an alternative mechanism for testicular development in 46,XX true hermaphrodites.

BackgroundHuman Papillomavirus (HPV) E2 plays several important roles in the viral cycle, including the transcriptional regulation of the oncogenes E6 and E7, the regulation of the viral genome replication by its association with E1 helicase and participates in the viral genome segregation during mitosis by its association with the cellular protein Brd4. It has been shown that E2 protein can regulate negative or positively the activity of several cellular promoters, although the precise mechanism of this regulation is uncertain. In this work we constructed a recombinant adenoviral vector to overexpress HPV16 E2 and evaluated the global pattern of biological processes regulated by E2 using microarrays expression analysis.ResultsThe gene expression profile was strongly modified in cells expressing HPV16 E2, finding 1048 down-regulated genes, and 581 up-regulated. The main cellular pathway modified was WNT since we found 28 genes down-regulated and 15 up-regulated. Interestingly, this pathway is a convergence point for regulating the expression of genes involved in several cellular processes, including apoptosis, proliferation and cell differentiation; MYCN, JAG1 and MAPK13 genes were selected to validate by RT-qPCR the microarray data as these genes in an altered level of expression, modify very important cellular processes. Additionally, we found that a large number of genes from pathways such as PDGF, angiogenesis and cytokines and chemokines mediated inflammation, were also modified in their expression.ConclusionsOur results demonstrate that HPV16 E2 has regulatory effects on cellular gene expression in HPV negative cells, independent of the other HPV proteins, and the gene profile observed indicates that these effects could be mediated by interactions with cellular proteins. The cellular processes affected suggest that E2 expression leads to the cells in to a convenient environment for a replicative cycle of the virus.