Standard protocols for the generation of adenoassociated virus type 2 (AAV-2)-based vectors for human gene therapy applications require cotransfection of cells with a recombinant AAV (rAAV) vector plasmid and a packaging plasmid that provides the AAV rep and cap genes. The transfected cells must also be overinfected with a helper virus, e.g., adenovirus (Ad), which delivers multiple helper functions necessary for rAAV production. Therefore, rAAV stocks produced using these protocols are contaminated with helper adenovirus. The generation of a novel packaging/helper plasmid, pDG, containing all AAV and Ad functions required for amplification and packaging of AAV vector plasmids, is described here. Cotransfection of cells with pDG and an AAV vector plasmid was sufficient for production of infectious rAAV, resulting in helper virus-free rAAV stocks. The rAAV titers obtained using pDG as packaging plasmid were up to 10-fold higher than those achieved using conventional protocols for rAAV production. Replacement of the AAV-2 p5 promoter by an MMTV-LTR promoter in pDG led to reduced expression of Rep78/68; however, expression of the VP proteins was significantly increased compared with VP levels from standard packaging plasmids. Immunofluorescence analyses showed that the strong accumulation of VP proteins in pDG-transfected cells resulted in enhanced AAV capsid assembly, which is limiting for efficient rAAV production. Furthermore, using a monoclonal antibody highly specific for AAV-2 capsids (A20), an rAAV affinity purification procedure protocol was established. The application of the tools described here led to a significant improvement in recombinant AAV vector production and purification.

Infection of cells with adeno-associated virus (AAV) type 2 (AAV-2) is mediated by binding to heparan sulfate proteoglycan and can be competed by heparin. Mutational analysis of AAV-2 capsid proteins showed that a group of basic amino acids (arginines 484, 487, 585, and 588 and lysine 532) contribute to heparin and HeLa cell binding. These amino acids are positioned in three clusters at the threefold spike region of the AAV-2 capsid. According to the recently resolved atomic structure for AAV-2, arginines 484 and 487 and lysine 532 on one site and arginines 585 and 588 on the other site belong to different capsid protein subunits. These data suggest that the formation of the heparin-binding motifs depends on the correct assembly of VP trimers or even of capsids. In contrast, arginine 475, which also strongly reduces heparin binding as well as viral infectivity upon mutation to alanine, is located inside the capsid structure at the border of adjacent VP subunits and most likely influences heparin binding indirectly by disturbing correct subunit assembly. Computer simulation of heparin docking to the AAV-2 capsid suggests that heparin associates with the three basic clusters along a channel-like cavity flanked by the basic amino acids. With few exceptions, mutant infectivities correlated with their heparin-and cell-binding properties. The tissue distribution in mice of recombinant AAV-2 mutated in R484 and R585 indicated markedly reduced infection of the liver, compared to infection with wild-type recombinant AAV, but continued infection of the heart. These results suggest that although heparin binding influences the infectivity of AAV-2, it seems not to be necessary.

We demonstrate the rapid and reliable quantification of the recombinant, but not in the wild-type AAV-2 prepphysical AAV-2 (adeno-associated virus type 2) particles arations. Moreover, additional expression of VP proteins via a novel ELISA based on a monoclonal antibody which during rAAV production was found to result in an excessive selectively recognizes assembled AAV-2 capsids. Titration capsid formation, whilst yielding only minor increases in of a variety of recombinant AAV-2 (rAAV) preparations DNA-containing or transducing rAAV particles. We conrevealed that at least 80% of all particles were empty, comclude that encapsidation of viral genomes rather than cappared with a maximum of 50% in wild-type AAV-2 stocks, sid assembly can be limiting for rAAV production, provided indicating that the recombinant genomes were less that a critical level of VP expression is maintained. The efficiently encapsidated. This finding was confirmed upon feasibility of quantifying AAV-2 capsid numbers via the titration of CsCl gradient fractions from recombinant and ELISA allows determination of physical to DNA-containing wild-type AAV-2 stocks. ELISA-based measurement of or infectious particle ratios. These are important paracapsid numbers revealed a large number of physical parmeters which should help to optimize and standardize the ticles with low densities corresponding to empty capsids in production and application of recombinant AAV-2.Keywords: AAV-2; recombinant AAV-2; AAV-2 titration; AAV-2 ELISA Gene therapy vectors derived from the human parvovirus AAV-2 (adeno-associated virus type 2) have gained attention owing to a unique combination of attractive features. Wild-type AAV-2 is nonpathogenic in humans and naturally defective, requiring coinfection with a helpervirus (eg adenovirus) for a productive infection. 1 Recombinant AAV-2 can infect both dividing and nondividing cells in vitro and in vivo [2][3][4][5][6] and have the potential for sitespecific integration into chromosome 19.7-9 Long-term expression of heterologous genes transduced by rAAV has been observed in a variety of human cells and tissues, such as muscle, 10,11 lung, 12 central nervous system, 13-15 retina 16 and liver. 17 An important prerequisite for the testing of AAV-2 vectors in preclinical and clinical studies is the accurate and reliable titration of the recombinant virus particles. Precise information on rAAV titers is not only crucial for the careful planning and execution of such studies, but also for comparing results amongst laboratories. In brief, presently available methods for rAAV titration can be divided into biological and physical assays. Biological assays rely on infection of cultured cells followed by events that depend on the biological functionality of the 18-20 These two types of assay yield titers of infectious or transducing particles, respectively. In contrast, physical methods are independent of biological functions of the recombinant viruses. Typically, viral DNA is extracted from the rAAV particles via enzymatic digestion ...

Using immunofluorescence and in situ hybridization techniques, we studied the intracellular localization of adeno-associated virus type 2 (AAV-2) Rep proteins, VP proteins, and DNA during the course of an AAV-2/ adenovirus type 2 coinfection. In an early stage, the Rep proteins showed a punctate distribution pattern over the nuclei of infected cells, reminiscent of replication foci. At this stage, no capsid proteins were detectable. At later stages, the Rep proteins were distributed more homogeneously over the nuclear interior and finally became redistributed into clusters slightly enriched at the nuclear periphery. During an intermediate stage, they also appeared at an interior part of the nucleolus for a short period, whereas most of the time the nucleoli were Rep negative. AAV-2 DNA colocalized with the Rep proteins. All three capsid proteins were strongly enriched in the nucleolus in a transient stage of infection, when the Rep proteins homogeneously filled the nucleoplasm. Thereafter, they became distributed over the whole nucleus and colocalized in nucleoplasmic clusters with the Rep proteins and AAV-2 DNA. While VP1 and VP2 strongly accumulated in the nucleus, VP3 was almost equally distributed between the nucleus and cytoplasm. Capsids, visualized by a conformationspecific antibody, were first detectable in the nucleoli and then spread over the whole nucleoplasm. This suggests that nucleolar components are involved in initiation of capsid assembly whereas DNA packaging occurs in the nucleoplasm. Expression of a transfected full-length AAV-2 genome followed by adenovirus infection showed all stages of an AAV-2/adenovirus coinfection, whereas after expression of the cap gene alone, capsids were restricted to the nucleoli and did not follow the nuclear redistribution observed in the presence of the whole AAV-2 genome. Coexpression of Rep proteins released the restriction of capsids to the nucleolus, suggesting that the Rep proteins are involved in nuclear redistribution of AAV capsids during viral infection. Capsid formation was dependent on the concentration of expressed capsid protein.

Adeno-associated viruses (AAV) have been developed and evaluated as recombinant vectors for gene therapy in many preclinical studies, as well as in clinical trials. However, only a few approaches have used recombinant AAV (rAAV) to deliver vaccine antigens. We generated an rAAV encoding the major capsid protein L1 (L1h) from the human papillomavirus type 16 (HPV16), aiming to develop a prophylactic vaccine against HPV16 infections, which are the major cause of cervical cancer in women worldwide. A single dose of rAAV5 L1h administered intranasally was sufficient to induce high titers of L1-specific serum antibodies, as well as mucosal antibodies in vaginal washes. Seroconversion was maintained for at least 1 year. In addition, a cellular immune response was still detectable 60 weeks after immunization. Furthermore, lyophilized rAAV5 L1h successfully evoked a systemic and mucosal immune response in mice. These data clearly show the efficacy of a single-dose intranasal immunization against HPV16 based on the recombinant rAAV5L1h vector without the need of an adjuvant.Genital infections with human papillomaviruses (HPV) are among the most common viral sexually transmitted infections in humans. It has been estimated that at least 50% of sexually active adults had a genital HPV infection. More than 120 different genotypes have been described thus far, 15 of which (i.e., HPV type 16 [HPV16] and HPV18) were identified as causative agents of at least 90% of cancers of the cervix and were also linked to more than the half of other anogenital cancers. Cervical cancer is the second most frequent malignant tumor in women worldwide (for a review, see reference 74).Several vaccination models against HPV have been evaluated aiming to generate neutralizing antibodies.

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MRCoNS from animal infection sites are genetically heterogeneous multidrug-resistant strains that represent a new challenge in the prevention and therapy of infections in veterinary clinics.

The proteins encoded by the adeno-associated virus type 2 (AAV-2) rep and cap genes obtained during a productive infection of HeLa cells with AAV-2 and adenovirus type 2 were fractionated according to solubility, cellular localization, and sedimentation properties. The majority of Rep and Cap proteins accumulated in the nucleus, where they distributed into a soluble and an insoluble fraction. Analysis of the soluble nuclear fraction of capsid proteins by sucrose density gradients showed that they formed at least three steady-state pools: a monomer pool sedimenting at about 6S, a pool of oligomeric intermediates sedimenting between 10 and 15S, and a broad pool of assembly products with a peak between 60 and 110S, the known sedimentation positions of empty and full capsids. While the soluble nuclear monomer and oligomer pool contained predominantly only two capsid proteins, the 30 to 180S assembly products contained VP1, VP2, and VP3 in a stoichiometry similar to that of purified virions. They probably represent different intermediates in capsid assembly, DNA encapsidation, and capsid maturation. In contrast, the cytoplasmic fraction of capsid proteins showed a pattern of oligomers continuously increasing in size without a defined peak, suggesting that assembly of 60S particles occurs in the nucleus. Soluble nuclear Rep proteins were distributed over the whole sedimentation range, probably as a result of association with AAV DNA. Subfractions of the Rep proteins with defined sedimentation values were obtained in the soluble nuclear and cytoplasmic fractions. We were able to coimmunoprecipitate capsid proteins sedimenting between 60 and 110S with antibodies against Rep proteins, suggesting that they exist in common complexes possibly involved in AAV DNA packaging. Antibodies against the capsid proteins, however, precipitated Rep78 and Rep68 predominantly with a peak around 30S representing a second complex containing Rep and Cap proteins.