Adeno-associated viruses (AAVs) are small, nonenveloped single-stranded DNA viruses that require helper viruses to facilitate efficient replication. Despite the presence of humoral responses to the wild-type AAV in humans, AAV remains one of the most promising candidates for therapeutic gene transfer to treat many genetic and acquired diseases. Characterization of the IgG subclass responses to AAV and study of the prevalence of both IgG and neutralizing factors to AAV types 1, 2, 5, 6, 8, and 9 in the human population are of importance for the development of new strategies to overcome these immune responses. Natural exposure to AAV types 1, 2, 5, 6, 8, and 9 can result in the production of antibodies from all four IgG subclasses, with a predominant IgG1 response and low IgG2, IgG3, and IgG4 responses. Prevalences of anti-AAV1 and -AAV2 total IgG determined by enzyme-linked immunosorbent assay were higher (67 and 72%) than those of anti-AAV5 (40%), anti-AAV6 (46%), anti-AAV8 (38%), and anti-AAV9 (47%). Furthermore, data showed that cross-reactions are important. The two highest neutralizing factor seroprevalences were observed for AAV2 (59%) and AAV1 (50.5%) and the lowest were observed for AAV8 (19%) and AAV5 (3.2%). Vectors based on AAV5, AAV8, and AAV9 may have an advantage for gene therapy in humans. Furthermore, among individuals seropositive for AAV5, AAV8, and AAV9, about 70-100% present low titers. Better characterization of the preexisting humoral responses to the AAV capsid and cross-reactivity will allow development of new strategies to circumvent AAV acquired immune responses.

Adeno-associated viruses (AAV) are small, nonenveloped single-stranded DNA viruses which require helper viruses to facilitate efficient replication. These recombinant viruses are some of the most promising candidates for therapeutic gene transfer to treat many genetic and acquired diseases. Nevertheless, the presence of humoral responses to the wild-type AAV common among humans is one of the limitations of in vivo transduction efficacy in humans using cognate recombinant vector. In this study, based on the serum samples that we were able to collect from various clinical situations, we studied the impact of one to five plasmapheresis (PP), at 1-5 day intervals on neutralizing factor (NAF) titers specific for AAV types 1, 2, 6, and 8 in seropositive patients with diverse pathologies and immunosuppressor treatments. We show that frequent sessions of PP result in drastic reduction of NAF specific for AAV1, 2, 6, and 8 to undetectable levels or titers <1:5, mainly when initial titers, i.e., before the first PP were ≤1:20. Altogether, these results show that the use of PP and its possible association with pharmacological immunosuppressive treatments may help to design optimal management of seropositive patients for AAV gene therapy treatments.

A major impediment to the use of adeno-associated virus (AAV)-mediated gene delivery to muscle in clinical applications is the pre-existing immune responses against the vector. Pre-existing humoral response to different AAV serotypes is now well documented. In contrast, cellular responses to AAV capsid have not been analyzed in a systematic manner, despite the risk of T cell reactivation upon gene transfer. AAV1 has been widely used in humans to target muscle. In this study, we analyzed PBMCs and sera of healthy donors for the presence of AAV1 capsid-specific T cell responses and AAV1 neutralizing factors. Approximately 30% of donors presented AAV1 capsid-specific T cells, mainly effector memory CD8+ cells. IFN-γ–producing cells were also observed among effector memory CD4+ cells for two of these donors. Moreover, to our knowledge, this study shows for the first time on a large cohort that there was no correlation between AAV1-specific T cell and humoral responses. Indeed, most donors presenting specific Ig and neutralizing factors were negative for cellular response (and vice versa). These new data raise the question of prescreening patients not only for the humoral response, but also for the cellular response. Clearly, a better understanding of the natural immunology of AAV serotypes will allow us to improve AAV gene therapy and make it an efficient treatment for genetic disease.

A major impediment to gene replacement therapy is immune elimination of genetically modified cells. In principle, this can be dealt with by inducing a strong, specific, and enduring tolerance through engraftment of transgene-modified autologous bone marrow (BM). Because usual myeloablation and/or immunosuppression are risk factors in most pathologies, we assessed the potential of monospecific CD4 ؉ CD25 ؉ regulatory T cells (Tregs) to engraft minor-mismatched BM without preconditioning. We found that as few as 5 ؋ 10 4 Tregs directed to the male DBY protein promote the engraftment of foreign male BM into sex-mismatched female hosts, establishing sustained chimerism in all hematopoeitic compartments. We achieved concomitantly strong tolerance to all foreign antigens expressed in the BM, likely occurring through induction of anergy and/or deletion of antidonor T cells. Chimerism was obtained in thymectomized mice too, underlining the major role of peripheral tolerance mechanisms in our system. This allowed us to engraft genemodified tissues while preserving full immunocompetence to third-party antigens. Our results demonstrate that very few donor-specific Tregs are effective as the sole conditioning to induce mixed molecular chimerism and long-term tolerance to multiple foreign antigens. IntroductionGene replacement therapy has raised considerable interest for the treatment of various metabolic diseases and genetic disorders such as muscular dystrophies 1 or hemophilia. 2 It is, however, very important to avoid deleterious immune responses. Major efforts have been devoted to improving gene delivery procedures, searching for the least immunogenic vectors, for tissue-specific promoters, and for appropriate injection routes and doses. 3,4 But these approaches face the risk of creating a status of immune ignorance, in which secondary inflammation and bystander pathogen signals may induce immune rejection of therapeutic gene products. 5 Induction of peripheral tolerance using immunosuppressive cotreatments or transient blockade of costimulation pathways, developed initially for transplantation or autoimmunity applications, have been successfully transposed to gene therapy, 6,7 but these procedures are accompanied by marked toxicity, increased risks of cardiovascular disease, opportunistic infections, and malignancy in humans. Transgene-specific tolerance approaches are needed to block initial rejection and induce long-term tolerance without affecting overall immune functions in patients. These requirements are met by expressing transgenes of interest in the hematopoietic system prior to gene therapy interventions.Mixed hematopoietic chimerism generates a robust donorspecific tolerance and represents an attractive method to obviate immune responses in organ transplantation 8 or for treatment of autoimmune diseases. 9,10 Mixed hematopoietic chimerism was recently used to induce tolerance to specific proteins by transferring genes into autologous hematopoietic stem cells (HSCs) for gene therapy. 11,12 However, induction ...

The discovery of tumor-associated antigens recognized by T lymphocytes opens the possibility of vaccinating cancer patients with defined antigens. However, one of the major limitation of peptide-based vaccines is the low immunogenicity of antigenic peptides. Interestingly, if these epitopes are directly delivered into the cytoplasm of antigen presenting cells, they can be efficiently presented via the direct MHC class I presentation pathway. To improve antigen entry, one promising approach is the use of cell penetrating peptides (CPPs). However, most studies use a covalent binding of the CPP with the antigen. In the present study, we focused on the C-terminal domain of Vpr which was previously demonstrated to efficiently deliver plasmid DNA into cells. We provide evidence that the peptides Vpr55-91 and Vpr55-82 possess the capacity of delivering proteins and epitopes into cell lines as well as into human primary dendritic cells, without the necessicity for a chemical linkage. Moreover, immunization of HLA-A2 transgenic mice with Vpr55-91 as the sole adjuvant is able to induce antigen-specific cytotoxic T lymphocytes against multiple tumor epitopes.