Over the past decade, vectors derived from adeno-associated virus (AAV) have established themselves as a powerful tool for in vivo gene transfer, allowing long-lasting and safe transgene expression in a variety of human tissues. Nevertheless, clinical trials demonstrated how B and T cell immune responses directed against the AAV capsid, likely arising after natural infection with wild-type AAV, might potentially impact gene transfer safety and efficacy in patients. Seroprevalence studies have evidenced that most individuals carry anti-AAV neutralizing antibodies that can inhibit recombinant AAV transduction of target cells following in vivo administration of vector particles. Likewise, liver- and muscle-directed clinical trials have shown that capsid-reactive memory CD8+ T cells could be reactivated and expanded upon presentation of capsid-derived antigens on transduced cells, potentially leading to loss of transgene expression and immune-mediated toxicities. In celebration of the 25th anniversary of the European Society of Gene and Cell Therapy, this review article summarizes progress made during the past decade in understanding and modulating AAV vector immunogenicity. While the knowledge generated has contributed to yield impressive clinical results, several important questions remain unanswered, making the study of immune responses to AAV a priority for the field of in vivo transfer.

Anti-transgene immune responses elicited after intramuscular (i.m.) delivery of recombinant adenoassociated virus (rAAV) have been shown to hamper long-term transgene expression in large-animal models of rAAV-mediated gene transfer. To overcome this hurdle, an alternative mode of delivery of rAAV vectors in nonhuman primate muscles has been described: the locoregional (LR) intravenous route of administration. Using this injection mode, persistent inducible transgene expression for at least 1 year under the control of the tetracycline-inducible Tet-On system was previously reported in cynomolgus monkeys, with no immunity against the rtTA transgene product. The present study shows the long-term follow-up of these animals. It is reported that LR delivery of a rAAV2/1 vector allows long-term inducible expression up to at least 5 years post gene transfer, with no any detectable host immune response against the transactivator rtTA, despite its immunogenicity following i.m. gene transfer. This study shows for the first time a long-term regulation of muscle gene expression using a Tet-On-inducible system in a largeanimal model. Moreover, these findings further confirm that the rAAV LR delivery route is efficient and immunologically safe, allowing long-term skeletal muscle gene transfer.

Pre-existing immunity to AAV capsid may compromise the safety and efficiency of rAAV-mediated gene transfer in patients. Anti-capsid cytotoxic immune responses have proven to be a challenge to characterize because of the scarcity of circulating AAV-specific CD8 + T lymphocytes which can seldom be detected with conventional flow cytometry or ELISpot assays. Here, we used fluorescent MHC class I tetramers combined with magnetic enrichment to detect and phenotype AAV8-specific CD8 + T cells in human PBMCs without prior amplification. We showed that all healthy individuals tested carried a pool of AAV8-specific CD8 + T cells with a CD45RA + CCR7 − terminally-differentiated effector memory cell (T EMRA) fraction. Ex vivo frequencies of total AAV-specific CD8 + T cells were not predictive of IFNγ ELISpot responses but interestingly we evidenced a correlation between the proportion of T EMRA cells and IFNγ ELISpot positive responses. T EMRA cells may then play a role in recombinant AAV-mediated cytotoxicity in patients with preexisting immunity. Overall, our results encourage the development of new methods combining increased detection sensitivity of AAV-specific T cells and their poly-functional assessment to better characterize and monitor AAV capsid-specific cellular immune responses in the perspective of rAAV-mediated clinical trials.

Preventing untoward immune responses against a specific antigen is a major challenge in different clinical settings such as gene therapy, transplantation, or autoimmunity. Following intramuscular delivery of recombinant adeno-associated virus (rAAV)-derived vectors, transgene rejection can be a roadblock to successful clinical translation. Specific immunomodulation strategies potentially leading to sustained transgene expression while minimizing pharmacological immunosuppression are desirable. Tolerogenic dendritic cells (TolDC) are potential candidates but have not yet been evaluated in the context of gene therapy, to our knowledge. Following intramuscular delivery of rAAV-derived vectors expressing an immunogenic protein in the nonhuman primate model, we assessed the immunomodulating potential of autologous bone marrow-derived TolDC generated in the presence of IL10 and pulsed with the transgene product. TolDC administered either intradermally or intravenously were safe and well tolerated. While the intravenous route showed a modest ability to modulate host immunity against the transgene product, intradermally delivery resulted in a robust vaccination of the macaques when associated to intramuscular rAAV-derived vectors-based gene transfer. These findings demonstrate the critical role of TolDC mode of injection in modulating host immunity. This study also provides the first evidence of the potential of TolDC-based immunomodulation in gene therapy.

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Background: Allergen-specific type 2 CD4 1 T H 2 cells are critically involved in the pathogenesis of IgE-mediated allergic diseases. However, the heterogeneity of the T H 2 response has only recently been appreciated. Objective: We sought to characterize at the single-cell level the ex vivo phenotype, transcriptomic profile, and T-cell receptor (TCR) repertoire of circulating CD4 1 T cells specific to the major dog allergens Can f 1, Can f 4, and Can f 5 in subjects with and without dog allergy. Methods: Dog allergen-specific memory CD4 1 T cells were detected ex vivo by flow cytometry using a CD154-based enrichment assay and single-cell sorted for targeted gene expression analysis and TCR sequencing. Results: Dog allergen-specific T-cell responses in allergic subjects were dominantly of T H 2 type. T H 2 cells could be phenotypically further divided into 3 subsets, which consisted ofcells. All these subsets were nonexistent within the allergen-specific T-cell repertoire of healthy subjects. Single-cell transcriptomic profiling confirmed the T H 2-biased signature in allergen-specific T cells from allergic subjects and revealed a T H 1/T H 17 signature in nonallergic subjects. TCR repertoire analyses showed that dog allergen-specific T cells were diverse and allergic subjects demonstrated less clonality compared to nonallergic donors.Finally, TCR and transcriptomic analyses revealed a close relationship between T H 2-like, T H 2, and T H 2A cells, with the last ones representing the most terminally differentiated and highly polarized subtype. Conclusions: Our study demonstrates heterogeneity within allergen-specific T H 2 cells at the single-cell level. The results may be utilized for improving immune monitoring after allergen immunotherapy and for designing targeted immunomodulatory approaches. (J Allergy Clin Immunol 2021;nnn:nnn-nnn.)

Context Validated assays to measure autoantigen-specific T cell frequency and phenotypes are needed for assessing the risk of developing diabetes, monitoring disease progression, evaluating responses to treatment, and personalizing antigen-based therapies. Objective Towards this end, we performed a technical validation of a tetramer assay for HLA-DRA-DRB1*04:01, a class II allele that is strongly associated with susceptibility to T1D. Methods HLA-DRA-DRB1*04:01-restricted T cells specific for immunodominant epitopes from islet cell antigens GAD65, IGRP, preproinsulin, and ZnT8, and a reference influenza epitope, were enumerated and phenotyped in a single staining tube with the tetramer assay. Single and multicenter testing was performed, using a clone-spiked specimen and replicate samples from T1D patients, with a target coefficient of variation (CV) < 30%. The same assay was applied to an exploratory cross-sectional sample set with 24 T1D patients to evaluate the utility of the assay. Results Influenza-specific T cell measurements had mean CVs of 6% for the clone-spiked specimen and 11% for T1D samples in single-center testing, and 20% and 31%, respectively, for multicenter testing. Islet-specific T cell measurements in these same samples had mean CVs of 14% and 23% for single-center and 23% and 41% for multicenter testing. The cross-sectional study identified relationships between T cell frequencies and phenotype and disease duration, sex, and autoantibodies. A large fraction of the islet-specific T-cells exhibited a naïve phenotype. Conclusions Our results demonstrated that the assay is reproducible and useful to characterize islet-specific T cells and identify correlations between T cell measures and clinical traits.

PBS-formulated rAAV2/8 PGK GFP-HY were injected subretinally in C57Bl/6 female mice. At day 7, mice were challenged subcutaneously with the UTY and DBY peptides adjuvanted in CFA, and the immune response was analyzed at day 14 by IFNγ ELISpot, cytokine titration and proliferation assays. Our results revealed that: (i) The subretinal injection of 10E8 to 2.10E9 vg/mouse of rAAV2/8 PGK GFP-HY did not induce a significant HY-specific peripheral immune modulation in contrast to the ACAID obtained after subretinal injection of UTY and DBY peptides (50µg each); (ii) Higher doses of rAAV2/8 PGK GFP-HY (5.10E10 vg/mouse) triggered increased Th1 and Tc1 cellular immune responses against the transgene product in peripheral lymphoid organs. Lower doses of vector did not increase peripheral immune responses toward the transgene product following the challenge. We have thus far failed to demonstrate the induction of ACAID by rAAV2/8 subretinal gene transfer. We show that rAAV2/8 vector-mediated subretinal gene transfer is not necessarily ignored at the immunological level. High doses of vector can effectively trigger anti-transgene T-cell responses with the potential for elimination of transgene-expressing cells. Clearly, anti-transgene-specific immune monitoring should be refined at least in preclinical models, to improve the biosafety and the long-term efficacy of rAAV-mediated ocular gene transfer.