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AAV capsid CD8+ T-cell epitopes are highly conserved across AAV serotypes.

Hui DJ, Edmonson SC, Podsakoff GM, Pien GC, Ivanciu L, Camire RM, Ertl H, Mingozzi F, High KA, Basner-Tschakarjan E - Mol Ther Methods Clin Dev (2015)

Bottom Line: We were able to identify immunodominant major histocompatibility complex (MHC) class I epitopes for common human leukocyte antigen (HLA) types by using spleens isolated from subjects undergoing splenectomy for non-malignant indications as a source of large numbers of lymphocytes and restimulating them with single AAV capsid peptides in vitro.Further experiments confirmed that these epitopes are naturally processed and functionally relevant.The design of more effective and less immunogenic AAV vectors, and precise immune monitoring of vector-infused subjects, are facilitated by these findings.

View Article: PubMed Central - PubMed

Affiliation: The Children's Hospital of Philadelphia, Division of Hematology , Philadelphia, Pennsylvania, USA.

ABSTRACT
Adeno-associated virus (AAV) has become one of the most promising vectors in gene transfer in the last 10 years with successful translation to clinical trials in humans and even market approval for a first gene therapy product in Europe. Administration to humans, however, revealed that adaptive immune responses against the vector capsid can present an obstacle to sustained transgene expression due to the activation and expansion of capsid-specific T cells. The limited number of peripheral blood mononuclear cells (PBMCs) obtained from samples within clinical trials allows for little more than monitoring of T-cell responses. We were able to identify immunodominant major histocompatibility complex (MHC) class I epitopes for common human leukocyte antigen (HLA) types by using spleens isolated from subjects undergoing splenectomy for non-malignant indications as a source of large numbers of lymphocytes and restimulating them with single AAV capsid peptides in vitro. Further experiments confirmed that these epitopes are naturally processed and functionally relevant. The design of more effective and less immunogenic AAV vectors, and precise immune monitoring of vector-infused subjects, are facilitated by these findings.

No MeSH data available.


Related in: MedlinePlus

AAV capsid transfected APCs induce the proliferation of effector cells that recognize and kill peptide-loaded target cells. HLA-matched and mismatched target cells were loaded with the peptides we determined for the corresponding AAV capsids (left four columns). Effector cells were generated in vitro as described, and cytotoxicity was measured as described. The right four columns show the fold-induction of cytotoxicity in the described CTL assay over background. CTL, cytotoxic T lymphocyte.
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fig5: AAV capsid transfected APCs induce the proliferation of effector cells that recognize and kill peptide-loaded target cells. HLA-matched and mismatched target cells were loaded with the peptides we determined for the corresponding AAV capsids (left four columns). Effector cells were generated in vitro as described, and cytotoxicity was measured as described. The right four columns show the fold-induction of cytotoxicity in the described CTL assay over background. CTL, cytotoxic T lymphocyte.

Mentions: As classic MHC class I presentation occurs with peptides synthesized by the presenting cell, first we addressed the question by nucleofection experiments followed by the previously described CTL assay as a read-out. Nucleofection with capsid encoding plasmids was used instead of infection with the different AAV serotypes in order to avoid influences of variability of infectivity and intracellular processing on the results. Splenocytes were depleted of CD8+ T cells (human CD8+ T-cell depletion kit by Miltenyi, San Diego, CA). The remaining cells were nucleofected (Amaxa Nucleofection kit; Lonza, Basel, Switzerland) with plasmids encoding AAV1, AAV2, or AAV8 capsid as well as a control GFP plasmid. Forty-eight hours after nucleofection, the cells were used as antigen-presenting cells (APCs) to generate effector T cells in in vitro expansions (see Materials and Methods). After two rounds of stimulation, the generated effector T cells were used in CTL assays. Target cells were hepatocyte cell lines loaded with the relevant peptide epitopes or an irrelevant control peptide. The experiments were conducted with HLA matched as well as mismatched (control) target cells. The results are depicted in a heat map, which shows induction of cytotoxicity over background. Significant cytotoxicity could only be detected when using HLA-matched target cells (Figure 5). The nucleofected APCs led to the expansion of functional effector cells that were able to recognize and kill target cells loaded with the identified peptides but not an irrelevant peptide. Consistent with the findings from the cytotoxicity assays, a striking amount of cross-reactivity between AAV serotypes could be detected. Expansion with the control protein GFP-nucleofected splenocytes did not create functional effector cells.


AAV capsid CD8+ T-cell epitopes are highly conserved across AAV serotypes.

Hui DJ, Edmonson SC, Podsakoff GM, Pien GC, Ivanciu L, Camire RM, Ertl H, Mingozzi F, High KA, Basner-Tschakarjan E - Mol Ther Methods Clin Dev (2015)

AAV capsid transfected APCs induce the proliferation of effector cells that recognize and kill peptide-loaded target cells. HLA-matched and mismatched target cells were loaded with the peptides we determined for the corresponding AAV capsids (left four columns). Effector cells were generated in vitro as described, and cytotoxicity was measured as described. The right four columns show the fold-induction of cytotoxicity in the described CTL assay over background. CTL, cytotoxic T lymphocyte.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588448&req=5

fig5: AAV capsid transfected APCs induce the proliferation of effector cells that recognize and kill peptide-loaded target cells. HLA-matched and mismatched target cells were loaded with the peptides we determined for the corresponding AAV capsids (left four columns). Effector cells were generated in vitro as described, and cytotoxicity was measured as described. The right four columns show the fold-induction of cytotoxicity in the described CTL assay over background. CTL, cytotoxic T lymphocyte.
Mentions: As classic MHC class I presentation occurs with peptides synthesized by the presenting cell, first we addressed the question by nucleofection experiments followed by the previously described CTL assay as a read-out. Nucleofection with capsid encoding plasmids was used instead of infection with the different AAV serotypes in order to avoid influences of variability of infectivity and intracellular processing on the results. Splenocytes were depleted of CD8+ T cells (human CD8+ T-cell depletion kit by Miltenyi, San Diego, CA). The remaining cells were nucleofected (Amaxa Nucleofection kit; Lonza, Basel, Switzerland) with plasmids encoding AAV1, AAV2, or AAV8 capsid as well as a control GFP plasmid. Forty-eight hours after nucleofection, the cells were used as antigen-presenting cells (APCs) to generate effector T cells in in vitro expansions (see Materials and Methods). After two rounds of stimulation, the generated effector T cells were used in CTL assays. Target cells were hepatocyte cell lines loaded with the relevant peptide epitopes or an irrelevant control peptide. The experiments were conducted with HLA matched as well as mismatched (control) target cells. The results are depicted in a heat map, which shows induction of cytotoxicity over background. Significant cytotoxicity could only be detected when using HLA-matched target cells (Figure 5). The nucleofected APCs led to the expansion of functional effector cells that were able to recognize and kill target cells loaded with the identified peptides but not an irrelevant peptide. Consistent with the findings from the cytotoxicity assays, a striking amount of cross-reactivity between AAV serotypes could be detected. Expansion with the control protein GFP-nucleofected splenocytes did not create functional effector cells.

Bottom Line: We were able to identify immunodominant major histocompatibility complex (MHC) class I epitopes for common human leukocyte antigen (HLA) types by using spleens isolated from subjects undergoing splenectomy for non-malignant indications as a source of large numbers of lymphocytes and restimulating them with single AAV capsid peptides in vitro.Further experiments confirmed that these epitopes are naturally processed and functionally relevant.The design of more effective and less immunogenic AAV vectors, and precise immune monitoring of vector-infused subjects, are facilitated by these findings.

View Article: PubMed Central - PubMed

Affiliation: The Children's Hospital of Philadelphia, Division of Hematology , Philadelphia, Pennsylvania, USA.

ABSTRACT
Adeno-associated virus (AAV) has become one of the most promising vectors in gene transfer in the last 10 years with successful translation to clinical trials in humans and even market approval for a first gene therapy product in Europe. Administration to humans, however, revealed that adaptive immune responses against the vector capsid can present an obstacle to sustained transgene expression due to the activation and expansion of capsid-specific T cells. The limited number of peripheral blood mononuclear cells (PBMCs) obtained from samples within clinical trials allows for little more than monitoring of T-cell responses. We were able to identify immunodominant major histocompatibility complex (MHC) class I epitopes for common human leukocyte antigen (HLA) types by using spleens isolated from subjects undergoing splenectomy for non-malignant indications as a source of large numbers of lymphocytes and restimulating them with single AAV capsid peptides in vitro. Further experiments confirmed that these epitopes are naturally processed and functionally relevant. The design of more effective and less immunogenic AAV vectors, and precise immune monitoring of vector-infused subjects, are facilitated by these findings.

No MeSH data available.


Related in: MedlinePlus