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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

MHC class I pentamer staining and polyfunctional analysis of capsid-specific CD8+ T cells expanded from splenocytes. (a) Capsid-specific MHC class I pentamer staining of HLA-A*0101 cells expanded without peptide, an irrelevant peptide (HIV-1 env gp120 848–856), or AAV capsid. Cells were gated on lymphocytes and CD14+, CD16+ and CD19+ cells were gated out. (b) Polyfunctional analysis of T-cell activation. Histograms show the fold increase in the percent of double-positive CD8+ cells (CD8+IFN-γ+CD107a+, CD8+IFN-γ+IL-2+, or CD8+IFN-γ+TNF-α+) over medium only control. SEB, Staphylococcal enterotoxin B (positive control). Cells were gated on lymphocytes and CD8+ T cells.
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fig2: MHC class I pentamer staining and polyfunctional analysis of capsid-specific CD8+ T cells expanded from splenocytes. (a) Capsid-specific MHC class I pentamer staining of HLA-A*0101 cells expanded without peptide, an irrelevant peptide (HIV-1 env gp120 848–856), or AAV capsid. Cells were gated on lymphocytes and CD14+, CD16+ and CD19+ cells were gated out. (b) Polyfunctional analysis of T-cell activation. Histograms show the fold increase in the percent of double-positive CD8+ cells (CD8+IFN-γ+CD107a+, CD8+IFN-γ+IL-2+, or CD8+IFN-γ+TNF-α+) over medium only control. SEB, Staphylococcal enterotoxin B (positive control). Cells were gated on lymphocytes and CD8+ T cells.

Mentions: MHC class I pentamer staining was performed in those samples for which these reagents were available to confirm the expansion of capsid-specific CD8+ T cells (Figure 2a). Polyfunctional analysis of markers of T-cell activation and function was performed after in vitro restimulation with AAV capsid; intracellular levels of IL-2, IFN-γ, TNF-α, and the degranulation marker CD107a in response to the AAV capsid antigen were normalized to background levels (medium only control). All positive samples presented a population of capsid-reactive CD8+ T cells secreting high levels of IL-2, IFN-γ, and TNF-α and displaying the degranulation marker CD107a (Figure 2b; Supplementary Figure S3).


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)

MHC class I pentamer staining and polyfunctional analysis of capsid-specific CD8+ T cells expanded from splenocytes. (a) Capsid-specific MHC class I pentamer staining of HLA-A*0101 cells expanded without peptide, an irrelevant peptide (HIV-1 env gp120 848–856), or AAV capsid. Cells were gated on lymphocytes and CD14+, CD16+ and CD19+ cells were gated out. (b) Polyfunctional analysis of T-cell activation. Histograms show the fold increase in the percent of double-positive CD8+ cells (CD8+IFN-γ+CD107a+, CD8+IFN-γ+IL-2+, or CD8+IFN-γ+TNF-α+) over medium only control. SEB, Staphylococcal enterotoxin B (positive control). Cells were gated on lymphocytes and CD8+ T cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: MHC class I pentamer staining and polyfunctional analysis of capsid-specific CD8+ T cells expanded from splenocytes. (a) Capsid-specific MHC class I pentamer staining of HLA-A*0101 cells expanded without peptide, an irrelevant peptide (HIV-1 env gp120 848–856), or AAV capsid. Cells were gated on lymphocytes and CD14+, CD16+ and CD19+ cells were gated out. (b) Polyfunctional analysis of T-cell activation. Histograms show the fold increase in the percent of double-positive CD8+ cells (CD8+IFN-γ+CD107a+, CD8+IFN-γ+IL-2+, or CD8+IFN-γ+TNF-α+) over medium only control. SEB, Staphylococcal enterotoxin B (positive control). Cells were gated on lymphocytes and CD8+ T cells.
Mentions: MHC class I pentamer staining was performed in those samples for which these reagents were available to confirm the expansion of capsid-specific CD8+ T cells (Figure 2a). Polyfunctional analysis of markers of T-cell activation and function was performed after in vitro restimulation with AAV capsid; intracellular levels of IL-2, IFN-γ, TNF-α, and the degranulation marker CD107a in response to the AAV capsid antigen were normalized to background levels (medium only control). All positive samples presented a population of capsid-reactive CD8+ T cells secreting high levels of IL-2, IFN-γ, and TNF-α and displaying the degranulation marker CD107a (Figure 2b; Supplementary Figure S3).

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