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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 reactivity of memory (CD45RO+) T cells. (a) Total splenocytes, (b) CD45RO–, or CD45RO+ T cells from an HLA-B*0702 donor were restimulated in vitro against the corresponding HLA-restricted AAV epitope (VPQYGYLTL) and tested on IFN-γ ELISpot assay. AAV, AAV whole capsids; Medium, negative control; PMA, phorbol 12-myristate 13-acetate (positive control); Sfu, spot-forming units.
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fig4: AAV capsid reactivity of memory (CD45RO+) T cells. (a) Total splenocytes, (b) CD45RO–, or CD45RO+ T cells from an HLA-B*0702 donor were restimulated in vitro against the corresponding HLA-restricted AAV epitope (VPQYGYLTL) and tested on IFN-γ ELISpot assay. AAV, AAV whole capsids; Medium, negative control; PMA, phorbol 12-myristate 13-acetate (positive control); Sfu, spot-forming units.

Mentions: The fact that T-cell responses are more frequent in the >5-years-old age cohort (presumably after initial infection) suggests that IFN-γ–producing cells might be derived from capsid-specific memory T cells (Table 1). To test this hypothesis, total splenocytes from an HLA-B*0702 reactive donor were sorted into two different fractions, CD4+-CD8+-CD45RO+ and CD4+-CD8+-CD45RO−. Cells were then restimulated in vitro with the HLA-B*0702–restricted epitope VPQYGYLTL and assayed in an IFN-γ ELISpot assay. The sorting resulted in the detection of a robust response to the peptide epitope and to the whole AAV capsid antigen only in total splenocytes and in the CD45RO+ fraction of splenocytes (Figure 4). Furthermore, CTL-mediated killing of AAV-transduced target cells was observed only when CD45RO+ cells of a reactive donor were used as effectors.


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 reactivity of memory (CD45RO+) T cells. (a) Total splenocytes, (b) CD45RO–, or CD45RO+ T cells from an HLA-B*0702 donor were restimulated in vitro against the corresponding HLA-restricted AAV epitope (VPQYGYLTL) and tested on IFN-γ ELISpot assay. AAV, AAV whole capsids; Medium, negative control; PMA, phorbol 12-myristate 13-acetate (positive control); Sfu, spot-forming units.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: AAV capsid reactivity of memory (CD45RO+) T cells. (a) Total splenocytes, (b) CD45RO–, or CD45RO+ T cells from an HLA-B*0702 donor were restimulated in vitro against the corresponding HLA-restricted AAV epitope (VPQYGYLTL) and tested on IFN-γ ELISpot assay. AAV, AAV whole capsids; Medium, negative control; PMA, phorbol 12-myristate 13-acetate (positive control); Sfu, spot-forming units.
Mentions: The fact that T-cell responses are more frequent in the >5-years-old age cohort (presumably after initial infection) suggests that IFN-γ–producing cells might be derived from capsid-specific memory T cells (Table 1). To test this hypothesis, total splenocytes from an HLA-B*0702 reactive donor were sorted into two different fractions, CD4+-CD8+-CD45RO+ and CD4+-CD8+-CD45RO−. Cells were then restimulated in vitro with the HLA-B*0702–restricted epitope VPQYGYLTL and assayed in an IFN-γ ELISpot assay. The sorting resulted in the detection of a robust response to the peptide epitope and to the whole AAV capsid antigen only in total splenocytes and in the CD45RO+ fraction of splenocytes (Figure 4). Furthermore, CTL-mediated killing of AAV-transduced target cells was observed only when CD45RO+ cells of a reactive donor were used as effectors.

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