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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

High-throughput mapping of AAV capsid T cell epitopes. (a, c, e) Single-peptide IFN-γ ELISpot on in vitro expanded splenocytes. Splenocytes from normal donors were restimulated in vitro with 15-mers derived from the AAV-2 (a, c) or AAV-1 (e) capsid VP-1 amino acid sequence. Each of the 145 peptides from the AAV libraries was used individually in the restimulation. After restimulation, cells were rechallenged with the same peptides in an ELISpot assay. Positive peptides were selected for further validation. Dashed line, threshold for positivity; numbers 1 through 145, 15-mers from the AAV peptide library; PMA, phorbol 12-myristate 13-acetate (positive control); Sfu, spot-forming units. *Peptides positive but not further confirmed. (b, d, f) Peptides identified with relative HLA restriction. N/D, not determined.
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fig1: High-throughput mapping of AAV capsid T cell epitopes. (a, c, e) Single-peptide IFN-γ ELISpot on in vitro expanded splenocytes. Splenocytes from normal donors were restimulated in vitro with 15-mers derived from the AAV-2 (a, c) or AAV-1 (e) capsid VP-1 amino acid sequence. Each of the 145 peptides from the AAV libraries was used individually in the restimulation. After restimulation, cells were rechallenged with the same peptides in an ELISpot assay. Positive peptides were selected for further validation. Dashed line, threshold for positivity; numbers 1 through 145, 15-mers from the AAV peptide library; PMA, phorbol 12-myristate 13-acetate (positive control); Sfu, spot-forming units. *Peptides positive but not further confirmed. (b, d, f) Peptides identified with relative HLA restriction. N/D, not determined.

Mentions: The large number of cells isolated and stored from each spleen, in combination with bioinformatics algorithms, allowed us to specify and confirm single 9-mer peptides as capsid T-cell epitopes experimentally (Figure 1; Supplementary Figure S1) and to define their predicted binding affinity to the cognate MHC class I molecules (Table 2). We also gained insight into the frequency of subjects responding to a specific epitope (Table 2; Supplementary Figure S2). All epitopes identified with the ELISpot assay exhibited a binding affinity to MHC class I of at least 50% of the maximum binding affinity of the consensus sequence defined by the position-specific scoring matrix (PSSM) for the cognate HLA allele.23 Most, but not all epitopes, could be confirmed, and there was no obvious relationship between the binding affinity score for a specific HLA allele and the frequency of subjects responding to the specific epitope (Table 2). Not all epitopes could be confirmed because bioinformatics analysis was restricted to HLA alleles present in the databases, and thus, further analysis and specification of some reactive peptides was not possible based on the rare haplotype (Table 2, footnote d). In several instances, subjects sharing the same HLA alleles showed reactivity to the same AAV-derived epitopes. In one instance, two different epitopes binding to the same HLA (HLA-B*51, Table 2) were identified.


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)

High-throughput mapping of AAV capsid T cell epitopes. (a, c, e) Single-peptide IFN-γ ELISpot on in vitro expanded splenocytes. Splenocytes from normal donors were restimulated in vitro with 15-mers derived from the AAV-2 (a, c) or AAV-1 (e) capsid VP-1 amino acid sequence. Each of the 145 peptides from the AAV libraries was used individually in the restimulation. After restimulation, cells were rechallenged with the same peptides in an ELISpot assay. Positive peptides were selected for further validation. Dashed line, threshold for positivity; numbers 1 through 145, 15-mers from the AAV peptide library; PMA, phorbol 12-myristate 13-acetate (positive control); Sfu, spot-forming units. *Peptides positive but not further confirmed. (b, d, f) Peptides identified with relative HLA restriction. N/D, not determined.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: High-throughput mapping of AAV capsid T cell epitopes. (a, c, e) Single-peptide IFN-γ ELISpot on in vitro expanded splenocytes. Splenocytes from normal donors were restimulated in vitro with 15-mers derived from the AAV-2 (a, c) or AAV-1 (e) capsid VP-1 amino acid sequence. Each of the 145 peptides from the AAV libraries was used individually in the restimulation. After restimulation, cells were rechallenged with the same peptides in an ELISpot assay. Positive peptides were selected for further validation. Dashed line, threshold for positivity; numbers 1 through 145, 15-mers from the AAV peptide library; PMA, phorbol 12-myristate 13-acetate (positive control); Sfu, spot-forming units. *Peptides positive but not further confirmed. (b, d, f) Peptides identified with relative HLA restriction. N/D, not determined.
Mentions: The large number of cells isolated and stored from each spleen, in combination with bioinformatics algorithms, allowed us to specify and confirm single 9-mer peptides as capsid T-cell epitopes experimentally (Figure 1; Supplementary Figure S1) and to define their predicted binding affinity to the cognate MHC class I molecules (Table 2). We also gained insight into the frequency of subjects responding to a specific epitope (Table 2; Supplementary Figure S2). All epitopes identified with the ELISpot assay exhibited a binding affinity to MHC class I of at least 50% of the maximum binding affinity of the consensus sequence defined by the position-specific scoring matrix (PSSM) for the cognate HLA allele.23 Most, but not all epitopes, could be confirmed, and there was no obvious relationship between the binding affinity score for a specific HLA allele and the frequency of subjects responding to the specific epitope (Table 2). Not all epitopes could be confirmed because bioinformatics analysis was restricted to HLA alleles present in the databases, and thus, further analysis and specification of some reactive peptides was not possible based on the rare haplotype (Table 2, footnote d). In several instances, subjects sharing the same HLA alleles showed reactivity to the same AAV-derived epitopes. In one instance, two different epitopes binding to the same HLA (HLA-B*51, Table 2) were identified.

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