Identification of mouse AAV capsid-specific CD8+ T cell epitopes Denise E. Sabatino, Federico Mingozzi, Daniel J. Hui, Haifeng Chen, Peter Colosi, Hildegund C.J. Ertl, Katherine A. High  Molecular Therapy  Volume 12, Issue 6, Pages 1023-1033 (December 2005) DOI: 10.1016/j.ymthe.2005.09.009 Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 1 Identification of the AAV-2 capsid epitope in C57BL/6 mice. (A) Intracellular cytokine staining of cells from C57BL/6 mice immunized with Ad-AAV-2 capsid. Cells were incubated with no peptide (negative control), six pools of peptides, and PMA/ionomycin (positive control) (not shown) to identify pool F as containing epitopes. Staining was done using an anti-mouse CD8+ antibody conjugated to FITC and an anti-mouse IFN-γ antibody conjugated to PE to determine frequencies of IFN-γ-producing CD8+ T cells by flow cytometry analysis. The % in each data plot represents the percentage of CD8+ T cells that secreted IFN-γ. (B) Intracellular cytokine staining of matrix pools of peptides from pool F. Eleven matrix pools were screened to identify the individual peptides that contain the epitope. (C) Matrix of pool F. Matrix pools 4, 5, and 10 implicate peptides 140 (PEIQYTSNYNKSVNV) and 141 (TSNYNKSVNVDFTVD) as containing the epitope. (D) To identify the epitope (9-mer), a panel of seven 9-mers offset by one amino acid was screened to identify SNYNKSVNV as the epitope. No peptide (negative control) and PMA/ionomycin (positive control) (36.6%) are shown. *Positive peptide. Molecular Therapy 2005 12, 1023-1033DOI: (10.1016/j.ymthe.2005.09.009) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 2 Identification of the AAV-2 capsid epitope in BALB/c mice. (A) ICS performed on cells from BALB/c mice immunized with Ad-AAV-2 capsid. Six pools containing the AAV-2 capsid peptide library were screened to identify one positive pool (pool D). (B) A matrix of pool D implicated peptides 74 and 75 as containing an epitope (data not shown). This was followed by a screen of 9-mers derived from the positive 15-mer sequences. The negative control was no peptide and the positive control was PMA/ionomycin (41.1%) (data not shown). Peptide VPQYGYLTL had the most robust response and represents the epitope. Staining was done using an anti-mouse CD8+ antibody conjugated to FITC and an anti-mouse IFN-γ antibody conjugated to PE to determine frequencies of IFN-γ-producing CD8+ T cells by flow cytometry analysis. The % in each data plot represents the percentage of CD8+ T cells that secreted IFN-γ. Molecular Therapy 2005 12, 1023-1033DOI: (10.1016/j.ymthe.2005.09.009) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 3 Identification of the AAV-8 capsid epitopes in C57BL/6 mice. (A) ICS was used to screen the entire AAV-8 peptide library using a matrix approach (data not shown). Eight peptides were screened individually by ICS. Staining was done using an anti-mouse CD8+ antibody conjugated to FITC and an anti-mouse IFN-γ antibody conjugated to PE. Peptides 171–174 had CD8+ T cells that were positive for IFN-γ after the initial screen. The % in each data plot represents the percentage of CD8+ T cells that secreted IFN-γ. (B) A panel of 9-mers contained within 171–174 was screened along with no peptide (negative control) and PMA/ionomycin (positive control) (21.8%), peptide 171–174 as a peptide pool, and the panel of 9-mers. This screen of 9-mers identified NSLANPGIA as having the most robust response. Molecular Therapy 2005 12, 1023-1033DOI: (10.1016/j.ymthe.2005.09.009) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 4 Identification of the AAV-8 capsid epitope in BALB/c mice. A peptide library was screened using a matrix approach to identify the AAV-8 capsid epitope in BALB/c mice. Peptides 124 and 125 were positive for IFN-γ secretion and CD8 after the screen of the matrix. Individual peptides (9-mers) were generated that are contained within the 124–125 peptide sequence and were screened along with no peptide (negative control), PMA/ionomycin (positive control) (data not shown), and the peptides 124 and 125 by ICS. Staining was done using an anti-mouse CD8+ antibody conjugated to FITC and an anti-mouse IFN-γ antibody conjugated to PE. The % in each data plot represents the percentage of CD8+ T cells that secreted IFN-γ. The peptide identified as the epitope with the most robust response was IPQYGYLTL. Molecular Therapy 2005 12, 1023-1033DOI: (10.1016/j.ymthe.2005.09.009) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 5 AAV-induced T cells cross-react with peptides for different serotypes. (A) BALB/c mice were immunized with Ad-AAV-2 capsid (top) or Ad-AAV-8 capsid (bottom). The splenocytes were harvested 9 days after immunization and were incubated with the identified epitope and the corresponding peptide sequence from the alternate serotype. That is, splenocytes from BALB/c mice immunized with Ad-AAV-2 were incubated with the AAV-2 epitope (VPQYGYLTL) and the corresponding AAV-8 peptide sequence (IPQYGYLTL). In this case the corresponding AAV-8 peptide sequence is also the AAV-8 capsid immunodominant epitope. (B) C57BL/6 mice were immunized with Ad-AAV-2 capsid (top) or Ad-AAV-8 capsid (bottom). The splenocytes from these mice were incubated with the identified epitope and the corresponding peptide sequence from the alternate serotype. (C) The specific peptide sequences used for the experiment are listed. Molecular Therapy 2005 12, 1023-1033DOI: (10.1016/j.ymthe.2005.09.009) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 6 Relative affinities of the AAV epitopes for the MHC I molecule as determined by concentration gradient. C57BL/6 and BALB/c mice were immunized with Ad-AAV-2 or Ad-AAV-8 and 9 days later ICS was performed on harvested splenocytes. The following peptide concentrations were analyzed: 5, 1, 0.2, 0.04, 0.008, 0.00016, 0.000032, and 0 μg/ml. The AAV-2 epitopes are identified as solid lines and the AAV-8 epitopes are identified as dashed lines. (A) Concentration gradient of C57BL/6 mouse AAV epitopes. The AAV-2 SNY (SNYNKSVNV) epitope had a high relative affinity, whereas the AAV-2 NGR (NGRDSLVNPGPAMAS) epitope did not demonstrate a pronounced affinity curve. The AAV-8 NSL (NSLANPGIA) epitope had a higher relative affinity than the AAV-8 LTS epitope (LTSEEEIKTTNPVAT); i.e., higher doses of peptides were needed to induce a response. (B) Concentration gradient of BALB/c AAV epitopes. The AAV-2 VPQ (VPQYGYLTL) epitope had higher relative affinity compared to the other epitopes, AAV-2 FRP (FRPKRLNFKLFNIQV) and AAV-2 HSQ (HSQSLDRLMNPLIDQ). The AAV-2 VPQ (VPQYGYLTL) and AAV-8 IPQ (IPQYGYLTL) epitopes that are homologous peptides had similar affinity for the MHC molecule. Molecular Therapy 2005 12, 1023-1033DOI: (10.1016/j.ymthe.2005.09.009) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions