Peripheral mononuclear cells (PBMCs) were collected from apheresis samples of 16 healthy donors under protocols approved by the National Institutes of Health Institutional Review Board (CBER-047). Samples were isolated using gradient-density separation by Ficoll- Hypaque (GE Healthcare, Chicago, Illinois) and frozen in liquid nitrogen using RPMI media (Thermo Fisher Scientific) supplemented with 10% heat-inactivated human AB serum (MilliporeSigma) and 7.5% DMSO (Thermo Fisher Scientific).

All peptides were purified to >95% homogeneity by high-performance liquid chromatography (HPLC) (GenScript Biotech, New Jersey), and their composition and deamidation were confirmed twice by mass spectrometry, once during manufacture release and once after DMSO reconstitution and a freeze thaw cycle. Peptides were resuspended 0.05% DMSO and RPMI in a concentration of 20 µg/ml.

PBMCs were thawed, resuspended in in RPMI media containing 5% heat-inactivated human serum, 1% Glutamax (Thermo Fisher Scientific), 1 mM sodium pyruvate (Thermo Fisher Scientific), 10 mM HEPES (Thermo Fisher Scientific), MEM Non-Essential Amino Acids (Thermo Fisher Scientific), and 1% penicillin/streptomycin (Thermo Fisher Scientific) at a concentration of 5×10⁶ cells/mL. Cells were stimulated with 5 μg/mL of WT or deamidated peptide pool and supplemented with 20 units of IL-2 (MilliporeSigma), 5 μg/mL of IL-7 (Biolegend, San Diego, CA), and 25 μg/mL of IL-15 (Biolegend) every 3-4 days for 10 days by gentle pipetting.

After the in vitro expansion step, cells were harvested, washed, and plated in ELISpot plates that were pre-coated with anti-human IL-2 or IFN-γ antibodies (Mabtech, Cincinnati, OH). Cells were re-stimulated with 5 μg/mL of WT or deamidated peptides and incubated for 24 hours at 37°C. Negative controls were treated with medium, and positive controls were treated with phytohemagglutinin (PHA, MilliporeSigma). Secretions of IL-2 and IFN-γ following stimulation with peptides were detected using a secondary biotinylated anti-IL-2 or anti-IFN-γ antibody (Mabtech) followed by streptavidin, alkaline phosphatase conjugate (SA-ALP) (Mabtech), nitro blue tetrazolium and 5-bromo-4-chloro-3’-indolyl phosphate (BCIP/NBT) substrate (KPL, Thermo Fisher Scientific). We used the computer software (Immunospot 7.0; Cellular Technology Limited, Cleveland, OH) to measure the number of spots forming cells (SFC). The assay was performed in triplicate and repeated at least once for each donor.

After the in vitro expansion step, cells were harvested, washed, and stained with Brilliant Violet (BV) 510-conjugated CD4 (clone SK3), fluorescein isothiocyanate (FITC)-conjugated CD3 (clone UCHT1), and R-phycoerythrin (R-PE)-conjugated CD154 (clone 24-31) for 20 minutes at 4°C. For monocyte isolation, freshly thawed PBMCs were stained with BV510-conjugated CD14 (clone M5E2). Cells were then washed twice and sorted using a BD FACSAria cell sorter by gating on CD3⁺CD4⁺CD154⁺ or CD14⁺ cells. Resulting cell fractions were washed and resuspended in 10% human serum containing media. In 96 well ELISpot plates, 25,000 of CD154⁺CD4⁺ T cells and 5000 of CD14⁺ cells were co-cultured with peptide stimulation, and ELISpot testing were performed as described above.

AAV9 vectors were produced in free style 293F cells (Thermo Fisher Scientific, San Jose, CA) using a triple transfection method as previously described (17) and purified with iodixanol ultracentrifugation methods. AAV vector titration and purity was performed by real-time quantitative PCR (qPCR) and ELISA (PRAAV9, Progen, PA), followed by silver staining (Thermo Fisher Scientific). For qPCR, the following primers and probes were used: EGFP (Forward: 5/- AGCAAAGACCCCAACGAGAA-3/, Reverse: 5/- GGCGGCGGTCACGAA-3/and Probe: 5/- 6FAM-CGCGATCACATGGTCCTGCTGG-TAMRA-3/.

Pre-existing anti-AAV9 antibodies in the healthy human serum were measured by standard ELISA. 96-well half area ELISA plates (Thermo Fisher) were coated overnight with WT or mutated (N57D, N329D, N452D, and N512D) AAV9 in boric acid buffer at a final concentration of 5 ×10¹⁰ vp/well. Plates were blocked with blocking buffer (PBS+3% bovine serum albumin) for 2 hours. Plates were then incubated with anti-human IgG antibodies conjugated with HRP (Jackson lab, Bar Harbor, ME) for 1 hour, followed by enzymatic development (3,3′,5,5′-tetramethylbenzidine (TMB)).

Data are represented as the group mean ± SD. Statistical significance (α=0.05) was tested using GraphPad Prism, version 7.0 (GraphPad Software, San Diego, CA). Differences between groups were examined for statistical significance using ANOVA with Tukey’s multiple comparisons test.

Prior studies have shown that recombinant AAV (rAAV) vectors undergo a high degree of spontaneous deamidation (16), and that this results in either reductions or enhancements in the immunogenicity of rAAV (17). This finding prompted us to investigate whether deamidated AAV cross-reacts with WT AAV. To investigate if T cells stimulated by deamidated peptides would cross-react and respond to WT peptides in an ELISpot assay, we enriched PBMCs from 16 healthy donors with WT or deamidated peptides and restimulated the cells with the reciprocal peptides. Figure 1 shows 4 representative responses from donor #17 ( Figure 1A ), #63 ( Figure 1B ), #4 ( Figure 1C ), and #36 ( Figure 1D ) that responded to T cell epitopes in deamidation sites (N57, N329, N452, and N512). In donor #17, PBMCs that were expanded with WT or deamidated peptides and restimulated with the same peptides had strong IL-2 and IFN-γ responses. However, these responses were significantly decreased when the same cells were restimulated with reciprocal peptides ( Figure 1A ). In donor #63, PBMCs that were expanded with WT peptides and restimulated with deamidated peptides showed substantially lower IL-2 responses, whereas the opposite setting resulted in no differences ( Figure 1B ). In the other donor (#4, Figure 1C ), there were significant differences when cells were expanded with deamidated peptides and restimulated with WT peptides. A different trend was observed in donor #36. PBMCs that were expanded with WT or deamidated peptides only responded to WT peptides, but not to deamidated peptides, regardless of the expansion ( Figure 1D ). This means that in cellular immune monitoring settings, donors #17 and #4 could have a false negative result to a deamidated AAV.

We further investigated the cross-reactivity of WT and deamidated peptides for other NG sites in other AAV serotypes. Deamidated peptides at N383 (from AAV1, 6, 8, 10) and N540 (from AAV8) showed low responses in IL-2 secretion in PBMC expanded with WT peptides, whereas WT peptides resulted in strong IL-2 production. There were no differences or responses in the opposite setting (data not shown).

To compare the responses of multiple donors, we calculated a cross-reactivity ratio (IL-2 spots after cognate stimulation/IL-2 secretion after reciprocal stimulation). A cross-reactivity ratio close to 1 indicates that WT peptides can successfully detect cellular response to WT or deamidated AAV. A cross-reactivity ratio that are close to 0 indicates that WT peptides do not cross react with deamidated peptides and that immune monitoring could miss a response to this peptide. Figure 2 and Table 1 show the cross-reactivity ratio for 16 samples. We observed low cross reactivity (<0.5) in the majority of the samples which indicates that a high level of deamidation of AAV therapy may result in underestimation or even failure to detect immune responses against these peptides during cellular immune monitoring.

We have reported that deamidation sites (NG) contain CD4 T cell epitopes (17). To determine whether AAV reactive CD4 T cell that were activated by deamidated peptides react with WT peptides, we isolated activated CD154⁺CD4⁺ T cells after stimulation with deamidated peptides and re-stimulated them with WT peptides or deamidated peptides. Figure 3 shows a representative response from donor #6. In this example, isolated CD154⁺CD4⁺ cells that were expanded with deamidated peptides and then restimulated with the same deamidated peptide showed 2.9-fold and 3.2-fold more spots than media control in IFN-γ and IL-2 ELISpot assays, respectively. Restimulating these cells with reciprocal WT peptides, on the other hand, did not show any significant responses, further indicating that WT peptides could fail to detect CD4 T cell response activated by deamidated epitopes in the capsid.

We hypothesize that the risk of a false negative response in cellular immune monitoring assays due to the low cross reactivity between WT and deamidated peptides can be mitigated by spiking the few deamidated peptides to the mixture of WT peptides. To test this hypothesis, we compared the cellular response to WT and deamidated peptides to a mixture of both WT and deamidated peptides in three, three, and five donors for each of the NG sites, N57, N329, and N452, respectively that previously demonstrated a low cross-reactivity ratio of <0.5 ( Figure 2 ). Figure 4 shows representative responses of three donor samples after stimulation with peptides N57, N329, and N452. In most of the donors, enriched PBMCs with deamidated peptide pools responded to a peptide mixture of WT and deamidated peptides at a level comparable to the deamidated peptide alone despite the fact that the response to WT peptides in these cells was significantly lower. These results suggest that including deamidated peptides in the peptide master pools used in immune monitoring assays would improve the assay’s sensitivity. Of note, there were a few donors with comparable cross-reactivity between WT and deamidated peptides (data not shown). This fact can be explained by donor-to-donor variation which can be attributed to different HLA background of some donors or prior exposure of these donors to WT AAV9 virus.

Testing pre-existing antibodies prior to gene transfer therapy is highly recommended due to the potential of antibody-mediated toxicity following AAV gene transfer. To investigate whether deamidated AAV could affect the sensitivity of detecting pre-existing anti-AAV9 antibodies, we compared the anti-AAV9 Ab titers in a binding ELISA assay using WT AAV9 and deamidated AAV9. To make 100% deamidated AAV9, four NG sites were mutated to DG (deamidated AAV9) ( Supplementary Figure 1 ). The amino acid mutations in the four NG sites dramatically decreased the yield of the recombinant deamidated AAV9 vectors compared to WT AAV9 capsid ( Supplementary Figure 1 ), as well as increased the ratio of empty and full AAV particles ( Supplementary Table 1 , Caspid/viral genome ratio) and its potency ( Supplementary Figure 2 ). Freshly made WT AAV9 and deamidated AAV9 were coated onto a plate within 5-7 days of production, serum samples from 16 healthy donors were then added and serially diluted to detect the pre-existing antibodies against AAV9. Figure 5A shows that most serum samples had pre-existing antibodies to AAV9 but there were no significant differences in the antibody titers between two assays using WT and deamidated AAV9 for most of the samples. Notably, if pre-existing Ab titers against WT AAV9 were high, its titer against mutant AAV were much higher ( Figure 5B ), implying several hypotheses: 1) natural AAV9 may be spontaneously deamidated, and 2) high anti-AAV9 titer against deamidated AAV was a result of antibody cross reactivity to other AAV serotypes that already have DG amino acid sequence such as AAV1, AAV2, AAV3, AAV6, AAV7, AAV8 or AAV13 ( Supplementary Figure 3 ).