Prevalence of Anti-Adeno-Associated Virus Immune Responses in International Cohorts of Healthy Donors

Abstract

Preexisting immunity against adeno-associated virus (AAV) is a major challenge facing AAV gene therapy, resulting in the exclusion of patients from clinical trials. Accordingly, proper assessment of anti-AAV immunity is necessary for understanding clinical data and for product development. Previous studies on anti-AAV prevalence lack method standardization, rendering the assessment of prevalence difficult. Addressing this need, we used clinical assays that were validated according to guidelines for a comprehensive characterization of anti-AAV1, -AAV2, -AAV5, and -AAV8 immunity in large international cohorts of healthy donors and patients with hemophilia B. Here, we report a higher than expected average prevalence for anti-AAV8 (∼40%) and anti-AAV5 (∼30%) neutralizing antibodies (NAbs), which is supported by strongly correlating anti-AAV IgG antibody titers. A similar anti-AAV8 NAb prevalence was observed in hemophilia B patients. In addition, a high co-prevalence of NAbs against other serotypes makes switching to gene therapy using another serotype difficult. As anti-AAV T cell responses are believed to influence transduction, we characterized anti-AAV T cell responses using interleukin-2 (IL-2) and interferon-γ (IFN-γ) ELISpot assays, revealing a similar prevalence of IFN-γ responses (∼20%) against different serotypes that did not correlate with NAbs. These data, along with the long-term stability of NAbs, emphasize the need to develop strategies to circumvent anti-AAV immunity.

Keywords: AAV; adeno-associated virus; anti-AAV T cell response; anti-AAV antibody response; anti-AAV prevalence; clinical assays; gene therapy; international cohorts.

Figure 1

Antibody Response against AAV8 Prevalence and titers of NAbs, IgG, and IgM against AAV8 in cohorts of healthy donors and patients with hemophilia B from different geographical regions. (A) NAb prevalence (% of donors) against AAV8 in 180 healthy donors (EU1 cohort: n = 60, EU2 cohort: n = 33, EU3 cohort: n = 27, and US1 cohort: n = 60) and 29 patients with hemophilia B (US hem cohort). (B) The percentage of NAb-positive donors with IgG or IgM detected by ELISA with a starting dilution of 1:20. For 1% IgM single positive (one donor), IgG was detectable in the IgG ELISA with 1:5 as a starting dilution. (C) IgG and IgM against AAV8 in the 180 healthy donors (group and n as in A), indicating that switched IgG antibodies were predominant. (D) Correlation between NAbs and IgG against AAV8 (linear regression: n = 180, R² = 0.8499, p < 0.0001). (E) Distribution of IgG subclasses. From 90 healthy donors, IgG subclasses were analyzed in all samples positive for NAb and IgG (EU1: n = 8, EU2: n = 10, and US1: n = 6). (F) Correlation between NAbs and IgG subclasses. Linear regression of NAb titers to IgG subclass titers was calculated; R² values and significance of the correlations are depicted in the graph; ****p < 0.0001; ns: not significant.

Figure 2

Co-prevalence of Antibody Responses against Different AAV Serotypes (A) Prevalence (% of donors) of NAbs against AAV8, AAV2, AAV5, and AAV1 in healthy donors and patients with hemophilia B from different regions. nd, not determined. (B) Co-prevalence (% of donors) of NAbs against AAV8, AAV2, and AAV5. The graph includes all donors from the EU1, EU2, EU3, and US2 cohorts from (A) that were positive for at least one of the serotypes. (C) The proportion of NAb-positive donors with IgG, suggesting that NAbs against AAV8 and AAV5 are always class switched. IgG ELISA was carried out with a starting dilution of 1:5. (D) Correlation between NAbs and IgG against AAV5 (linear regression: n = 180, R² = 0.8652, p < 0.0001).

Figure 3

Prevalence of T Cell Responses against Different AAV Serotypes Frequency of capsid-specific (A) IFN-γ- or (B) IL-2-secreting T cells detected using ELISpot analysis in PBMCs from 90 healthy donors. PBMCs were stimulated with AAV8, AAV5, or AAV2 peptide pools for 18 to 24 h. The percentage of donors positive for each peptide pool is depicted, showing a similar prevalence for all serotypes. (C) Table showing percentage of donors with detectable IFN-γ and IL-2 T cell responses against AAV2, AAV5, and AAV8.

Figure 4

Correlation between T Cell and Antibody Responses against Different AAV Serotypes AAV-specific T cells were analyzed using an IFN-γ ELISpot assay in PBMCs from 90 healthy donors (the same donors as in Figure 3). The prevalence of T cells directed against AAV8, AAV5, and AAV2 was compared between (A) NAb-positive and NAb-negative or (B) IgG-positive and IgG-negative donors, indicating no correlation between T cell and antibody responses against AAV. The tables depict the proportion of IFN-γ-positive donors from each column of the figure.

Figure 5

Development of Anti-AAV8 NAb Titers over Time (A) NAb titers from 30 healthy donors were analyzed at least three times during the course of up to 3 years. The figure shows the serological status of donors at the first time point. Within the donors that did not seroconvert (n = 24), 16 donors were negative, 2 donors had borderline NAb titers, and 6 had NAb titers >1:10 at the first time point. In contrast, none of the donors that seroconverted (n = 6) had an NAb titer >1:10. One of these donors was negative and 5 had borderline NAb titers at the first time point. (B) Fluctuations in NAb titer of the donors who seroconverted over time. The dashed line shows the seroconversion threshold (assay cutoff).