Systemic and local immune responses to intraocular AAV vector administration in non-human primates

Abstract

Positive clinical outcomes in adeno-associated virus (AAV)-mediated retinal gene therapy have often been attributed to the low immunogenicity of AAVs and immune privilege of the eye. However, several recent studies have shown potential for inflammatory responses. The current understanding of the factors contributing to inflammation, such as the pre-existence of serum antibodies against AAVs and their contribution to increases in antibody levels post-injection, is incomplete. The parameters that regulate the generation of new antibodies in response to the AAV capsid or transgene after intraocular injections are also insufficiently described. This study is a retrospective analysis of the pre-existing serum antibodies in correlation with changes in antibody levels after intraocular injections of AAV in non-human primates (NHPs) of the species Macaca fascicularis. In NHP serums, we analyzed the binding antibody (BAB) levels and a subset of these called neutralizing antibodies (NABs) that impede AAV transduction. We observed significantly higher pre-existing serum BABs against AAV8 compared with other serotypes and a dose-dependent increase in BABs and NABs in the serums collected post-injection, irrespective of the serotype or the mode of injection. Lastly, we were able to demonstrate a correlation between the serum BAB levels with clinical grading of inflammation and levels of transgene expression.

Keywords: AAVs; BABs; NABs; adeno-associated viruses; binding antibodies; gene therapy; neutralizing antibodies; non-human primates.

Graphical abstract

Figure 1

Higher basal levels of BABs against AAV8 and AAV9 in NHP serums Mean ± SD of the concentration of BABs against AAV2 (green), AAV5 (red), AAV8 (yellow), and AAV9 (blue) in serums from 41 NHPs before injection. Significance between individual serotypes was tested using Student’s t test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

Figure 2

Dose-dependent increase in levels of BABs and NABs post-injection (A) Schematic representation of the experimental protocol showing the serum collection points before injection (BI) and post-injection (PI) from which the total binding antibodies (BABs) are isolated for testing by ELISA, and a subset of these, the neutralizing antibodies (NABs), are tested by NAB assay. (B) Mean fold change in the concentration of BABs against AAV in n = 26 NHPs at a high dose, n = 9 NHPs at a medium dose, and n = 4 NHPs at a low dose. (C–G) Change in the levels of anti-AAV2 NAB levels and (C′–G′) BAB levels in (C and C′) n = 3 NHPs with high-dose response type 1, (D and D′) n = 16 NHPs with high-dose response type 2, (E and E′) n = 3 NHPs with medium-dose response type 1, (F and F′) n = 4 NHPs with medium-dose response type 2, and (G and G′) n = 2 NHPs with low-dose response. (H) Serum dilution at which 50% of the AAVs are neutralized (H1 and H2: NHPs with high-dose response types 1 and 2, respectively; M1 and M2: NHPs with medium-dose response types 1 and 2, respectively, L: NHPs with low dose). The values for BABs are normalized relative to the BI level (set to 100) and are shown as mean ± SD. The values for NABs at each dilution are normalized relative to the negative control (set to 100) and are shown as mean ± SD. Significance between individual time points was tested using Student’s t test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). High dose: 10–60 × 10¹¹ vg, medium dose: 1–6 × 10¹¹ vg, low dose: 0.1–0.6 × 10¹¹ vg.

Figure 3

Increase in the serum levels of BABs co-relates with clinical signs of inflammation (A and B) OCT images of the retina of (A) NHP 39 that received a high-dose injection and (D) NHP 27 that received a medium-dose injection. (A and B) Fundus image of the injected area, cross-section of the retina close to the fovea indicated by the green line, and the inset demarcated by the dotted lines is shown at day of injection (M0), 1 month (M1), 2 months (M2), and 4 months (M4) or 5 months (M5) post-injection as indicated; arrows point to disruptions in the outer retina. (C) Clinical grading of ocular immune response by evaluation of anterior chamber cells, vitreous cells, opacity, and posterior uveitis from the day of injection (D0) to 5 months post-injection (5M). (D) Serum levels of anti-AAV BABs in NHPs BI and PI grouped by the level of ocular inflammation observed. (E) Serum levels of anti-AAV BABs in NHPs grouped by the level of ocular inflammation and transgene expression (NHPs from Table 1). The values for BABs are shown as mean ± SD. Significance between individual time points was tested using Student’s t test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

Figure 4

No impact of the mode of ocular injection on the serum levels of BABs and NABs (A and B) Schematic representation of the modes of injection. SR, subretinal, IVT, intravitreal. (B–F) Anti-AAV2 NAB levels in (B) n = 2 NHPs that received SR injections, (C and D) n = 12 NHPs that received IVT injections, and (F) n = 2 NHPs that received a combination of SR and IVT injections. (C) BAB levels in n = 6 NHPs that received SR injections, n = 12 animals that received IVT injections, and n = 4 NHPs that received SR + IVT injections. (E) Serum dilution at which 50% of the AAVs are neutralized. The values for NABs are normalized relative to the negative control (set to 100) and are shown as mean ± SD. The values for BABs are normalized relative to the BI level (set to 100) and are shown as mean ± SD. Significance between individual time points was tested using Student’s t test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

Figure 5

Effect of pre-existing antibodies on BAB and NAB production (A–C) Serum concentration of BABs against (A) AAV2 in n = 22 NHPs without (–) and n = 5 NHPs with (+) pre-existing BABs, (B) against AAV5 in n = 7 NHPs without (–) and n = 2 NHPs with (+) pre-existing BABs, and (C) against AAV9 in n = 4 NHPs without (–) and n = 7 NHPs with (+) pre-existing BABs. (D and E) Serum concentration of NABs and BABs against AAV2 in (D) NHP18 and (E) NHP33. (F and G) Serum concentration of NABs and BABs against AAV9 in (F) NHP9 and (G) NHP8. In each case, the BABs and NABs are tested against the same serotype that the animals were injected with. The values for BABs are shown as mean ± SD. The values for NABs at each dilution are normalized relative to the negative control (set to 100) and are shown as mean ± SD. Significance between individual time points was tested using Student’s t test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

Figure 6

Cross-reactivity across serotypes (A–C) NHPs injected with AAV2 and tested BI and PI for serum concentration of binding antibodies (A) against AAV5 in n = 18 NHPs without (–) and n = 9 NHPs with (+) pre-existing BABs, (B) anti-AAV8 in n = 27 NHPs with (+) pre-existing BAB,s and (C) anti-AAV9 in n = 7 NHPs without (–) and n = 20 NHPs with (+) pre-existing BABs. (D–G) NABs against AAV2 and (E and G) BABs against AAV2, AAV5, AAV8, and AAV9 in (D and E) NHP8 and (F and G) NHP9 BI and PI of AAV5 + AAV9. Shown are mean values ± SD. Significance at individual time points was tested using Student’s t test (∗p < 0.05). (H) Phylogeny tree generated from AAV capsid sequences of different serotypes, where the branch lengths are proportional to the evolutionary change (calculated using ClustalW).