AAV-mediated delivery of optogenetic constructs to the macaque brain triggers humoral immune responses

Abstract

Gene delivery to the primate central nervous system via recombinant adeno-associated viral vectors (AAV) allows neurophysiologists to control and observe neural activity precisely. A current limitation of this approach is variability in vector transduction efficiency. Low levels of transduction can foil experimental manipulations, prompting vector readministration. The ability to make multiple vector injections into the same animal, even in cases where successful vector transduction has already been achieved, is also desirable. However, vector readministration has consequences for humoral immunity and gene delivery that depend on vector dosage and route of administration in complex ways. As part of optogenetic experiments in rhesus monkeys, we analyzed blood sera collected before and after AAV injections into the brain and quantified neutralizing antibodies to AAV using an in vitro assay. We found that injections of AAV1 and AAV9 vectors elevated neutralizing antibody titers consistently. These immune responses were specific to the serotype injected and were long lasting. These results demonstrate that optogenetic manipulations in monkeys trigger immune responses to AAV capsids, suggesting that vector readministration may have a higher likelihood of success by avoiding serotypes injected previously.NEW & NOTEWORTHY Adeno-associated viral vector (AAV)-mediated gene delivery is a valuable tool for neurophysiology, but variability in transduction efficiency remains a bottleneck for experimental success. Repeated vector injections can help overcome this limitation but affect humoral immune state and transgene expression in ways that are poorly understood. We show that AAV vector injections into the primate central nervous system trigger long-lasting and serotype-specific immune responses, raising the possibility that switching serotypes may promote successful vector readministration.

Keywords: AAV vectors; neutralizing antibodies; nonhuman primate; optogenetics.

Fig. 1.

Blood draw and adeno-associated viral vector (AAV) injection timeline. Three rhesus monkeys received AAV vector injections (white triangles; also see Table 1) as part of optogenetic experiments. Blood samples were drawn (black triangles) before and after injections, and sera were collected for testing. Sera contributing to the data shown in Figs. 3–5 are highlighted (black dots).

Fig. 2.

Flow cytometry methods. Cells were analyzed by fluorescence activated cell sorting. A: events were gated to exclude cellular debris and large aggregates of cells (gray polygon). B: events were also gated to exclude doublets of cells (gray rectangle). C: events that passed both gating procedures were analyzed for green fluorescent protein (GFP) signal; those that achieved a criterion level of GFP expression (gray line) were classified as GFP-positive.

Fig. 3.

Comparing neutralizing antibodies (NAbs) with AAV before and after vector injections. Sera collected before and after injections into the brain of 3 monkeys were analyzed for NAbs to the AAV serotype that was injected. A–G: preinjection sera (gray) tested negative for NAbs to AAV. The only exception was the preinjection serum for monkey S (in D), which was collected ~3 yr after an injection of AAV1. Postinjection sera (black) contained NAbs to AAV. We quantified NAbs in each serum using the serum dilution corresponding to a 50% decrement in the percentage of GFP-positive cells from its maximal value (D₅₀) (triangles, see materials and methods). Error bars represent SD.

Fig. 4.

Serotype specificity of NAbs. A–C: sera collected before and after injections into the brain of 2 monkeys were analyzed for NAbs to AAV serotypes that were not injected. Plotting conventions are as in Fig. 3.

Fig. 5.

Testing for cross reactivity of NAbs to AAV2. A–C: sera collected before and after injections into the brain of 3 monkeys were analyzed for NAbs to AAV2, a serotype that was not injected. Plotting conventions are as in Fig. 3.

Fig. 6.

Time course of AAV immune responses. Sera collected from 3 monkeys at different time points relative to a single vector injection, or repeated vector injections (triangles; only for monkey F), were analyzed for NAbs to the serotype that was injected.