Circulating anti-wild-type adeno-associated virus type 2 (AAV2) antibodies inhibit recombinant AAV2 (rAAV2)-mediated, but not rAAV5-mediated, gene transfer in the brain

Abstract

Epidemiological studies report that 80% of the population maintains antibodies (Ab) to wild-type (wt) adeno-associated virus type 2 (AAV2), with 30% expressing neutralizing Ab (NAb). The blood-brain barrier (BBB) provides limited immune privilege to brain parenchyma, and the immune response to recombinant AAV (rAAV) administration in the brain of a naive animal is minimal. However, central nervous system transduction in preimmunized animals remains unstudied. Vector administration may disrupt the BBB sufficiently to promote an immune response in a previously immunized animal. We tested the hypothesis that intracerebral rAAV administration and readministration would not be affected by the presence of circulating Ab to wt AAV2. Rats peripherally immunized with live wt AAV2 and naive controls were tested with single intrastriatal injections of rAAV2 encoding human glial cell line-derived neurotrophic factor (GDNF) or green fluorescent protein (GFP). Striatal readministration of rAAV2-GDNF was also tested in preimmunized and naive rats. Finally, serotype specificity of the immunization against wt AAV2 was examined by single injections of rAAV5-GFP. Preimmunization resulted in high levels of circulating NAb and prevented transduction by rAAV2 as assessed by striatal GDNF levels. rAAV2-GFP striatal transduction was also prevented by immunization, while rAAV5-GFP-mediated transduction, as assessed by stereological cell counting, was unaffected. Additionally, inflammatory markers were present in those animals that received repeated administrations of rAAV2, including markers of a cell-mediated immune response and cytotoxic damage. A live virus immunization protocol generated the circulating anti-wt-AAV Ab seen in this experiment, while human titers are commonly acquired via natural infection. Regardless, the data show that the presence of high levels of NAb against wt AAV can reduce rAAV-mediated transduction in the brain and should be accounted for in future experiments utilizing this vector.

Figures

FIG. 1.

(A to C) Experimental design and order of treatments. The different treatments are presented in chronological order as indicated for each experiment. Each experimental treatment group included immunized animals as well as naive controls. The multiple survival intervals shown here were necessary to control for the possibility that rAAV-mediated GDNF expression levels increase over time. Blood was drawn for NAb titer assay at each time point indicated. (A) Single-administration experimental design. All of the animals (n = 40) in this portion of the study received a single 2-μl striatal injection of rAAV-GDNF or sterile saline, as described in Materials and Methods. (B) Readministration experimental design. The animals in this portion of the study (n = 38) received a single 2-μl striatal injection of rAAV-GDNF followed by an identical injection of rAAV-GDNF or sterile saline 2 weeks later in the opposite hemisphere, as described in Materials and Methods. (C) Serotype specificity experimental design. The animals in this portion of the study (n = 26) received a single injection of 2 μl of either rAAV2-GFP or rAAV5-GFP, as described in Materials and Methods section. (D and E) Vector purification. Viral vector stocks were analyzed for the presence of cellular proteins and contaminants by PAGE analysis with silver staining. Lanes MW, molecular weight ladder (in thousands) for identification. VP1, -2, and -3 are the three AAV capsid proteins. (D) Right lane, rAAV-GDNF vector after purification; middle lane, crude stock. (E) Second lane, PBS and buffer as a negative control; third lane, standard rAAV2-CBA-GFP preparation as a positive control; fourth lane, rAAV2-GFP vector after purification; right lane, rAAV5-GFP vector after purification.

FIG. 2.

NAb titers. Individual animal serum samples were serially diluted and incubated with a standard amount of rAAV2 expressing GFP. GFP fluorescence intensity was quantified 28 h after transduction in HeLa cells. Pretreatment sera served as the control for each animal. NAb titers are expressed as the reciprocal of the serum dilution required to exceed 50% GFP expression of the identically diluted control serum. (A) Presurgical baseline titers for both the naive and immunized animals. Each of the animals included in the immunized groups displayed high levels of circulating NAb at the time of surgery, while the groups that were not immunized had very low titers. The median titer for the naive animals was 50 (the lowest level of detection), while the immunized animals had a median titer of 51,200. (B) The postsurgical titers for the single-administration study were similar to the presurgical titers, with the median titer of the single-naive group remaining unchanged at 50 and that the immune-single group also remaining at 51,200. (C) The median postsurgical titers for the repeat-administration study were unchanged at 51,200 and 50 for the immune-repeat group and naive-repeat groups, respectively; however, 5 of 10 naive-repeat animals developed a moderate NAb titer after the second vector administration. (D) The median postsurgical titers for the serotype specificity experiment were 50 in the naive groups and 51200 in the immunized groups.

FIG. 3.

Intrastriatal GDNF expression as determined by ELISA and immunohistochemistry. (A) Single-administration experiment. Immunization with wt AAV2 completely blocked GDNF expression at both the 2- and 4-week time points after rAAV2-GDNF striatal transduction. This is demonstrated by the lack of measurable GDNF in the immunized group, compared to the robust expression seen in the naive animals (the asterisk indicates statistical significance between results for naive and immunized animals [P < 0.0001]). Error bars indicate standard errors of the mean. (B) Repeat-administration experiment. Immunization with wt AAV2 completely blocked GDNF expression in both the rAAV2-GDNF readministered and rAAV2-GDNF-saline groups. This is demonstrated by the lack of measurable GDNF in both hemispheres of the immunized group, compared to the robust expression seen in the naive animals (the asterisk indicates statistical significance between naive and immunized animals [P < 0.0001]). (C to E) Immunohistochemistry of GDNF expression in repeat-administration experiment. Representative striatal sections were immunostained for human GDNF and are oriented with the first injection side to the right. Bar, 1 mm (applies to all panels). (C) Section from immunized animal after rAAV2-GDNF injection in right striatum, followed by a second administration of rAAV2-GDNF in left striatum 2 weeks later. In agreement with the data in panel B, GDNF expression was undetectable. (D) Section from naive animal after rAAV2-GDNF injection in right striatum, followed by a second administration of rAAV2-GDNF in left striatum 2 weeks later. (E) Section from naive animal 4 weeks after rAAV2-GDNF injection in right striatum, with no further treatment. Right and left are reversed in panels D and E.

FIG. 4.

GFP expressed in striatal neurons. (A to D) Native fluorescence of transduced neurons in the striatum. Forty-micrometer tissue sections were mounted on subbed slides for GFP visualization. Representative pictures from each treatment group are presented. (A) Animals that received intrastriatal rAAV2-GFP administration after peripheral immunization with wt AAV2 demonstrated very little to no GFP expression. The bar applies to panels A to D. (A′) Magnified view of the highlighted box in panel A, revealing few fluorescent neurons. (B) Naive animals that received intrastriatal rAAV2-GFP administration after no peripheral immunization demonstrated high levels of GFP expression. (B′) Magnified view of the highlighted box in panel B, showing a dense population of fluorescent neurons. The bar applies to panels A′ to D′. (C) Animals that received intrastriatal rAAV5-GFP administration after peripheral immunization with wt AAV2 also demonstrated high levels of GFP expression. (C′) The magnified view of the highlighted box in panel C is similar to that shown in panel B′, demonstrating numerous GFP-positive neurons. (D) Naive animals that received intrastriatal rAAV5-GFP administration again demonstrated high levels of GFP expression, identical to those seen in panel C. (D′) The magnified view of the highlighted box in panel D matches that shown in panels B′ and C′, with a dense population of GFP-positive neurons. (E and F) Quantification of GFP-positive striatal neurons by stereological cell counts. (E) Immunization with wt AAV2 almost completely blocked GFP expression after rAAV2-GFP striatal transduction. This is demonstrated by the significant reduction in GFP-positive neurons in the immunized group, compared to the robust expression seen in the naive animals (the asterisk indicates statistical significance between naive and immunized animals [P < 0.0001]). Error bars indicate standard errors of the means. (F) Immunization with wt AAV2 had no effect on GFP expression after rAAV5-GFP striatal transduction. This is demonstrated by equal numbers of GFP-expressing cells in the immunized group and the naive animals (P > 0.99).

FIG. 5.

Striatal sections immunostained with DARPP-32 and counterstained with hematoxylin. The brown reaction product is DARPP-32, a specific striatal neuronal marker, which is reduced in response to striatal damage. Hematoxylin is a classical nuclear stain used to detect infiltrating leukocytes. Bar in panel D, 500 μm (also applies to panels A, B, E, G, and H). Bar in panel F, 100 μm (also applies to panels C and I). The white boxes in panels B, E, and H illustrate the areas of higher magnification shown in panels C, F, and I, respectively. (A to C) Immunized animal that received rAAV2-GDNF injection in the right striatum (A), followed by a second administration of rAAV2-GDNF in the left striatum 2 weeks later (B). Significant leukocyte infiltration is seen in both hemispheres in immunized animals. Note the association of the infiltrate with blood vessels. Likewise, the DARPP-32 staining is greatly diminished in the injection site (seen in panels A to C compared to D to F and G to I), indicating profuse tissue damage. (D to F) Section from a naive animal after rAAV2-GDNF injection in the right striatum (D), followed by a second administration of rAAV2-GDNF in the left striatum 2 weeks later (E). Leukocyte infiltration is more prevalent in the second injected hemisphere in naive animals. As in panel C, the infiltration seen in panel F is associated with a blood vessel (top of panel). Similarly, DARPP-32 staining was reduced only in the area of the second injection seen in panel F. (G to I) Section from an immunized animal after rAAV2-GDNF injection in the right striatum (G), followed by an injection of sterile saline in the left striatum 2 weeks later (H). Minimal infiltration accompanied the needle tract, and no reduction of DARPP-32 is visible. No sections from the single-injection study are depicted here, but all of the animals (both immunized and naive) had staining similar to that for the saline injection pictured in panels G and H. Left and right are reversed in panels A, B, D, E, G, and H.

FIG. 6.

Striatal sections immunostained for GFAP. The brown reaction product is GFAP, a specific marker for reactive astrocytes that are normally activated in response to inflammation. Significant tissue reaction was observed only in the animals in the readministration experiment; therefore, all sections presented here are representative of the readministration study. Bar in panel D, 500 μm (also applies to panels A, B, E, G, and H). Bar in panel F, 100 μm (also applies to panels C and I). The black boxes in panels B, E, and H illustrate the areas of higher magnification shown in panels C, F, and I, respectively. (A to C) Immunized animal that received rAAV2-GDNF injection in the right striatum (A), followed by a second administration of rAAV2-GDNF in the left striatum 2 weeks later (B). Significant reactive astrocytosis is seen in the second injection site in the immunized animals and radiates from the area of the injection. (D to F) Section from a naive animal after rAAV2-GDNF injection in the right striatum (D), followed by a second administration of rAAV2-GDNF in the left striatum 2 weeks later (E). Significant reactive astrocytosis is again prevalent in the second injected hemisphere in naive animals. (G to I) Section from a naive animal after rAAV2-GDNF injection in the right striatum (G), followed by an injection of sterile saline in the left striatum 2 weeks later (H). Reactive astrocytosis, in this case, is limited to the needle tract. Left and right are reversed in panels A, B, D, E, G, and H.

FIG. 7.

Striatal sections immunostained for activated microglia (OX-42). The brown reaction product is OX-42, a specific marker for activated microglia observed in response to inflammation. Significant tissue reaction was observed only in the animals in the readministration experiment; therefore, all sections presented here are representative of the readministration study. Bar in panel D, 500 μm (also applies to panels A, B, E, G, and H). Bar in panel F, 100 μm (also applies to panels C and I). The white boxes in panels B, E, and H illustrate the areas of higher magnification shown in panels C, F, and I, respectively. (A to C) Immunized animal that received rAAV2-GDNF injection in the right striatum (A), followed by a second administration of rAAV2-GDNF in the left striatum 2 weeks later (B). Significant reactive microgliosis is seen in both hemispheres in immunized animals and radiates from the area of the injection. (D to F) Section from a naive animal after rAAV2-GDNF injection in the right striatum (D), followed by a second administration of rAAV2-GDNF in the left striatum 2 weeks later (E). Significant reactive microgliosis is more prevalent in the second injected hemisphere in naive animals. (G to I) Section from a naive animal after rAAV2-GDNF injection in the right striatum (G), followed by an injection of sterile saline in the left striatum 2 weeks later (H). Reactive microgliosis, in this case, is limited to the needle tract. Left and right are reversed in panels A, B, D, E, G, and H.

FIG. 8.

Striatal sections immunostained for MHC1, double stained for CD8α, and imaged with confocal microscopy. The green label is rt1-a, a rat-specific MHC1 marker. MHC1 is normally upregulated in virally infected cells. Activated cytotoxic T cells expressing CD8 (alpha subunit), a coreceptor for MHC1 complexes, are labeled in red. All areas injected with rAAV were positive for MHC1 in various degrees, with prominent double staining observed only in the readministration study. For consistency with other figures, all sections presented here are representative of the readministration study. Bar in panel D, 500 μm (also applies to panels A, B, E, G, and H). Bar in panel F, 100 μm (also applies to panels C and I). The white boxes in panels B, E, and H illustrate the areas of higher magnification shown in panels C, F, and I, respectively. (A to C) Immunized animal that received rAAV2-GDNF injection in the right striatum (A), followed by a second administration of rAAV2-GDNF in the left striatum 2 weeks later (B). Significant MHC1 expression and CD8+ infiltration persists in both hemispheres in immunized animals. Note the association of the infiltrate with blood vessels. (D to F) Section from a naive animal after rAAV2-GDNF injection in the right striatum (D), followed by a second administration of rAAV2-GDNF in the left striatum 2 weeks later (E). Significant MHC1 expression and CD8+ infiltration is more prevalent in the second injected hemisphere in naive animals. As in panel C, the infiltration seen in panel F is associated with a blood vessel (just to the right of the white box). (G to I) Section from a naive animal after rAAV2-GDNF injection in the right striatum (G), followed by an injection of sterile saline in the left striatum 2 weeks later (H).