The brain is distinguished by its relative immune privilege, including the presence of the BBB and lack of formal antigen-presenting cells (28
). Thus, it provides a unique setting in which to study the effects of preimmunization. Like in the periphery, rAAV has been shown to achieve long-term expression in the brain with negligible inflammation in naive animals (27
). However, readministration studies reveal differences between the brain and the periphery. While readministration in the periphery may not be possible without immune suppression (17
), our data and those of others show that rAAV can be readministered in the brain with no reduction of transgene expression, despite low-level production of Ab to the transgene and the capsid (27
; C. S. Peden and R. J. Mandel unpublished observations). Taken together, our previous experience and the relative immune privilege of the brain led us to doubt that preimmunization or readministration would pose any immunologically relevant risk to rAAV gene transfer in the brain. On the contrary, our data indicate a more significant involvement of the immune system in the brain than previously thought. In this study, immunization completely abolished transduction in all preimmunized animals, regardless of the transgene used. Quantified GDNF production levels demonstrate unilateral transduction failure in preimmunized animals relative to their naive counterparts. Immunohistochemistry verified that GDNF expression was present only in naive animals, with staining absent in immunized animals. Stereological cell counts of rAAV2-induced GFP-positive cells also revealed transduction failure in immunized animals compared to naive controls.
The immunization protocol employed here reproduced accepted immunization paradigms in the human population to induce humoral immunity in an already well-studied animal model. The use of wt virus was important to mimic the types of Ab generated in a natural infection despite the “unnatural” inoculation methods of immunization. Transduction failure occurred in response to immunization with wt virus irrespective of which transgene was present in the recombinant viruses and is therefore a likely result of an immune response, in the form of NAb to the capsid proteins and structure, which are identical in both the recombinant and the wt viruses. This conclusion is also supported by the differential blockade of two recombinant vectors, rAAV2 and rAAV5, bearing identical genomes and differing only in capsid composition. rAAV5-GFP was able to infect cells successfully despite the presence of high levels of NAb to rAAV2-GFP, while rAAV2-GFP was not. The complete blockade of transduction in the absence of active inflammation or any indication of a CMI response is thought to be due entirely to the presence of NAb, as demonstrated by a quantified NAb assay. This is entirely feasible because the intracerebral injection disrupts the BBB for 24 to 48 h, providing high levels of NAb immediate access to the virus, which completes infection in less than 3 h (1
). Some studies that also report failure in peripheral gene transfer protocols allege that NAb generated by preimmunization or previous administration are responsible for the lack of transduction. To date, however, no study could correlate Ab titer with the level of transgene production. Likewise, attempts to correlate NAb titer with levels of GDNF or GFP production in this study have not proved successful, but this may be due to a threshold effect of NAb. Additionally, the circulating NAb are polyclonal and, while collectively quantifiable, may not reflect the same reactivity in individual animals. Finally, future studies will require administration in the presence of various levels of NAb to specifically determine the threshold for transduction.
In the readministration experiment, all groups that expressed GDNF had an equal probability of developing anti-GNDF Ab, but any anti-GDNF Ab produced did not reduce the level of protein in the naive readministration group, which showed equal levels of GDNF at both time points. This is in agreement with the results of Lo et al. (27
), who reported that the presence of low-level Ab generated in response to the transgene protein after intracerebral injection had a minimal effect on protein expression. Additionally, the effective Ab responses for transgene and capsid can be distinguished, because GDNF expression was allowed only in naive animals and immunized animals exhibited no GDNF expression. Furthermore, the virtual homology between human and rat GDNF makes it less likely that GDNF itself would be the target of an Ab response. Similarly, in the rAAV-GFP experiment, anti-GFP Ab could have been formed in any animals that produced GFP. Nonetheless, high numbers of GFP-positive cells were found in all animals except the group that was preimmunized against wt AAV2 and then received an injection of rAAV2-GFP. Therefore, almost certainly the confirmed high levels of NAb to the capsid caused the nearly complete transduction failure of the immunized groups. Consequently, as vector persistence and gene transfer success are paramount in most gene therapy protocols, these data suggest that screening for anti-AAV capsid NAb of specific serotypes should be considered in CNS rAAV-based clinical experiments.
The efficacy issues raised in response to a preimmunization paradigm are entirely separate from the inflammation and possible safety concerns inherent in the readministration results. The lack of inflammation in the single-injection groups was in stark contrast to the significant brain inflammation observed in those animals that received two sequential rAAV administrations. Inflammation was present only in those animals with multiple injections, irrespective of GDNF expression, and similarly, those animals that have a single injection do not have inflammation regardless of GDNF expression. Therefore, inflammation does not correlate with GDNF expression in any way. While the levels of transgene expression were unaffected by readministration, there was significant inflammation at the second surgical site for both naive and immunized animals, with inflammation persisting at the first injection sites of the immunized animals. A recent study does report a significant decrease in transgene expression when readministration was performed 2 weeks later (32
); however, these disparate results may be due to differences in the expression profiles of luciferase and GDNF. While luciferase turns over rapidly, GDNF has an extremely long half-life in the brain (35
). The stability of GDNF may explain the failure to detect a reduction in striatal transgene expression in spite of the presence of significant inflammation as observed in this study. However, transgene expression in the presence of a robust inflammatory response in the brain has been shown repeatedly for adenoviral vectors (6
). In addition, Mastakov et al. (32
) failed to find brain inflammation 4 weeks after their second rAAV injection. Differences in the inflammation profiles in the studies could be due to the longer survival time after the second injection in their study (4 weeks) compared to the survival time after the second injection in the present study (2 weeks). Indeed, we have previously observed that inflammation induced by readministration of rAAV in immunologically naive animals was undetectable by 4 weeks after the second injection (Peden and Mandel, unpublished observations).
The present study revealed aspects of both an innate and an adaptive immune response. Staining for markers of innate immunity in the single-injection groups revealed the absence of astrocytosis and microgliosis, while identical immunohistochemical staining procedures in the repeat-injection groups revealed significant innate inflammation. This is also in agreement with a recent study that investigated innate immune responses to adenovirus and AAV, which found that a single injection of rAAV was not sufficiently immunogenic to elicit a significant, persistent innate immune response (46
). Moreover, the present study shows that the adaptive arm of the immune response also plays a significant role. This is supported by the substantial infiltration of lymphocytes comprised partly of activated cytotoxic T cells, as well as tissue damage revealed by a reduction of DARPP-32 staining precisely in those areas. The juxtaposition of CD8+
T cells and neurons expressing upregulated MHC1 complexes solely in the readministration groups strongly supports a T-cell-mediated response to the rAAV injection with the potential for cytotoxic damage.
Inflammation was limited to both readministration groups irrespective of successful intrastriatal rAAV-mediated transduction. Furthermore, significant inflammation was present in both hemispheres of the immunized animals, while it was present only in the second sites of the naive readministration animals. These observations suggest that the NAb in the immune-single animals immediately bound the virus, thereby preventing transduction, but, lacking a second insult to the BBB, did not encounter enough T cells to prime a response to the future introduction of rAAV. In contrast, in the repeat-administration study, the BBB was disrupted a second time, allowing for the priming of a cellular immune response by the Ab bound to the rAAV at both injection sites in the immunized animals. Likewise, the naive-repeat animals would not have had NAb present at the first injection site. However, in accordance with previous studies (27
), the first rAAV administration could have induced Ab to either the transgene or capsid that would then be available at the second injection to activate T cells. The activated T cells would then be available to induce an inflammatory response in the second injection site. Confirming this, 5 of the 10 naive-repeat animals developed moderate NAb titers by the time of sacrifice, including those depicted in Fig. to . Correspondingly, in the animals with an immune system primed by a previous intracerebral rAAV injection, astrocytosis and microgliosis appear as an innate response to cytokines secreted by the infiltrating activated T cells. Regardless, it is important to clarify that the immune response in the presence of repeated administrations is not observed in those animals that received only a single vector injection, whether preimmunized or not. Therefore, the presence of inflammation should be attributed not to prior immunization status but to the effects of repeated intrastriatal administration within a short time interval.
Despite the high prevalence of wt AAV2 infection in the human population and the predominance of people with NAb titers (3
), there are no rAAV-based CNS gene transfer studies with animals that parallel this situation. While this question has been addressed in peripheral gene transfer paradigms, nothing has been done to address this issue for the brain. We and others have shown that GDNF may be therapeutic in Parkinson's disease in a gene therapy setting (16
). In light of this and other potential or existing clinical trials, this question is now of particular importance.
Addressing these questions in an animal model, while not directly comparable to the human population, is an important first step in designing future trials, including studies using naturally infected, nonhuman primates. Likewise, the human NAb titers presented in the literature to date cannot be compared to NAb levels found in the animal models in this and other studies, because previous methods of determination have not been standardized and the studies were performed by using subjective methods (3
). Moreover, there may be qualitative differences in circulating NAb derived from an immunization protocol, such as that employed here, as opposed to a naturally acquired respiratory infection in childhood.
It will be important to directly compare these titers to those seen in the human population, and by using the same automated and quantifiable NAb assay outlined in these experiments, it will be possible to do so in future studies. Currently unpublished data from our laboratory indicate that these titer levels are present in the human population, but the frequency is still unknown. In any event, we have shown in this study that immunization can radically reduce transduction efficiency but that utilization of an alternate serotype can circumvent the effect. It is presumable that in addition to utilization of alternative serotypes, capsid alterations and/or immunosuppression at the time of administration may also be helpful in permitting successful gene transfer in the brain of a preimmunized patient. Regardless, knowledge of a patient's immune status will enable treatment strategies to be customized to provide optimal safety and efficacy for the individual.
Thus, the present data unequivocally show that immune status is vitally important to brain transduction, and these novel findings suggest that the immunization status of potential human subjects should be taken into account. AAV's lack of immunogenicity in the brain has been well documented in many studies utilizing single-injection protocols. However, these studies have not attempted to model the case of an AAV-immunized patient and furthermore do not reflect the possibility of an immune response generated after repeated administrations. Therefore, with impending clinical trials it becomes necessary to utilize well-tested methods to clarify these newly identified potential complications. Additional studies specified here, including the use of various titers, transient immunosuppression, and alternate animal models, such as nonhuman primates, become warranted and even compulsory in light of these data.