Host immune responses to vector capsid proteins have created a significant barrier to repeated delivery of Ad vectors in a number of animal models (40
). The data presented here indicate that host immune responses to rAAV vectors delivered to the rabbit airway are sufficiently compartmentalized to allow for effective repeated gene transfer. More specifically, our results show that expression may occur from repeated airway doses of rAAV vectors despite the development of neutralizing antibodies in the serum.
There are several possible explanations for the finding that serum neutralizing antibodies do not prevent reinfection with rAAV vectors. Our neutralization assay measured a total serum antibody response of which IgG is the predominant immunoglobulin. In the absence of inflammation and capillary leakage of serum proteins, IgG is generally not available on the large airway surface to neutralize foreign antigen. The predominant surface immunoglobulin is secretory IgA (39
). Although we did not measure secretory IgA directly, our ability to reinfect the animals strongly suggests the absence of such a response.
Early studies of AAV seroepidemiology showed that antibody to AAV was common in the serum of children and young adults independently of the individual’s exposure to Ad and that wild-type AAV infection was not prevented by preexisting serum neutralizing antibodies (10
). This is consistent with our findings which show that preexisting serum neutralizing antibodies to AAV2 did not prevent direct airway reinfection with either rAAV2- or rAAV3-encapsidated vectors. Our findings that vector DNA or protein expression was detected in a given rabbit sample despite elevated levels of serum neutralizing antibody at the time of delivery indicates that serum neutralizing anti-AAV antibody does not play a significant role in neutralization of rAAV vectors in the normal airway.
The data in this study are consistent with previous reports (13
) that a single dose of rAAV-CFTR vector to the airway does not elicit an inflammatory response and is not associated with lymphocytic inflammation. We have also shown here that repeated airway delivery of rAAV-CFTR does not instigate inflammatory changes or induce clearance of rAAV-mediated transgene expression. Our findings are also similar to those in muscle where rAAV-mediated transgene expression has been particularly long term, with inflammation being notably absent (31
). The mechanism by which AAV evades immunologic responses following injection into muscle has been studied by Jooss et al., who showed that rAAV was unable to efficiently transduce antigen presenting cells (dendritic cells) in muscle, thus accounting for the lack of CTL against rAAV vector transgene and the long-term persistence (28
). In our study, endobronchial delivery targeted vector primarily to the segmental branches of the airway where pulmonary dendritic cells lace the submucosal area of the epithelium and function as antigen-presenting cells (46
). Since AAV is inefficient at transducing dendritic cells, perhaps the lack of immune response in the airways affords AAV the same protection as the muscle. This possibility is supported by our observations that readministration of highly purified AAV vector yields substantial expression when delivered to an immunologically compartmentalized site, such as the lung.
Host immune responses may be more readily mounted against vectors that can replicate. If AAV replication took place, the immune response would likely be enhanced due to the processing of synthesized capsid proteins through the MHC class I, CD8+ cell-mediated pathway, leading to CTL destruction of cells expressing vector-derived proteins, as well as boosting the humoral arm. The rAAV vectors used in our experiments did not contain the Cap gene and were free of detectable replication-competent forms of both Ad and AAV, thus making it unlikely for replication and augmentation to take place.
In contrast, a study by Halbert et al. reported failure to transduce rabbit lung upon readministration of homologous serotype rAAV vector in association with the development of neutralizing antibodies to AAV (25
). First, it is possible that greater airway trauma was produced in their study since an intralumenal balloon catheter rather than a fiberoptic bronchoscope was used to deliver vector. This procedure might have allowed for additional protein leakage across the airway surface or more efficient antigen presentation at the site of delivery. Second, in the same study, vector preparations were reported to be contaminated with wild-type AAV, which correlated with decreased transduction efficiency following the first dose compared to less contaminated preparations. Low-level Rep and Cap expression from wild-type AAV contamination might have augmented the development of neutralizing antibodies and adversely influenced uptake from a second dose of same serotype vector. Alternatively, expression of Rep proteins from wild-type AAV might have down-regulated expression from rAAV. In a second study reporting failure to transduce mouse lung following repeated aspiration of homologous serotype rAAV vectors (26
), the alveolar region was transduced following the first but not second administration of rAAV vector in association with the development of serum neutralizing anti-AAV antibodies. In that study (26
), the second dose may have been neutralized by plasma derived IgG, since IgG is predominantly distributed in the alveolar region of the lung (44
We examined the strategy of switching vector capsid by using AAV2 and AAV3 and were able to develop a wild-type-free packaging system for rAAV3 vectors that produced infectious pseudotyped rAAV3 vectors with physical and biological titers equivalent to those of rAAV2 vectors. The similar rise in titer after the third dose of either rAAV3 or rAAV2 in this study reflects the cross-reactivity between the two serotypes (compare Fig. A to Fig. B). Our findings are consistent with those of earlier investigators who found that antibody to AAV2 does not prevent reinfection with either AAV2 or AAV3 (9
). Following the third dose, the rAAV3-GFP-treated group tended to have a higher percentage of GFP-expressing cells per sample than the rAAV2-GFP-treated group, but there was no difference in the number of rabbits expressing GFP between the groups. The difference in the range of positive cells between the groups likely reflects the sampling variability in this model rather than a true difference between the groups. The yield of vector mRNA detection was low despite the presence of GFP expression as detected by FACS. The FACS analysis was performed on a specific population of targeted epithelial cells and accounts for the higher level of GFP detection. In contrast, for the RT-PCR, RNA was isolated from whole lung homogenates; thus, the percentage of isolated mRNA specifically from targeted epithelial cells is small relative to the total amount of RNA in each sample and accounts for the lower level of detection. Following repeated delivery of rAAV2-CFTR vector to the airway, both rAAV2- and rAAV3-encapsidated vectors yielded substantial expression of transgene, and switching from serotype 2 to serotype 3 did not provide a further advantage in this setting.
The presence of hCFTR vector DNA may be a cumulative phenomenon, as demonstrated by the comparison of DNA transfer in the thrice-dosed RLL (40%) as opposed to the once-dosed LLL (10%). The increased detection of hCFTR following three doses may reflect incremental increase in the number of transduced cells or proliferation of transduced progenitor cells. Alternatively, the number of hCFTR genomes per cell may have increased over time due to concatemer formation as noted in other studies (1
). Most of the animals in this group were not brushed, and so selective epithelial trauma from the brushing technique does not explain the discrepancy between the right and left lobes. Although the study design did not allow us to determine from which dose(s) the hCFTR vector DNA resulted and the sampling methods did not allow for precise quantification of vector transfer, the consistent results upon repeated testing suggest there may be an incremental increase in vector hCFTR DNA after three doses. In any case, there was no evidence of inflammation or lymphocytic destruction of epithelial cells in the lobes positive for hCFTR vector DNA. We have shown persistence of rAAV-CFTR genomes for at least 17 weeks following repeated bronchoscopic delivery, indicating that repeated airway delivery of homologous rAAV vectors is a feasible approach to maintain rAAV-mediated expression in the airway of an individual.
In summary, we have demonstrated that the repeated bronchoscopic administration of highly purified rAAV vectors to the rabbit airway results in incremental expression of transgene without detectable toxicity. Furthermore, we detected no toxicity from either GFP expression or repeated exposure to rAAV-CFTR. Repeated bronchoscopic administration is one way to maintain persistent rAAV-hCFTR in the airways. As rAAV-CFTR clinical applications are developed, future studies should consider that mucosal immunity might be influenced by the purity of vector, the targeted area of delivery, and the condition of the delivery surface. Pseudotyping of rAAV vectors with serologically distinct capsid proteins may then be necessary to circumvent host responses. However, in the setting of a normal airway, repeated bronchoscopic delivery of highly purified rAAV vectors is successful.