Transient Immunosuppression Stops Rejection of Virus-Transduced Enhanced Green Fluorescent Protein in Rabbit Retina

Abstract

The expression of lentivirus-transduced enhanced green fluorescent protein (EGFP) was detectable in rabbit retinal pigment epithelium (RPE) within 3 to 5 days after subretinal injection of the vector. Within 2 to 3 weeks, EGFP-expressing cells were eliminated by rejection. In the current experiments, we monitor serum antibody titers for EGFP before and after transduction and determine whether systemic immunosuppression prevents recognition of EGFP by the immune system. While all control rabbits developed antibodies against EFGP and showed signs of rejection, no such evidence was observed with animals which received immunosuppression. One month of systemic immunosuppression permanently prevented rejection of RPE with EGFP expression. Fluorescence has been maintained for more than a year. If a control eye was injected with the same virus after terminating immunosuppression, both eyes showed signs of rejection. The lack of rejection is not due to tolerance but to a failure of the animals to detect the foreign protein. Detection must depend upon a brief window of time after surgery needed to introduce the vector, perhaps related to a concurrent but transient inflammation. This strategy may be useful in managing other types of rejection in the retina.Numerous investigators have used viral vectors to transduce retinal cells with enhanced green fluorescent protein (EGFP), with no evidence of rejection of this foreign protein (2,3,15,20,21). Similar results have been reported with other reporter genes that express foreign proteins in the retina, such as LacZ (6,8,14). We have found that with relatively high retinal expression of EGFP, transduced by lentivirus, rejection will occur (7). GFP has been shown to be immunogenic (9) and will be rejected in mice (19). In order to examine this phenomenon more completely, we have determined whether antibodies to EGFP can be detected in rabbits during the course of rejection in the retina. We have also determined whether this rejection can be prevented by immunosuppression. Our results indicate that serum antibodies to EGFP are detectable concurrent with retinal rejection of this fluorescent protein. If the rabbits are immunosuppressed, rejection does not occur. Surprisingly, if immunosuppression is stopped after 1 month, rejection never occurs and antibodies to EGFP are not detectable. These results imply that foreign proteins transduced in the retina are only detectable immunologically during a brief period of time after the surgical procedure needed to introduce the viral vector into the retina. MATERIALS AND METHODSPreparation of viral stocks. The viral stocks were produced as described previously (16) by cotransfection of human kidney-derived 293T cells with three plasmids by the calcium phosphate method (4). The packaging construct designated pCMV⌬R8.2 contained the cytomegalovirus (CMV) promoter and the insulin polyadenylation signal to express all of the viral proteins in trans form, except the envelope and Vpu. The second plasmid, pHRЈ-CMV-GFP, provided a vector with all ...

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