Cytotoxic T Lymphocyte Responses to Proteins Encoded by Heterologous Transgenes TransferredIn Vivoby Adenoviral Vectors

Abstract

Although replication-deficient adenovirus (Ad) vectors are efficient vehicles for in vivo gene transfer, persistence of expression of the Ad genome is limited in immunocompetent hosts by cellular immunity directed against the gene product of the vector. While most attention has been focused on cytotoxic T lymphocytes (CTL) directed against the low-level early and late Ad gene expression in the Ad vector-infected target cells, significant cellular immunity is likely also directed against the product of heterologous transgenes. To evaluate this concept, in vivo generation of CTL was evaluated in C57B1/6 and BALB/c mice with Ad vectors expressing a variety of heterologous transgenes, including Escherichia coli chloramphenicol acetyl transferase (CAT), beta-galactosidase (beta-Gal), cytosine deaminase, and human thrombopoietin (hTPO), with an Ad vector expressing no transgene ("null") as a control. Following intravenous administration of Ad vectors, spleen cells were harvested 2 weeks later, stimulated for 5 days with syngeneic cells infected with various Ad vectors, and then evaluated for CTL activity using 51Cr-release from syngeneic Ad vector-infected targets. In all cases, CTL directed against the heterologous transgene products was observed, although there were differences in the amounts of transgene-specific CTL. CTL directed against the transgene were also observed with other routes of administration, including intratracheal, subcutaneous, and intraperitoneal administration. These observations suggest that inclusion of a heterologous transgene in Ad vectors enhances the elimination of vector-infected cells, a circumstance that will be partially circumvented using autologous genes. For some applications, specific immune responses to products of transgenes delivered by Ad vectors might be exploited for therapeutic purposes.

Reduced toxicity, attenuated immunogenicity and efficient mediation of human p53 gene expression in vivo by an adenovirus vector with deleted E1-E3 and inactivated E4 by GAL4-TATA promoter replacement

“…[1][2][3][4][5] However, recent studies have demonstrated that the replication defect in E1-deleted adenoviruses could be overcome in cultured cells at high multiplicities of infection (MOI) 6,7 and that the early and late viral gene products can be expressed independently of E1a function following adenovirus-mediated gene transfer in vivo. [8][9][10] The expressed viral genes may elicit a host immune response in the transduced cells and lead to toxicity, 11,12 limit the duration of transgene expression, [13][14][15][16] and consequently hinder the application of these vectors for human gene therapy. To overcome these drawbacks, further modifications have been targeted to the E4 and E2 regions of adenovirus, which are critical for viral DNA synthesis and late gene production, to diminish the residual DNA replication of viral vectors and reduce their potential for viral gene expression.…”

Development and application of adenoviral vectors for gene therapy of cancer

“…76 -78 The expression of viral proteins and transgene products in Ad vector-transduced cells can stimulate destructive cellular immune responses, which leads to the coordinate loss of transgene expression and to the development of inflammation. Support for this observation has been derived from experiments performed in the nonhuman primate lung, 79 mouse lung, 80 liver, 71,81 spleen, 82 and cotton rat lung. 83 Ad vector-induced cellular immune responses clearly require redesign of the Ad vectors to eliminate the sources of the problem, although it was demonstrated that Ad vector-infected cells could escape Ad antigen (Ag)-specific cytotoxic T lymphocyte (CTL) killing in vivo.…”

Regional treatment of hepatic micrometastasis by adenovirus vector-mediated delivery of interleukin-2 and interleukin-12 cDNAs to the hepatic parenchyma

“…Mice were sacrificed at 12 days after vector delivery, and splenic mononuclear cells were isolated as described previously. 45 Briefly, spleens harvested from each group were minced and ground, sheared with a 19-gauge needle, and passed through a 200-m mesh to remove fibrous tissue. Live lymphocytes were separated from dead cells and red blood cells using FicollPaque (Pharmacia, Piscataway, NJ) density separation, washed, and resuspended in Dulbecco's modified Eagle's medium.…”