Biology of E1-deleted adenovirus vectors in nonhuman primate muscle

Abstract

Adenovirus vectors have been studied as vehicles for gene transfer to skeletal muscle, an attractive target for gene therapies for inherited and acquired diseases. In this setting, immune responses to viral proteins and/or transgene products cause inflammation and lead to loss of transgene expression. A few studies in murine models have suggested that the destructive cell-mediated immune response to virally encoded proteins of E1-deleted adenovirus may not contribute to the elimination of transgene-expressing cells. However, the impact of immune responses following intramuscular administration of adenovirus vectors on transgene stability has not been elucidated in larger animal models such as nonhuman primates. Here we demonstrate that intramuscular administration of E1-deleted adenovirus vector expressing rhesus monkey erythropoietin or growth hormone to rhesus monkeys results in generation of a Th1-dependent cytotoxic T-cell response to adenovirus proteins. Transgene expression dropped significantly over time but was still detectable in some animals after 6 months. Systemic levels of adenovirus-specific neutralizing antibodies were generated, which blocked vector readministration. These studies indicate that the cellular and humoral immune response generated to adenovirus proteins, in the context of transgenes encoding self-proteins, hinders long-term transgene expression and readministration with first-generation vectors.

FIG. 1

Serum levels of growth hormone (GH). Rhesus monkeys were administered Ad-rhGH intramuscularly, and serum was obtained at various time points and analyzed for vector-induced GH levels as described in the text. Serum levels following vector administration are presented both before and after octreotide administration. Postoctreotide levels indicate vector-induced growth hormone.

FIG. 2

Serum erythropoietin (Epo) levels (A) and hematocrits (B). Rhesus monkeys were administered Ad-rhEPO intramuscularly, and blood obtained at various time points was analyzed for serum Epo levels or hematocrits.

FIG. 3

Lymphoproliferative responses following adenovirus vector administration in muscle. Rhesus monkeys were administered either Ad-rhGH (RQ1828 and RQ1735) or Ad-rhEPO (animals RQ154 and RQ1748) intramuscularly as described in the text. Heparinized blood was drawn prior to and on various days following vector administration. PBMC were isolated by Ficoll-Hypaque density gradient centrifugation and cultured in medium alone or in the presence of adenovirus antigens for 6 days. Responses were measured by [³H]thymidine incorporation and expressed as the stimulation index (see text). The top panel shows responses in animals administered Ad-rhGH, and the lower panel shows responses in animals administered Ad-rhEPO.

FIG. 4

Cytokine secretion profiles following intramuscular administration of adenovirus vectors in rhesus monkeys. Rhesus monkeys were administered either Ad-rhGH (animals RQ1828 and RQ1735) or Ad-rhEPO (animals RQ1564 and RQ1748) intramuscularly as described in the text on day 1. For induction of cytokines, PBMC obtained on various days were cultured with adenovirus antigens for 48 h. Culture supernatants were collected and measured in duplicate for the presence of IFN-γ, IL-2, IL-4, and IL-10 using commercial ELISA kits (BioSource International) as described by the manufacturer. No significant IL-4 levels could be measured in the culture supernatants, although the extent of cross-reactivity of this human-based assay with rhesus IL-4 is unknown.

FIG. 5

CTL responses to adenovirus proteins in rhesus monkeys. CTL responses to hexon, penton, and fiber were measured using autologous herpesvirus papio-transformed B lymphocytes from rhesus monkey RQ1828, which received Ad-rhGH. Effector T cells were generated by culturing PBMC for 14 days in the presence of Ad-GFP and IL-2. Autologous target cells were infected with vaccinia virus expressing hexon, penton, or fiber. Specific lysis was measured as described in the text.

FIG. 6

NAB in serum following intramuscular administration of adenovirus vectors in rhesus monkeys. Sera were obtained from animals administered either Ad-rhGH (RQ1828 and RQ1735) or Ad-rhEPO (RQ1564 and RQ1748) on various days. Animals RQ1564 and RQ1748 were readministered Ad-rhGH on day 210. NAB were measured by the ability of sera to interfere with the infection of HeLa cells with adenovirus expressing GFP. Various dilutions of serum preincubated with the reporter virus for 1 h at 37°C were added to 90% confluent HeLa cell cultures. Cells were incubated for 24 h, and expression of GFP was measured by fluoroimaging. The neutralizing titer of the serum was calculated as the highest dilution at which 50% of the cells turned green.

FIG. 7

Adenovirus vector-specific immunoglobulin isotypes in serum following intramuscular administration of adenovirus vectors in rhesus monkeys. Sera obtained from animals administered Ad-rhGH or Ad-rhEPO were analyzed for the presence of adenovirus-specific immunoglobulin isotypes IgM, IgG1, IgG2, and IgG4 by ELISA as described in the text. Results are expressed as optical density (O.D.).

FIG. 8

Analyses of antibodies to viral capsid proteins. Sera obtained from animals administered either Ad-rhGH or Ad-rhEPO intramuscularly were analyzed for the presence of antibodies to various components of adenovirus capsid proteins by Western blot analyses. Adenovirus antigens were electrophoresed on a 10% polyacrylamide gel and probed with serum obtained preadministration on various days (d) after. Reactivities were visualized by treatment with peroxidase-conjugated goat anti-human IgG followed by chemiluminescence. The positions of hexon, penton, and fiber proteins were determined by using antibodies directed against individual components and are indicated on the figure (data not shown). The lower-molecular-weight proteins comprise late gene proteins associated with the vector particle. Sizes are shown in kilodaltons.

FIG. 9

Lack of antibodies to growth hormone (GH) and erythropoietin (EPO) in sera from animals administered adenovirus vectors. Sera obtained from animals administered either Ad-rhGH (animals RQ1828 and RQ1735) or Ad-rhEPO (animals RQ1564 and RQ1748) intramuscularly were analyzed for the presence of antibodies to growth hormone (α-GH) or erythropoietin (α-EPO) by Western blot analyses as described in the text. The positions of the proteins were determined using polyclonal antibodies obtained from mice injected with Ad-rhGH or Ad-rhEPO. Sizes are shown in kilodaltons.