Attenuated, replication-competent herpes simplex virus type 1 mutant G207: safety evaluation of intracerebral injection in nonhuman primates

Abstract

This study examined the safety of intracerebral inoculation of G207, an attenuated, replication-competent herpes simplex virus type 1 (HSV-1) recombinant, in nonhuman primates. Sixteen New World owl monkeys (Aotus nancymae [karyotype 1, formerly believed to be A. trivirgatus]), known for their exquisite susceptibility to HSV-1 infection, were evaluated. Thirteen underwent intracerebral inoculation with G207 at doses of 10(7) or 10(9) PFU, two were vehicle inoculated, and one served as an infected wild-type control and received 10(3) PFU of HSV-1 strain F. HSV-1 strain F caused rapid mortality and symptoms consistent with HSV encephalitis, including fever, hemiparesis, meningitis, and hemorrhage in the basal ganglia. One year after G207 inoculation, seven of the animals were alive and exhibited no evidence of clinical complications. Three deaths resulted from nonneurologic causes unrelated to HSV infection, and three animals were sacrificed for histopathologic examination. Two animals were reinoculated with G207 (10(7) PFU) at the same stereotactic coordinates 1 year after the initial G207 inoculation. These animals were alive and healthy 2 years after the second inoculation. Cerebral magnetic resonance imaging studies performed both before and after G207 inoculation failed to reveal radiographic evidence of HSV-related sequelae. Despite the lack of outwardly observable HSV pathology, measurable increases in serum anti-HSV titers were detected. Histopathological examination of multiple organ tissues found no evidence of HSV-induced histopathology or dissemination. We conclude that intracerebral inoculation of up to 10(9) PFU of G207, well above the efficacious dose in mouse tumor studies, is safe and therefore appropriate for human clinical trials.

FIG. 1

Brains from G207-injected animals. (A) Brain from T9205 at time of necropsy, 6 months after injection of 10⁷ PFU of G207, with frontal lobes at the bottom and brain stem at the top. (B) Coronal cut through the brain from T910805 at the injection site 4 months after injection of 10⁷ PFU of G207. In both panels, the left side of the brain is on the right side of the figure, and the injection site is marked (>). Hatch marks on rulers are in millimeters. Photographic slides were scanned with a Polaroid SprintScan 35, and Adobe Photoshop was used to adjust the contrast and brightness of the images.

FIG. 2

Histology of brain from G207 injection site. Brain tissue was obtained at necropsy from animals T1122 (A) and T894 (B) 3 months and 25 days, respectively, after inoculation of 10⁷ PFU of G207. Ten-micrometer paraffin-embedded sections were stained with hematoxylin and eosin. Mild perivascular inflammation adjacent to the injection site is visible (arrows).

FIG. 3

Histology and immunohistochemistry of brain tissue from HSV-1 strain F injection. Brain tissue was obtained at necropsy from animal T1057 5 days after inoculation of 10³ PFU of F. Ten-micrometer paraffin-embedded sections were stained with hematoxylin and eosin (A, B, and C) or were immunohistochemically stained with anti-HSV antibody (D). Changes consistent with HSV encephalitis are evident. (A) Meningitis, with thickened meninges, and inflammatory cell infiltrate (between arrows) as opposed to the normal condition of single-cell thickness. (B) Hemorrhage in the basal ganglia. (C) Cowdry A eosinophilic intranuclear inclusions (10) in the ipsilateral (arrowheads) and contralateral hemispheres. (D) HSV-immunoreactive cells (black) are seen throughout the cortex, in both ipsilateral and contralateral hemispheres. Photographic slides were scanned with a Polaroid SprintScan 35, and Adobe Photoshop was used to adjust the contrast and brightness of the images. Bar, 0.08 mm (A and D), 0.07 mm (B), and 0.03 mm (C).

FIG. 4

Cerebral MRI studies obtained after G207 inoculation. Sagittal T1-weighted MRIs were obtained after the administration of contrast dye. Images were obtained from T894 (A), T9205 (B), and T348 (C) 10 days, 1 month, and 10 months, respectively, after inoculation of 10⁷ PFU of G207 and from T558 (D) 6 months after inoculation of 10⁹ PFU of G207. The left frontal region of the brain (*) and the site of G207 injection (arrow) are indicated in each panel. Normal postoperative incisional scarring and inflammation of the skin overlying the inoculation site are present in all panels but are best seen in panel C. There were no changes, such as edema, hemorrhage, or necrosis, that would indicate the presence of HSV encephalitis.

FIG. 5

Serum anti-HSV antibody levels. Anti-HSV antibody titers in serum were determined by ELISA (Table 1) at various times (months) pre- and post-G207 injection. Optical density (OD) values of ≥0.4 are positive, and values between 0.17 and 0.39 are equivocal. All preinjection sera had OD values of 0.