Replication-competent viral vectors that are cytotoxic have a number of attractive features for tumor therapy, such as the abilities to generate new infectious virus in situ while killing tumor cells and to spread within the tumor (49
). However, as a result of these properties, they are potentially more pathogenic than replication-defective viral vectors. When clinical trials with such viruses are being considered, it is imperative that adequate safeguards be engineered into the vector and that rigorous safety testing be performed. As an example of such a viral vector, we developed an attenuated, replication-competent HSV-1 vector, G207, as a therapeutic agent for brain tumors. G207 is derived from HSV-1 strain F, one of the least virulent laboratory strains (13
). It contains deletions of both copies of the γ34.5 gene, the major viral determinant for neurovirulence (6
), and a lacZ
insertion inactivating the ICP6 gene. ICP6 is required for efficient viral replication in nondividing cells (16
) and also is a determinant of neurovirulence (5
). The presence of multiple mutations is important for safety, even with deletion mutants, because second-site suppressor mutations (41
) or novel recombinants could arise through selective pressure during replication in the tumor or brain.
HSV has evolved elaborate mechanisms to survive in the nervous system, its natural reservoir in humans and the site where it causes the most serious disease. In humans, illness resulting from HSV can be manifested by a variety of syndromes, including disseminated virus; CNS infections; and exanthema localized to the eyes, skin, and mouth (9
). CNS infections, usually due to HSV-1, often cause a devastating and rapidly fatal encephalitis. Death occurs in approximately 70% of those who do not receive treatment (66
). The signs and symptoms of HSV encephalitis include fever, mental status changes (altered consciousness and personality changes), headache, nausea, and focal neurological findings (i.e., seizures and hemiparesis) indicative of the infected brain area, usually the temporal lobes (9
). In neonates, about half the infections involve encephalitis, alone or with disseminated disease, and death is often associated with brain stem involvement (55
). Acyclovir is the treatment of choice. However, even when adequate antiviral treatment is rendered, approximately 20% of patients die and the majority of survivors suffer significant neurologic impairment (35
). Since G207 is a derivative of HSV-1, it is important to demonstrate that intracerebral inoculation with G207 does not pose a potential health hazard.
is an excellent animal model for characterizing the pathogenesis of HSV. These nonhuman primates are extremely sensitive to HSV infection and disease (24
), similar to human neonates and immunocompromised patients (37
). They develop clinical symptoms of HSV infection similar to those of humans. Aotus
has a somewhat elevated and variable body temperature compared to humans, which might affect the pathogenicity of G207. However, strain F has a temperature sensitivity similar to that of G207 (27
), and the animal inoculated with 103
PFU of strain F rapidly developed an encephalitis so severe that euthanasia was required within 5 days of inoculation. An examination of the brain tissues from this animal revealed typical histologic findings of HSV infection, including meningitis, inflammatory infiltrates, neuronal necrosis, Cowdry A intranuclear inclusions, and hemorrhage (24
). In contrast, a single intracerebral inoculation with 107
PFU of G207 produced no neurologic symptoms, and some animals were still alive and healthy 41 months after injection. One animal had a brief seizure 5 days after its inoculation with 109
PFU of G207; this could have been due to cortical irritation along the needle track, the location of the inoculation, the volume inoculated, or the virus. However, the symptoms were self-limiting and required no treatment, and no viral pathology was detected in the brain when it was examined 7 months after inoculation.
Noninvasive diagnostic tests for HSV encephalitis include electroencephalography, computerized tomography, single-photon emission-computed tomography, and MRI (14
). MRI is highly effective at detecting the changes in brain water content that accompany brain tissue inflammation and is particularly useful for detecting the early changes associated with HSV encephalitis (28
). We obtained MRI scans from 10 days to 1 year following G207 inoculation, and no radiographic evidence of encephalitis was found.
All animals tested developed a detectable but equivocal serum anti-HSV antibody response after intracerebral inoculation of G207. The two animals that received a second G207 inoculation 1 year after the first developed a rapid boost in antibody titer that lasted for at least two more years. In humans experiencing a primary genital infection (HSV-1 or -2), anti-HSV serum antibodies detected by complement fixation, radioimmunoprecipitation, or ELISA are generally not detected in the acute phase of infection (within 1 to 2 weeks) but are present in the early convalescent phase (2 to 8 weeks) after the onset of symptoms (2
). In a 9-month-old child that developed HSV encephalitis, neutralizing serum antibody was not detected on day 5 after disease onset but was detected on day 9, increasing to a maximum at days 14 and 26 and then decreasing to about half this level in 14 months (32
). While intracerebral inoculation is not a reasonable approach for vaccination, these studies suggest that G207 may be a useful live, attenuated strain for HSV vaccination in the periphery.
These studies demonstrate that G207 can be inoculated safely into the brain at doses of up to 109 PFU, at least 6 log units higher than a lethal dose of wild-type HSV-1. Even animals with humoral immunity due to prior exposure to G207 experienced no adverse consequences, and neither demyelination nor other neurologic sequelae occurred following a repeat intracerebral inoculation. This extensive neural toxicity testing suggests that G207 should be safe for intracerebral use in humans.