Examination of the potential interactions between herpes simplex virus vectors and replication-competent virus <I>in vitro</I> and <I>in vivo</I>

Abstract

In this paper, we have studied the potential interactions of replication incompetent herpes virus vectors with replication-competent virus both in vitro and in vivo. This might be thought to be particularly important for the use of HSV as compared to other virus vectors, as humans often harbour latent HSV. A vector virus carrying a transgene could interact with endogenous wild-type virus when used in gene therapy procedures. For this study we constructed four recombinant viruses containing marker genes to allow interactions between replication competent and disabled vector viruses to be explored. Recombination of viruses under replicating conditions was assessed in vitro and in vivo in the peripheral nervous system following the inoculation of combinations of viruses into the hindpaw of BALB/c mice and the examination of virus content in the dorsal root ganglia. Recombination between the viruses, when co-administered was found to occur such that transgene-bearing replication-competent viruses were generated only when the transgene was inserted at a non-essential site in the HSV genome. When the transgene was inserted into an essential site in the disabled virus, or when disabled and non-disabled virus were administered separately to the same site, transgene-bearing replication-competent recombinants were not observed. In the central nervous system, the ability of disabled, LacZ containing virus to reactivate latent replication-competent virus was examined. CNS latency was established by infecting BALB/c mice with a replication competent, GFP containing virus by the intra nasal route. After the establishment of latency, disabled virus was injected intra-cerebrally. Reactivation could not be detected as evidenced by a lack of GFP expression and replicating virus even though robust LacZ expression from the incoming vector virus could be detected. A similar lack of reactivation occurred when vector virus was inoculated into the footpad following the establishment of latency in the PNS by a replication competent virus. These experiments show (i) that insertion of the transgene in an essential site of the viral genome prevents its incorporation into an hazardous replication-competent recombinant derivative, indicating that non-homologous recombination between disabled and replication competent viruses does not occur at the level of sensitivity of the in vitro/in vivo assays used here, (ii) even homologous recombination in vivo only occurs at detectable levels when vector and replication competent virus are co-administered, and (iii) inoculation of vector HSV into the nervous system is very unlikely to reactivate latent wild-type virus that may be present.