Replication-Competent Controlled Herpes Simplex Virus

Abstract

We present the development and characterization of a replication-competent controlled herpes simplex virus 1 (HSV-1). Replication-essential ICP4 and ICP8 genes of HSV-1 wild-type strain 17syn؉ were brought under the control of a dually responsive gene switch. The gene switch comprises (i) a transactivator that is activated by a narrow class of antiprogestins, including mifepristone and ulipristal, and whose expression is mediated by a promoter cassette that comprises an HSP70B promoter and a transactivator-responsive promoter and (ii) transactivator-responsive promoters that drive the ICP4 and ICP8 genes. Singlestep growth experiments in different cell lines demonstrated that replication of the recombinant virus, HSV-GS3, is strictly dependent on an activating treatment consisting of administration of a supraphysiological heat dose in the presence of an antiprogestin. The replication-competent controlled virus replicates with an efficiency approaching that of the wild-type virus from which it was derived. Essentially no replication occurs in the absence of activating treatment or if HSV-GS3-infected cells are exposed only to heat or antiprogestin. These findings were corroborated by measurements of amounts of viral DNA and transcripts of the regulated ICP4 gene and the glycoprotein C (gC) late gene, which was not regulated. Similar findings were made in experiments with a mouse footpad infection model. IMPORTANCEThe alphaherpesviruses have long been considered vectors for recombinant vaccines and oncolytic therapies. The traditional approach uses vector backbones containing attenuating mutations that restrict replication to ensure safety. The shortcoming of this approach is that the attenuating mutations tend to limit both the immune presentation and oncolytic properties of these vectors. HSV-GS3 represents a novel type of vector that, when activated, replicates with the efficiency of a nonattenuated virus and whose safety is derived from deliberate, stringent regulation of multiple replication-essential genes. By directing activating heat to the region of virus administration, replication is strictly confined to infected cells within this region. The requirement for antiprogestin provides an additional level of safety, ensuring that virus replication cannot be triggered inadvertently. Replication-competent controlled vectors such as HSV-GS3 may have the potential to be superior to conventional attenuated HSV vaccine and oncolytic vectors without sacrificing safety.

Immunization by replication-competent controlled herpesvirus vectors

“…The two-component HSP promoter-based gene switch employed in the vectors of the present study relied on transactivator GLP65 that is activated by a narrow class of antiprogestins including mifepristone and ulipristal (31-34). GLP65 is a chimeric protein consisting of a DNA-binding domain from yeast transcription factor GAL4, a truncated ligand-binding domain from a human progesterone receptor and a transcriptional activation domain from human NFkappaB protein P65.…”

“…Replication of HSV-GS3 had previously been shown to be strictly dependent on activation by heat and antiprogestin in single-step growth experiments (34). When activated, the recombinant replicated nearly as efficiently as wildtype virus 17 syn +.…”

“…We next examined in a similar experiment whether induction of enhanced protective immunity was caused by an activation of the heat- and antiprogestin-dependent gene switch that controls the expression of ICP4 and ICP8 in the HSV-GS3 recombinant. In this and all subsequent experiments, ulipristal was utilized instead of mifepristone, because the former compound is a more active gene switch co-activator than the latter compound (34). Furthermore, the heat treatment regimen was changed to heat exposure at 45°C for 10 min to achieve greater reproducibility between experiments.…”

“…In secondarily infected cells (in which HSF1 is not activated), HSV-GS7 was expected to express all viral proteins except the regulated capsid protein at approximately normal levels. In contrast, HSV-GS3 all but lacks ICP4 expression in the absence of activation and exhibits dramatically curtailed expression of other viral proteins (34). The finding that HSV-GS3 and HSV-GS7 induced comparable protective responses suggested that viral proteins expressed in secondarily infected cells did not contribute in a relevant fashion to the immune response.…”

“…(A) Illustration of the operation of the dual-responsive gene switch employed to control replication of the viral vectors of the present study. (Copyright © American Society for Microbiology, (34)) eHSF1: endogenous heat shock transcription factor; HSP70B: HSP70B/HSPA7 promoter; TRP: transactivator-responsive promoter; TA: transactivator; VG: replication-essential viral gene; SMR: small-molecule regulator. (B) Diagrams of viral vectors HSV-GS3 (top of panel) and HSV-GS7 (bottom of panel).…”