Replication-inducible vaccinia virus vectors with enhanced safety in vivo.

Vaccinia virus (VACV) has been used extensively as the vaccine against smallpox and as a viral vector for the development of recombinant vaccines and cancer therapies. Replication-competent, non-attenuated VACVs induce strong, long-lived humoral and cell-mediated immune responses and can be effective oncolytic vectors. However, complications from uncontrolled VACV replication in vaccinees and their close contacts can be severe, particularly in individuals with predisposing conditions. In an effort to develop replication-competent VACV vectors with improved safety, we placed VACV late genes encoding core or virion morphogenesis proteins under the control of tet operon elements to regulate their expression with tetracycline antibiotics. These replication-inducible VACVs would only express the selected genes in the presence of tetracyclines. VACVs inducibly expressing the A3L or A6L genes replicated indistinguishably from wild-type VACV in the presence of tetracyclines, whereas there was no evidence of replication in the absence of antibiotics. These outcomes were reflected in mice, where the VACV inducibly expressing the A6L gene caused weight loss and mortality equivalent to wild-type VACV in the presence of tetracyclines. In the absence of tetracyclines, mice were protected from weight loss and mortality, and viral replication was not detected. These findings indicate that replication-inducible VACVs based on the conditional expression of the A3L or A6L genes can be used for the development of safer, next-generation live VACV vectors and vaccines. The design allows for administration of replication-inducible VACV in the absence of tetracyclines (as a replication-defective vector) or in the presence of tetracyclines (as a replication-competent vector) with enhanced safety.

Antibiotic-dependent expression of early transcription factor subunits leads to stringent control of vaccinia virus replication.

The use of vaccinia virus (VACV) as the vaccine against variola virus resulted in the eradication of smallpox. VACV has since been used in the development of recombinant vaccine and therapeutic vectors, but complications associated with uncontrolled viral replication have constrained its use as a live viral vector. We propose to improve the safety of VACV as a live-replicating vector by using elements of the tet operon to control the transcription of genes that are essential for viral growth. Poxviruses encode all enzymes and factors necessary for their replication within the host cell cytoplasm. One essential VACV factor is the vaccinia early transcription factor (VETF) packaged into the viral core. This heterodimeric protein is required for expression of early VACV genes. VETF is composed of a large subunit encoded by the A7L gene and a small subunit encoded by the D6R gene. Two recombinant VACVs were generated in which either the A7L or D6R gene was placed under the control of tet operon elements to allow their transcription, and therefore viral replication, to be dependent on tetracycline antibiotics such as doxycycline. In the absence of inducers, no plaques were produced but abortively infected cells could be identified by expression of a reporter gene. In the presence of doxycycline, both recombinant viruses replicated indistinguishably from the wild-type strain. This stringent control of VACV replication can be used for the development of safer, next-generation VACV vaccines and therapeutic vectors. Such replication-inducible VACVs would only replicate when administered with tetracycline antibiotics, and if adverse events were to occur, treatment would be as simple as antibiotic cessation.

A vaccinia virus renaissance: new vaccine and immunotherapeutic uses after smallpox eradication.

In 1796, Edward Jenner introduced the concept of vaccination with cowpox virus, an Orthopoxvirus within the family Poxviridae that elicits cross protective immunity against related orthopoxviruses, including smallpox virus (variola virus). Over time, vaccinia virus (VACV) replaced cowpox virus as the smallpox vaccine, and vaccination efforts eventually led to the successful global eradication of smallpox in 1979. VACV has many characteristics that make it an excellent vaccine and that were crucial for the successful eradication of smallpox, including (1) its exceptional thermal stability (a very important but uncommon characteristic in live vaccines), (2) its ability to elicit strong humoral and cell-mediated immune responses, (3) the fact that it is easy to propagate, and (4) that it is not oncogenic, given that VACV replication occurs exclusively within the host cell cytoplasm and there is no evidence that the viral genome integrates into the host genome. Since the eradication of smallpox, VACV has experienced a renaissance of interest as a viral vector for the development of recombinant vaccines, immunotherapies, and oncolytic therapies, as well as the development of next-generation smallpox vaccines. This revival is mainly due to the successful use and extensive characterization of VACV as a vaccine during the smallpox eradication campaign, along with the ability to genetically manipulate its large dsDNA genome while retaining infectivity and immunogenicity, its wide mammalian host range, and its natural tropism for tumor cells that allows its use as an oncolytic vector. This review provides an overview of new uses of VACV that are currently being explored for the development of vaccines, immunotherapeutics, and oncolytic virotherapies.