A Novel Approach on Immunization by Replication- Competent, Controlled Viral Pathogen: Exploring for Diseases Refractory to Effective Conventional Vaccination?

The first vaccine that targeted small pox in 1796 was studied by Edward Jenner. Overall, for disease prevention, vaccination has been a very good method. However, illnesses and disorders that can not be successfully avoided or treated by vaccines still occur more than two centuries after the first use of the vaccine. Unfortunately, global diseases, including influenza / flu, tuberculosis , malaria, HIV / AIDS, and oral and genital herpes, are diseases that have remained refractory to vaccination or are insufficiently protected against by vaccination. Although efforts to develop vaccines based on the “classical” model (described below) continue, it may also be necessary to consider novel approaches that may have the potential to induce superior immune responses qualitatively and/or quantitatively.

Our failure to recognise the molecular basis of current liveattenuated vaccine attenuation and immunogenicity is a key constraint in the development of novel vaccines against today and tomorrow’s challenging infectious diseases. This is partly due to the empirical existence of their growth [1,2]. The majority of vaccines currently approved are either subunit vaccines (comprising isolated proteins or protein fractions) or attenuated types of microorganisms that cause disease. Pathogens are attenuated by their killing or by hereditary deficiency of their replicative ability. Although attenuation provides the protection needed, the ability of so-modified pathogens to induce robust inflammatory responses is almost universally compromised, which translates into suboptimal humoral and T-cell responses[3-6]. The comparison of immune responses elicited by a wild type pathogenic agent vs. those caused by an attenuated agent is obviously not feasible. Many studies, however, contrasted immune responses elicited by viral vectors that were attenuated but retained residual capacity to replicate and corresponding vectors that were incompetent for replication[7-10]. Results showed that attenuated viruses retaining some replication capacity induced immune responses that were more complete and more potent than non-replicating comparison viruses.

On the basis of these considerations and observations, we hypothesised that a genetically engineered viral pathogen that can be triggered transiently in an inoculation site region to (local) replicate will be a superior immunisation agent to a traditional vaccine with an efficiency approaching that of the corresponding wild type agent[11]. Here, replication is understood as agent propagation. Bramson[12] endorsed that full immune response can be obtained from immunisation with a disease-causing virus modified to subject replication-essential genes to the control of a non-lethal heat-activated gene switch in the presence of a drug-like compound.

How does one restrict a viral pathogen’s replication to the area of the inoculation site as well as limit the length of its replication? It is well known that in the area in which they have been administered, viruses will not live, but will disperse inside the host organism. Therefore, in the inoculation site region but not elsewhere, an appropriate control mechanism will need to be capable of activating replication. It is believed that a physical “signal” that can be targeted at the inoculation site region will need to be responded to by such a control mechanism. The use of a highly heat-inducible heat shock protein gene (HSP) promoter to regulate the expression of replication-essential genes of a pathogenic virus is one possibility, representing perhaps the only possible solution available at this time. Some HSP promoters, such as the human HSP70B promoter, have very low basal activity, which can be induced by heat activation several thousand times [13,14]. Rohmer et al.[15] have produced novel gene transfer vectors for adenovirus that feature enhanced and strict control of transgene expression using promoter insulation from the HSP70B promoter. For gene therapy applications benefiting from external regulation of therapeutic gene expression or combination therapy with hyperthermia, these vectors have potential[16]. In all mammalian cell types, the promoters appear to be capable of being activated. A heat dose that is basically beyond the physiological range but can be easily tolerated by a human subject (44-450C for 5-10min) involves activation of the HSP70B promoter. If this is needed, the promoter could be changed so that it reacts to a lower heat dose[17]. In several ways, heat can be targeted. A simple and robust solution will include applying a heating pack in the case of intradermal or subcutaneous inoculation. Using well-known and inexpensive technology (of the kind used in commercial products such as ThermaCare), such a pack may be manufactured.

Safety from accidental transient activation or, worse, run-away activation will be the most critical problem with an immunisation agent that can be activated to replicate with near-wild type efficiency. The mode of regulation of HSP promoters emerges from a practical (not safety-related) issue that also needs to be answered. Exposure of a cell to heat (even prolonged heat) results in a heat shock transcription factor 1 (HSF1) transient activation that then binds to and mediates HSP promoter transcription. Within a few hours of heat activation at most, HSF1 returns to an inactive form. As a result, HSP promoters per se are not well suited to the control of a pathogen ‘s genes whose operation is required during most of the replication cycle or which need to be active at different cycle times. It has been suggested that adequate safety from inadvertent activation, whether due to exceptional circumstances ( e.g. ischemia, intoxication, etc.) or recombination events, could result from the use of a dual-responsive gene switch to control at least two viral pathogen replication-essential genes to be used as immunisation agents[11]. Dual-responsive gene switches activated by a combination of heat and a small-molecule regulator (SMR) have been previously described and are known to strictly control both in vitro and in vivo target gene expression [18-20]. The gene switches consist of I an SMR-activated transactivator expressed from a heat- and transactivator-activated promoter cassette, and (ii) a transactivator-responsive promoter to drive a gene of interest. Fig. represents a viral pathogen with two replication-essential genes controlled by a dual-responsive gene switch. 1A, and how the gene switch functions is shown in Fig. 1B. Upon administration to a selected inoculation region of such a replication-competent regulated pathogen, replication of the agent is activated by localised heat treatment, e.g. the application of a heating pack, in the presence of SMR. The SMR may be systemically administered or can be co-administered with the immunising agent. In the infected cells, the triggered gene switch will remain active until clearance of the SMR has occurred or the replicating agent has lysed the cells. The agent is safely disabled thereafter. Intentional re-activation of replication would be possible for as long as the agent remains in the inoculation zone.

Is building such replication-competent regulated viruses feasible? Are they likely to cause better immune reactions than traditional vaccines? We placed under the control of a dual-responsive gene switch (manuscript in preparation *) two replication-essential genes of a wild type strain of Herpes Simplex Virus 1 ( HSV-1). Antiprogestin mifepristone and closely related compounds (but not progestins) were activated by the transactivator incorporated in the latter gene switch. In vitro, in the presence of antiprogestin, the recombinant virus replicated nearly as effectively as the wild-type virus after heat activation, but not in the absence of either antiprogestin or heat treatment. In the mouse, replication of the virus was also rigorously regulated. The once-activated recombinant virus was substantially more protective than the corresponding non-replicating virus in immunisation / challenge experiments. These results encourage and require an examination of the full potential of the proposed approach to immunisation.

Agent Potential use
HSV * regulated by replication-competent Therapeutic or preventive immunisation toward Oncolytic  Treatment HSV


HSV * regulated by replication-competent Heterologous antigens are expressed (e.g., from HIV, the flu, M. tuberculosis) Anti-HIV / AIDS immunisation, flu, Via tuberculosis, etc.
Replication-competent HSV controlled * + pathogen co-infecting the virus bacterial pathogens that contain at least one viral pathogen   The replication-essential gene that is regulated by the  The transfer of dual-responsive genes (present in the HSV that is controlled) Immunization against the infectious

(e.g. adenovirus, papilloma, polyoma, infecting other herpesvirus etc.)

Replication-competent regulated HSV * expressing a replication-essential gene of a viral pathogen co-infecting under gene switch  regulation + viral pathogen co-infecting with disabled  replication-essential genetic control Immunization against the viral pathogen co-infecting (basically any viral pathogen capable of co-infection)


How broadly will this technology be applied? Only if the pathogen normally engages the host transcriptional machinery for the expression of its genes can replication of a viral pathogen be regulated by the above-described dual-responsive gene shift. At first sight, this limitation is less significant than one would expect (Table 1). There has been a long history of the use of viral vectors (replication-defective and attenuated) to express heterologous antigens [21,22]. As a potent immunisation platform, a replication-competent regulated virus can serve. Expression of heterologous antigens in the sense of vigorous viral replication is expected to result in superior immune responses for the reasons which were addressed at the beginning of this correspondence. In addition, via complementation, viral pathogens that do not use the host transcriptional machinery can be regulated. A replication-essential gene of such a virus and a co-infecting (replication-disabled or replication-competent-controlled) virus that expresses the replication-essential gene under the control of a dual-responsive gene switch can disable the gene product of such a virus. It is noted that if viral vectors are used as immunisation or oncolytic agents, the issue arises as to whether the effectiveness of these agents would be significantly diminished by pre-existing immunity. For HSV, our preferred backbone for managed virus construction, several studies have addressed this problem (cited in ref.9). The prevailing answer is that pre-existing immunity has little or only relatively minor effects on the immune response to antigens delivered by HSV or on the oncolytic HSV anti-tumor efficacy.

Author(s) Details

Richard Voellmy
Department of Physiological Sciences, University of Florida College of Veterinary Sciences, Gainesville, FL, USA and HSF Pharmaceuticals SA, La Tour-de-Peilz, Switzerland.

David C. Bloom
Department of Molecular Genetics & Microbiology, University of Florida College of Medicine, Gainesville, FL, USA.

Nuria Vilaboa
Hospital Universitario La Paz-IdiPAZ, Madrid, Spain and CIBER de Bioingenieria, Biomateriales y Nanomedicina, CIBER-BBN, Spain.

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