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 Anti-HIV
/ AIDS immunisation, flu,
Heterologous
antigens are expressed (e.g., from Via
tuberculosis, etc.
HIV, the flu, M.
tuberculosis)
|
|
Replication-competent
HSV controlled * + Immunization
against the infectious pathogen co-infecting the virus (e.g.
adenovirus, papilloma, polyoma, infecting bacterial pathogens that contain at other
herpesvirus etc.)
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)
|
|
Replication-competent
regulated HSV * Immunization
against the viral pathogen expressing a replication-essential gene co-infecting
(basically any viral pathogen
of a viral pathogen co-infecting under gene capable
of co-infection)
switch regulation + viral pathogen
co-infecting
with disabled replication-essential
genetic control
|
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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