Recombinant Herpes Simplex Virus Vectors Research Articles

IL-4 mediated by HSV vector suppresses morphine withdrawal response and decreases TNFα, NR2B, and pC/EBPβ in the periaqueductal gray in rats.

Chronic opiates induce the development of physical dependence. Opioid physical dependence characterized by withdrawal symptoms, may have very long-lasting effects on the motivation for reward, including the incubation of cue-induced drug-seeking behavior. Elucidation of the mechanisms involved in physical dependence is crucial to developing more effective treatment strategies for opioid dependence. Chronic morphine induces production of proinflammatory cytokines in regional-specific sites of the brain. Interleukin-4 (IL-4) is a prototypical anti-inflammatory cytokine that globally suppresses proinflammatory cytokines. Here, we used recombinant herpes simplex virus vector S4IL4 that encode mouse il4 gene to evaluate the therapeutic potential of IL-4 in naloxone-precipitation morphine withdrawal (MW). One week after microinjection of the vector S4IL4 into the PAG LacZ or mouse IL-4 immunoreactivity in the vlPAG was visualized. ELISA assay showed that vector S4IL4 into the PAG induced the expression of IL-4. S4IL4 blunted the morphine withdrawal syndrome. S4IL4 suppressed the upregulated TNFα, NR2B and pC/EBPβ in the PAG induced by MW. These results show that inhibition of proinflammatory factor in the PAG suppressed MW. This study may provide a novel therapeutic approach to morphine physical withdrawal symptoms.

Ability of herpes simplex virus vectors to boost immune responses to DNA vectors and to protect against challenge by simian immunodeficiency virus

The immunogenicity and protective capacity of replication-defective herpes simplex virus (HSV) vector-based vaccines were examined in rhesus macaques. Three macaques were inoculated with recombinant HSV vectors expressing Gag, Env, and a Tat-Rev-Nef fusion protein of simian immunodeficiency virus (SIV). Three other macaques were primed with recombinant DNA vectors expressing Gag, Env, and a Pol-Tat-Nef-Vif fusion protein prior to boosting with the HSV vectors. Robust anti-Gag and anti-Env cellular responses were detected in all six macaques. Following intravenous challenge with wild-type, cloned SIV239, peak and 12-week plasma viremia levels were significantly lower in vaccinated compared to control macaques. Plasma SIV RNA in vaccinated macaques was inversely correlated with anti-Rev ELISPOT responses on the day of challenge ( P value < 0.05), anti-Tat ELISPOT responses at 2 weeks post challenge ( P value < 0.05) and peak neutralizing antibody titers pre-challenge ( P value 0.06). These findings support continued study of recombinant herpesviruses as a vaccine approach for AIDS.

Flip-Flop HSV-BAC: bacterial artificial chromosome based system for rapid generation of recombinant herpes simplex virus vectors using two independent site-specific recombinases

BackgroundOncolytic herpes simplex virus (HSV) vectors that specifically replicate in and kill tumor cells sparing normal cells are a promising cancer therapy. Traditionally, recombinant HSV vectors have been generated through homologous recombination between the HSV genome and a recombination plasmid, which usually requires laborious screening or selection and can take several months. Recent advances in bacterial artificial chromosome (BAC) technology have enabled cloning of the whole HSV genome as a BAC plasmid and subsequent manipulation in E. coli. Thus, we sought a method to generate recombinant oncolytic HSV vectors more easily and quickly using BAC technology.ResultsWe have developed an HSV-BAC system, termed the Flip-Flop HSV-BAC system, for the rapid generation of oncolytic HSV vectors. This system has the following features: (i) two site-specific recombinases, Cre and FLPe, are used sequentially to integrate desired sequences and to excise the BAC sequences, respectively; and (ii) the size of the HSV-BAC-insert genome exceeds the packaging limit of HSV so only correctly recombined virus grows efficiently. We applied this to the construction of an HSV-BAC plasmid that can be used for the generation of transcriptionally-targeted HSV vectors. BAC sequences were recombined into the UL39 gene of HSV ICP4-deletion mutant d120 to generate M24-BAC virus, from which HSV-BAC plasmid pM24-BAC was isolated. An ICP4 expression cassette driven by an exogenous promoter was re-introduced to pM24-BAC by Cre-mediated recombination and nearly pure preparations of recombinant virus were obtained typically in two weeks. Insertion of the ICP4 coding sequence alone did not restore viral replication and was only minimally better than an ICP4-null construct, whereas insertion of a CMVIE promoter-ICP4 transgene (bM24-CMV) efficiently drove viral replication. The levels of bM24-CMV replication in tumor cells varied considerably compared to hrR3 (UL39 mutant).ConclusionOur Flip-Flop HSV-BAC system enables rapid generation of HSV vectors carrying transgene inserts. By introducing a tumor-specific-promoter-driven ICP4 cassette into pM24-BAC using this system, one should be able to generate transcriptionally-targeted oncolytic HSV vectors. We believe this system will greatly facilitate the screening of a plethora of clinically useful tumor-specific promoters in the context of oncolytic HSV vectors.

Treatment of Schwannomas with an Oncolytic Recombinant Herpes Simplex Virus in Murine Models of Neurofibromatosis Type 2

Gene therapy for schwannomas was evaluated in two mouse models of neurofibromatosis type 2 (NF2): (1) a transgenic model in which mice express a dominant mutant form of merlin and spontaneously develop schwannomas, and (2) a xenograft model in which human schwannoma tissue is implanted subcutaneously into immune- compromised mice. In both models, schwannoma volumes were monitored by magnetic resonance imaging (MRI) and showed strong gadolinium enhancement typical of these tumors in humans. Both types of tumor were positive for the Schwann cell marker S100, and highly infectable with herpes simplex virus (HSV) vectors. Schwannomas were injected with an oncolytic HSV-1 recombinant virus vector, G47Delta, which has deletions in genes for ribonucleotide reductase (ICP6), gamma34.5, and ICP47. In the NF2 transgenic model, schwannomas were reduced by more than half their original size by 10 days after infection. In the case of subcutaneous schwannoma xenografts, reduction in size after infection occurred more slowly, with a mean reduction of onethird by 42 days after treatment. Schwannomas injected with control vehicles continued to grow slowly over time in both schwannoma models. These studies demonstrate the ability of an oncolytic recombinant HSV vector to reduce the volume of schwannoma tumors in NF2 tumor models in mice and extend the possible therapeutic applications of oncolytic vectors for benign tumors to reduce mass while minimizing nerve damage.

Herpesvirus-based infectious titering of recombinant adeno-associated viral vectors

Studies in animals and human clinical trials demonstrate the safety and persistence of recombinant adeno-associated viral (rAAV) serotype 2 vectors in a variety of tissues. rAAV vectors of other serotypes are also being developed for efficient gene transfer. To date, the literature describing these vectors has relied on physical or transducing titers to determine dose, but few, if any, infectious titers have been presented. This is due in large part to the lack of reagents and methods that would facilitate the infectious titering of vectors other than serotype 2. Here, we describe reagents and methods for infectious titering of AAV2 ITR-containing vectors pseudotyped with other AAV capsid serotypes and demonstrate their utility by titering pseudotyped rAAV1 or rAAV5 vectors. Cell lines are screened for optimal transduction using a vector of a particular serotype that expresses a marker transgene. Once a cell line and vector serotype are matched, a recombinant herpes simplex virus vector expressing AAV2 rep and cap genes provides helper functions that amplify the rAAV vector genome. The vector genomes are then detected and a titer is calculated. These methods generate reliable infectious titers for AAV vectors of different serotypes, thus enhancing product characterization and reducing risk in future clinical applications.

Infection with an H2 recombinant herpes simplex virus vector results in expression of MHC class I antigens on the surfaces of human neuroblastoma cells in vitro and mouse sensory neurons in vivo.

The majority of neurons in herpes simplex virus (HSV)-infected murine sensory ganglia are transiently induced to express MHC-I antigens at the cell surface, whereas only a minority are themselves productively infected. The aim of the current work was to determine whether MHC-I antigens can be expressed on the surfaces of infected neurons in addition to their uninfected neighbours. To address this aim a recombinant HSV type 1 strain, S-130, was used to deliver a mouse H2K(d) gene, under control of the HCMV IE-1 promoter/enhancer, into human neuroblastoma cells in vitro and mouse primary sensory neurons in vivo. S-130 expressed H2K(d) antigens on the surfaces of IMR-32 cells, a human neuroblastoma cell line that expresses very low levels of MHC-I constitutively. In K562 cells, which do not express MHC-I constitutively, H2K(d) and beta(2)-microglobulin (beta(2)m) were shown to be co-expressed at the cell surface following S-130 infection. This observation was taken as evidence that class I heavy chain (alphaC) molecules encoded by the expression cassette in the HSV genome were transported to the cell surface as stable complexes with beta(2)m. Significantly, after introduction of S-130 into flank skin, H2K(d) antigens were detected on the surfaces of primary sensory neurons in ganglia innervating the inoculation site. Our data show that HSV-infected murine primary sensory neurons and human neuroblastoma cells are capable of expressing cell-surface MHC-I molecules encoded by a transgene. From this, we infer that up-regulation of alphaC expression is, in principle, sufficient to overcome potential impediments to neuronal cell surface expression of MHC-I complexes.

Long-term gene expression in the anterior horn motor neurons after intramuscular inoculation of a live herpes simplex virus vector.

To clarify the feasibility of the herpes simplex virus (HSV) vector in expressing the foreign gene in the motor neuron, we inoculated a live attenuated HSV expressing beta-galactosidase (beta-gal) activity under a latency-associated transcript promoter in the right gastrocnemius muscle of rats. Expression of beta-gal activity was observed 5 days after inoculation in the bilateral anterior horn cells of the spinal cord that innervates the inoculation muscle. However, the spread of beta-gal activity was not observed in the inoculation muscle. Without significant pathological changes, the spread of beta-gal-expressing neurons was observed in the lumbosacral spinal cord until 14 days after inoculation with staining concentrated in the anterior horn cells. Ninety percent of the anterior horn motor neurons expressed beta-gal activity with expression continuing to at least 182 days after inoculation. Thus beta-gal activity was expressed in the bilateral anterior horn cells at the lumbosacral spinal cord that innervates the inoculated muscle for a long time, possibly a life-long period. This indicates that this recombinant HSV vector system to motor neurons may further improve the understanding and treatment of neurological diseases in motor neurons of the spinal cord.

Inhibition of rat vascular smooth muscle cell proliferation in vitro and in vivo by recombinant replication-competent herpes simplex virus.

The proliferation of vascular smooth muscle cells (VSMCs) is a common feature associated with vascular proliferative disorders such as atherosclerosis and restenosis after balloon angioplasty. We examined the antiproliferative effects of recombinant replication-competent herpes simplex virus (HSV), hrR3, to proliferative VSMCs both in vitro and in vivo. Early passages of Sprague-Dawley rat VSMCs were infected with hrR3 at a low multiplicity of infection (0.01 to 1.0) to examine the in vitro cytotoxic activity of this recombinant HSV to VSMCs in a proliferative state. Sprague-Dawley rats underwent balloon dilatation injury of the left carotid artery to induce neointimal formation. The injured carotid arteries were infected with hrR3 five days after balloon injury. Two weeks after injury, the left carotid arteries were fixed, and the areas of the neointimal and medial layers were analyzed microscopically. Because the reporter Escherichia coli lacZ gene in hrR3 is expressed only in infected cells in which the virus is actively replicating, virus replication was confirmed by X-gal staining. A morphometric analysis revealed that there were significant differences in the intima/media ratio between the HSV-treated group and mock-infected group (0. 354+/-0.068 and 1.08+/-0.055, respectively). In the histological study (X-gal staining), positive X-gal staining was observed chiefly in the VSMCs in the medial layer just beneath the internal elastic lamina, indicating active viral replication. Virus-mediated cytocidal therapy using recombinant HSV vector is a promising modality for the treatment of the restenosis after balloon angioplasty.

Overexpression in Neurons of Human Presenilin-1 or a Presenilin-1 Familial Alzheimer Disease Mutant Does Not Enhance Apoptosis

Programmed cell death, or apoptosis, has been implicated in Alzheimer's disease (AD). DNA damage was assessed in primary cortical neurons infected with herpes simplex virus (HSV) vectors expressing the familial Alzheimer's disease (FAD) gene presenilin-1 (PS-1) or an FAD mutant of this gene, A246E. After infection, immunoreactivity for PS-1 was shown to be enhanced in infected cells. The infected cells exhibited no cytotoxicity, as evaluated by trypan blue exclusion and mitochondrial function assays. Quantitative analysis of cells that were immunohistochemically labeled using a Klenow DNA fragmentation assay or the TUNEL method revealed no enhancement of apoptosis in PS-1-infected cells. This result was confirmed using assays for chromatin condensation and for DNA fragmentation. Expression of PS-1 protected against induction of apoptosis in the cortical neurons by etoposide or staurosporine. The specificity of this phenotype was demonstrated by the fact that cortical cultures infected with recombinant HSV vectors expressing the amyloid precursor protein (APP-695) showed, in contrast, a significant increase in the number of apoptotic cells and an increase in DNA fragmentation for all parameters tested. Our results indicate that overexpression of wild-type or A246E mutant PS-1 does not enhance apoptosis in postmitotic cortical cells and suggest that the previously reported enhancement of apoptosis by presenilins may be dependent on cell type.

Mechanisms of trophoblast-virus interaction

The human placenta serves as a barrier to the transmission of some viruses, but allows others to reach the fetal circulation. The resistance and permissiveness of the placenta to viral transmission appears to be determined in large part by the placental trophoblast. In order to define the mechanisms by which human trophoblast cells influence vertical transmission of viruses, we have studied the interaction of replication-deficient recombinant viral vectors with transformed human choriocarcinoma (BeWo) cells and primary trophoblast cell cultures. Recombinant adenovirus vectors efficiently transduce BeWo cells and primary trophoblast cells. However, as BeWo cells differentiate after treatment with cAMP analogs, they become relatively resistant to adenovirus-mediated transduction due to diminished uptake of the virus particles. This differentiation-dependent loss of adenovirus transduction may be related to the down-regulation of the coxsackie adenovirus receptor, which we have detected in undifferentiated trophoblast cells. Recombinant herpes simplex virus vectors also transduce undifferentiated BeWo cells and isolated cytotrophoblast cells, but transduction by herpes simplex virus vectors declines with cAMP-induced trophoblast differentiation, apparently due to reduced viral uptake. In contrast, cAMP treatment augments trophoblast transduction by recombinant adeno-associated virus, a member of the parvovirus family. This augmentation appears to be due to increased virus uptake by a yet to be determined mechanism. The understanding of the molecular basis of these interactions between recombinant viral vectors and trophoblast cells may yield strategies to prevent vertical transmission of viruses as well as to create opportunities to modify trophoblast function through gene transfer using viral vectors.

Introduction of the glutamate receptor subunit 1 into motor neurons in vitro and in vivo using a recombinant herpes simplex virus

We developed and characterized a recombinant herpes simplex virus vector and used it to introduce the complementary DNA encoding glutamate receptor subunit 1 flip into postmitotic motor neurons. Infection of purified motor neurons in vitro with this vector resulted in selective, high-level expression of glutamate receptor subunit 1 immunoreactivity in nearly 100% of the neurons. Patch-clamp experiments demonstrated that the protein product of the glutamate receptor subunit 1 flip transgene assembles into functional α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor channels. Herpes simplex virus-glutamate receptor subunit 1 flip was introduced into spinal cord cells by direct injection into the ventral horn and selectively into motor neurons by sciatic nerve injection. High levels of expression were sustained for at least one week and were accompanied by changes in the ionic permeability of AMPA receptors in transgene-expressing neurons. Throughout the first week of infection, there was little evidence for toxicity. Herpes simplex virus provides a versatile tool for manipulating the glutamate receptor phenotype of postmitotic neurons and will permit study of the role of individual glutamate receptor subunits in neuronal physiology and pathophysiology.