Use Of Herpes Simplex Virus Research Articles

Use of Blood Polymerase Chain Reaction Testing for Diagnosis of Herpes Simplex Virus Infection

The use of herpes simplex virus (HSV) polymerase chain reaction for diagnosis of HSV disease involving the central nervous system has not translated into widespread use for the detection of DNAemia. We report our 6-year experience using blood polymerase chain reaction testing for HSV infection in neonates and older children with HSV disease.

HSV Recombinant Vectors for Gene Therapy

The very deep knowledge acquired on the genetics and molecular biology of herpes simplex virus (HSV), has allowed the development of potential replication-competent and replication-defective vectors for several applications in human healthcare. These include delivery and expression of human genes to cells of the nervous systems, selective destruction of cancer cells, prophylaxis against infection with HSV or other infectious diseases, and targeted infection to specific tissues or organs. Replication-defective recombinant vectors are non-toxic gene transfer tools that preserve most of the neurotropic features of wild type HSV-1, particularly the ability to express genes after having established latent infections, and are thus proficient candidates for therapeutic gene transfer settings in neurons. A replication-defective HSV vector for the treatment of pain has recently entered in phase 1 clinical trial. Replication-competent (oncolytic) vectors are becoming a suitable and powerful tool to eradicate brain tumours due to their ability to replicate and spread only within the tumour mass, and have reached phase II/III clinical trials in some cases. The progress in understanding the host immune response induced by the vector is also improving the use of HSV as a vaccine vector against both HSV infection and other pathogens. This review briefly summarizes the obstacle encountered in the delivery of HSV vectors and examines the various strategies developed or proposed to overcome such challenges.

Subcutaneous Peripheral Injection of Cationized Gelatin/DNA Polyplexes As a Platform for Non-viral Gene Transfer to Sensory Neurons

Selective modulation of sensory neuron gene expression could have numerous applications for the peripheral nervous system. Here, we report that subcutaneous peripheral injection of plasmid DNA complexed with a non-viral cationized gelatin (CG) vector led to transgene expression in rat lumbar dorsal root ganglia (DRGs). CG/DNA polyplexes appeared to undergo rapid retrograde transport through sciatic and spinal nerves, with reporter gene messenger RNA (mRNA) expression detectable in L4 and L5 DRGs within 60 hours. Maximum transgene expression was observed for polyplexes formed at 7.5:1 CG-to-DNA weight ratio under salt-free conditions, which generated 615 +/- 112 nm nanoparticles with zeta-potential of 9.4 +/- 0.19 mV. Six days after injection of the CG/DNA polypex, reporter gene protein immunofluorescence was observed in 1,164 +/- 176 DRG neurons, representing an estimated transfection rate of 47% of targeted neurons. Reporter gene expression was not detected in heart, lung, or liver tissues, suggesting a lack of systemic uptake. Measurements of tactile sensitivity indicate that CG/DNA injection did not cause behavioral toxicity. The injection platform was further used for plasmid-driven short hairpin RNA-mediated suppression of glyceraldehyde-3-phosphate dehydrogenase. This non-invasive gene delivery system could be used for the mechanistic study and targeted molecular evaluation of peripheral nervous system pathologies such as neuropathic pain.

Use of herpes simplex virus and pseudorabies virus chimeric glycoprotein D molecules to identify regions critical for membrane fusion.

Membrane fusion induced by herpes simplex virus (HSV) requires the action of four viral membrane glycoproteins (gB, gD, gH, and gL) and the binding of gD to one of its receptors, such as the herpesvirus entry mediator or nectin-1. The related animal herpesvirus, pseudorabies virus (PRV), encodes a homologous set of glycoproteins and its gD can also use nectin-1 as an entry receptor. We show here that PRV gD, when coexpressed with HSV gB, gH, and gL, cannot substitute for HSV gD in inducing fusion with target cells expressing nectin-1. Chimeric gD molecules composed of HSV and PRV sequences can substitute, provided the first 285 aa are from HSV gD. Because the first 261 aa were sufficient for receptor binding, this suggested that amino acids 262-285 contain a region required for cell fusion but not for receptor binding. Deletions from amino acids 250-299 failed to identify a specific subregion critical for cell fusion, except possibly for amino acids 250-255, which also influenced receptor binding. Instead, presence of a flexible stalk between the membrane and receptor-binding domain appears to be required, perhaps to enable conformational changes in gD on receptor binding and subsequent interactions of undefined regions of gD with the other glycoproteins required for membrane fusion.

HSV amplicon-mediated Aβ vaccination in Tg2576 mice: differential antigen-specific immune responses

Given the participation of amyloid beta (Aβ) in Alzheimer’s disease (AD) pathogenesis the derivation of experimental therapeutics to prevent Aβ fibrillogenesis and/or enhance removal of parenchymal amyloid deposits represent viable disease-modifying approaches. Active Aβ-based immunotherapies have shown promise in mouse AD models, but application in human trials was accompanied by moderate brain inflammation in a subset of patients. Immune-shaping vaccine platforms may mitigate adverse effects. Herein, we describe the use of herpes simplex virus (HSV)-derived amplicons to elicit distinctive immune responses against Aβ. Two vaccine vectors were constructed: one expressing Aβ 1–42 alone (HSVAβ), and a second expressing Aβ 1–42 fused with the molecular adjuvant tetanus toxin Fragment C (HSVAβ/TtxFC). Peripheral administration of these vaccines augmented humoral responses to Aβ and reduced CNS Aβ deposition in Tg2576 AD mice. Interestingly and unexpectedly, HSVAβ vaccination was uniquely toxic and incited the expression of pro-inflammatory molecule transcripts within the hippocampi of Tg2576 mice, suggesting that this paradigm may serve as a relevant model to study Aβ vaccine-elicited CNS inflammatory syndromes.

42. AAV-mediated gene therapy causes functional improvements in a murine model of infantile neuronal ceroid lipofuscinosis (INCL, Batten Disease)

Active Aβ peptide-based immunotherapies have shown promise in preclinical mouse Alzheimer's disease (AD) models, but application in human trials was accompanied by brain inflammation in a subset of trial participants. Immune response-shaping vaccine platforms may impart therapeutic benefit and simultaneously mitigate adverse effects. Herein, we describe the use of herpes simplex virus (HSV)-derived amplicons to elicit distinctive immune responses against Aβ. Two vaccine vectors were constructed: one expressing Aβ1–42 alone (HSVAβ), and a second expressing Aβ1–42 fused in-frame with the molecular adjuvant tetanus toxin Fragment C (HSVAβ/TtxFC). Peripheral administration of HSVAβ/TtxFC augmented humoral responses to Aβ and reduced CNS Aβ deposition in Tg2576 AD mice. Interestingly and unexpectedly, HSVAβ vaccination was uniquely toxic, and incited the expression of pro-inflammatory molecule transcripts within the hippocampi of Tg2576 mice. However, the few surviving HSVAβ-vaccinated Tg2576 mice exhibited the most dramatic reduction in Aβ deposit numbers as compared to HSVAβ/TtxFC-inoculated counterparts. Vaccination of non-transgenic littermates with either amplicon vector produced detectable humoral responses but resulted in no observable toxicity. This paradigm may serve as a relevant model to study the inflammatory component presenting in Aβ peptide-vaccinated human subjects. Ongoing and future studies involve assessment of vaccine-elicited cellular immune responses, effects on brain amyloid deposition and memory behavior, and employment of co-delivered immunomodulatory genes to shape the immune responses elaborated to the Aβ antigen. A detailed characterization of CNS immunological responses in the setting of AD is highly relevant to development of safe vaccines.

Transfection-free and scalable recombinant AAV vector production using HSV/AAV hybrids.

Adeno-associated virus (AAV) vectors are highly efficient tools for use in gene therapy. Current production methods rely on plasmid transfection and are not generally considered amenable to scale-up. To improve recombinant AAV (rAAV) vector production in terms of both final titre and simplicity, we constructed recombinant herpes simplex virus (HSV) vectors, either disabled (ICP27 deleted) or nondisabled, encoding the AAV rep and cap genes. We also integrated an rAAVGFP construct into the nondisabled vector and also into a second pair of HSV vectors (disabled and nondisabled) not expressing rep and cap. Transgene incorporation and expression was confirmed by Southern and Western blot, respectively. Optimal double-infection ratios were established for disabled and nondisabled pairs of rep/cap-expressing and rAAVGFP-containing vectors, resulting in up to 1.55 x 10(12) rAAV capsids and 4 x 10(8) expression units from approximately 1 x 10(7) BHK producer cells. Functionality of the prepared vector was confirmed by the detection of abundant green fluorescent protein (GFP) expression following injections of rAAV preparations into the rat brain. This paper therefore describes a simple, efficient, and transfection-free rAAV production process based on the use of HSV and not relying on a proviral cell line that, with appropriate scale-up, could yield quantities of rAAV sufficient for routine clinical use.

Virus Vectors for use in the Central Nervous System: Problems in the Use of Herpes Simplex Virus as a Vector

Publisher Summary This chapter describes problems in the use of herpes simplex virus (HSV) as a vector for expression of foreign gene. The use of HSV1 as a vector has been investigated by a number of groups and these studies have included both replicating and nonreplicating viruses. The use of HSV1 as a vector became a practical goal with the discovery that the ICP4 gene could be deleted and the virus grown as a host range mutant. It is found that functional promoter expression during latency may require two elements, and that the upstream latency associated transcript (LAT) promoter and the long terminal repeats (LTR) together provide these functions. It is observed that as specific regions of the upstream part of the LAT promoter were removed, particularly between −330 and −250, expression in neurons decreased. It is suggested that targeted expression is nothing more than making a good match between transcription factor binding sites on the promoter and high levels of the same transcription factor in a given cell type. A reasonable explanation for the lack of expression of 8117-43 in the hypoglossal nucleus may have been a lack of transcription factors that could bind to either the LTR or the LAT enhancer. It is suggested that in neurons of the dorsal root ganglia, where the factors can bind to the LAT enhancer, expression is high, and in other neurons it is lower or not observed.

First episodes of genital herpes in a Swedish STD population: a study of epidemiology and transmission by the use of herpes simplex virus (HSV) typing and specific serology. Lowhagen GB,∗ Tunback P, Andersson K, Bergstrom T, Johannisson G. Sex Transm Infect 2000;76:179–82.

First episodes of genital herpes in a Swedish STD population: a study of epidemiology and transmission by the use of herpes simplex virus (HSV) typing and specific serology. Lowhagen GB,∗ Tunback P, Andersson K, Bergstrom T, Johannisson G. Sex Transm Infect 2000;76:179–82.

First episodes of genital herpes in a Swedish STD population: a study of epidemiology and transmission by the use of herpes simplex virus (HSV) typing and specific serology

Objectives: To determine the proportion of herpes simplex virus type 1 (HSV-1) and HSV type 2 (HSV-2) in first episodes of genital herpes. To evaluate the use of HSV specific...

Use of herpes simplex virus (HSV) type 1 ISCOMS 703 vaccine for prophylactic and therapeutic treatment of primary and recurrent HSV-2 infection in guinea pigs.

The effect of subunit vaccination on the incidence and severity of primary and recurrent genital herpes was investigated in the female guinea pig model of herpes simplex virus (HSV) type 2 genital infection. After prophylactic immunization with zwitterionic detergent-solubilized HSV-1 glycoproteins formulated with alhydrogel or as immunostimulating complex particles, significant reductions in the incidence and severity of primary herpetic illness were observed in both vaccinated groups compared with immunization-naive controls. There was a significant reduction in the incidence of spontaneous herpetic recurrences after administration of HSV-1 antigens formulated as immunostimulatory complexes to guinea pigs in a prophylactic mode (P<.01). Increased levels of both postimmunization and postchallenge ELISA and neutralizing antibodies were significant correlates of protection against primary herpetic disease in a prophylactic scenario. However, no correlation was observed between elevated ELISA or neutralizing antibody levels and protection against recurrent disease following prophylactic or therapeutic administration of HSV-1 subunit vaccines.

Pseudotyping of glycoprotein D-deficient herpes simplex virus type 1 with vesicular stomatitis virus glycoprotein G enables mutant virus attachment and entry.

The use of herpes simplex virus (HSV) vectors for in vivo gene therapy will require the targeting of vector infection to specific cell types in certain in vivo applications. Because HSV glycoprotein D (gD) imparts a broad host range for viral infection through recognition of ubiquitous host cell receptors, vector targeting will require the manipulation of gD to provide new cell recognition specificities in a manner designed to preserve gD's essential role in virus entry. In this study, we have determined whether an entry-incompetent HSV mutant with deletions of all Us glycoproteins, including gD, can be complemented by a foreign attachment/entry protein with a different receptor-binding specificity, the vesicular stomatitis virus glycoprotein G (VSV-G). The results showed that transiently expressed VSV-G was incorporated into gD-deficient HSV envelopes and that the resulting pseudotyped virus formed plaques on gD-expressing VD60 cells, albeit at a 50-fold-reduced level compared to that of wild-type gD. This reduction may be related to differences in the entry pathways used by VSV and HSV or to the observed lower rate of incorporation of VSV-G into virus envelopes than that of gD. The rate of VSV-G incorporation was greatly improved by using recombinant molecules in which the transmembrane domain of HSV glycoprotein B or D was substituted for that of VSV-G, but these recombinant molecules failed to promote virus entry. These results show that foreign glycoproteins can be incorporated into the HSV envelope during replication and that gD can be dispensed with on the condition that a suitable attachment/entry function is provided.

Equine herpesvirus 1 gene 12 can substitute for vmw65 in the growth of herpes simplex virus (HSV) type 1, allowing the generation of optimized cell lines for the propagation of HSV vectors with multiple immediate-early gene defects.

Herpes simplex virus (HSV) has often been suggested for development as a vector, particularly for the nervous system. Considerable evidence has shown that for use of HSV as a vector, immediate-early (IE) gene expression must be minimized or abolished, otherwise such vectors are likely to be highly cytotoxic. Mutations of vmw65 which abolish IE promoter transactivating activity may also be included to reduce IE gene expression generally. However, when vmw65 mutations are combined with an IE gene deletion, such viruses are hard to propagate, even on cells which otherwise complement the IE gene deletion effectively. We have found that vmw65 mutants can be effectively grown on cell lines expressing equine herpesvirus 1 gene 12, a non-HSV homologue of vmw65 with little sequence similarity to its HSV counterpart. This prevents repair by homologous recombination of vmw65 mutations in the virus, which would occur if mutations were complemented by vmw65 itself. The gene 12 protein is not packaged into HSV virions, which is important if viruses grown on such cells are to be used as vectors. These results not only further strengthen the evidence for direct functional homology between and similar modes of action of the two proteins but have allowed the generation of gene 12-containing cell lines in which ICP4 and ICP27 expression is induced by virus infection (probably by ICP0) and which give efficient growth of viruses deficient in ICP27, ICP4, and vmw65 (the viruses also have ICP34.5/ORFP deleted). Efficient growth of such viruses has not previously been possible. As these viruses are highly deficient in IE gene expression generally, such virus-cell line combinations may provide an alternative to HSV vectors with deletions of all four of the regulatory IE genes which, for optimal growth, require cell lines containing all four IE genes but which are hard to generate due to the intrinsic cytotoxicity of each of the proteins.

Efficient infection of mature skeletal muscle with herpes simplex virus vectors by using dextran sulfate as a co-receptor.

The use of herpes simplex virus (HSV) vectors for gene delivery to skeletal muscle is hampered by a maturation-dependent loss of muscle fiber infectivity. Previous studies of HSV type 1 (HSV-1) infection in the rodent show that the loss of infectivity may be due, at least in part, to the development of the basal lamina throughout the course of maturation, which may block the initial events in HSV infection. To initiate infection, HSV normally attaches to cell surface heparan sulfate, which stabilizes the virus such that it can interact with secondary protein receptors required for entry into host cells. In this study, we demonstrate that heparan sulfate biosynthesis is downregulated during skeletal muscle maturation. When myofibers were treated with a variety of enzymes, including collagenase type IV or chondroitin ABC lyase, HSV infection was restored, which suggests that virus secondary receptors were present but not readily accessible to the virus in the intact myofiber. Surprisingly, we also found that HSV-1 infectivity could be restored in vitro and in vivo by exposing myofibers to low concentrations of the glycosaminoglycan analog dextran sulfate, which appears to act as a surrogate receptor to stabilize the virus at the myofiber surface such that HSV can engage additional receptors. This demonstration that the basal lamina is not an absolute block to HSV-1 infection is remarkable because it allows for the nondestructive targeting of HSV-1 to mature myofibers and greatly expands the usefulness of HSV as a gene therapy vector for the treatment of inherited and acquired diseases.

High efficiency gene transfer to the central nervous system of rodents and primates using herpes virus vectors lacking functional ICP27 and ICP34.5.

The safe and efficient use of herpes simplex virus (HSV)-based vectors to deliver genes of potentially therapeutic benefit to the central nervous system will require their effective disablement by the inactivation of viral genes required for lytic growth. Here we report that viruses lacking functional genes for ICP27 (which is required for growth in all cell types) and ICP34.5 (which is required for growth in nondividing cell types) can deliver a marker gene to both the rodent and primate CNS with high efficiency whilst producing relatively minimal damage and having no effect on sodium currents in dorsal root ganglion neurons. Such viruses paradoxically deliver genes at much higher efficiency than the less disabled single mutant lacking ICP34.5 alone and also, as expected, produce less damage in vivo. Moreover, unlike the single mutant lacking ICP27 the double mutant viruses cannot revert to wild-type by acquistion of complimenting gene sequences during growth of virus stocks in vitro on dividing cells expressing ICP27 since artificial expression of ICP34.5 in these cells is not required. Such ICP27-; ICP34.5- viruses thus offer a platform for the development of vectors which are sufficiently safe for ultimate use in human gene therapy.

Persistence and expression of the herpes simplex virus genome in the absence of immediate-early proteins.

The immediate-early (IE) proteins of herpes simplex virus (HSV) function on input genomes and affect many aspects of host cell metabolism to ensure the efficient expression and regulation of the remainder of the genome and, subsequently, the production of progeny virions. Due to the many and varied effects of IE proteins on host cell metabolism, their expression is not conducive to normal cell function and viability. This presents a major impediment to the use of HSV as a vector system. In this study, we describe a series of ICP4 mutants that are defective in different subsets of the remaining IE genes. One mutant, d109, does not express any of the IE proteins and carries a green fluorescent protein (GFP) transgene under the control of the human cytomegalovirus IE promoter (HCMVIEp). d109 was nontoxic to Vero and human embryonic lung (HEL) cells at all multiplicities of infection tested and was capable of establishing persistent infections in both of these cell types. Paradoxically, the genetic manipulations that were required to eliminate toxicity and allow the genome to persist in cells for long periods of time also dramatically lowered the level of transgene expression. Efficient expression of the HCMVIEp-GFP transgene in the absence of ICP4 was dependent on the ICP0 protein. In d109-infected cells, the level of transgene expression was very low in most cells but abundant in a small subpopulation of cells. However, expression of the transgene could be induced in cells containing quiescent d109 genomes weeks after the initial infection, demonstrating the functionality of the persisting genomes.

Herpes simplex viruses.

Herpes simplex virus (HSV) infections of humans have been documented since the advent of writing. The spectrum of disease was expanded to include primary and recurrent infections of mucous membranes (gingivostomatitis, herpes labialis, and genital HSV infections), keratoconjunctivitis, neonatal HSV infection, visceral HSV infections of the immunocompromised host, HSV encephalitis, Kaposi's varicella-like eruption, and an association with erythema multiforme. Cumulative experience suggests that factors associated with pregnancy may place both the mother and fetus at increased risk for severe infection, possibly because of altered cell-mediated immunity. The major risk to the fetus is with primary or initial genital HSV infection of the mother. PCR evaluation of cerebrospinal fluid can be utilized to monitor therapeutic outcome in patients with herpes simplex encephalitis. The use of HSV for gene therapy heralds a new era of herpes biology, the conversion of a hazardous foe into a user-friendly surgical tool. Asymptomatic shedding of virus can continue despite clinically effective suppression with acyclovir, so the possibility of person-to-person transmission persists. Newborns with HSV infections can be classified as having disease that is localized to the skin, eyes, and mouth; affects the central nervous system (CNS); or is disseminated. Drug resistance was considered rare and resistant isolates were thought to be less pathogenic until a series of acyclovir-resistant HSV isolates from patients with AIDS were characterized. The risk of nephrotoxicity can be minimized by administering acyclovir by slow infusion and ensuring adequate hydration.

Potential use of herpes simplex virus (HSV) vectors for gene therapy of neurological disorders.

Advances in molecular biology and virus genetics have allowed the possibility of gene therapy using viral vectors for a variety of neurological diseases in which the genetic or biochemical basis is understood. A number of such vectors have now been constructed, including those derived from herpes simplex virus (HSV), adenovirus, retrovirus and adeno-associated virus, and used in preliminary in vitro experiments and in animal models. It is possible to package a foreign gene into such a vector which can then be targeted to specific regions of the nervous system. HSV is particularly appropriate for delivering genes to neurons in view of its ability to establish latent infection in these cells. Viral vectors have the potential to be used to treat such neurological conditions as malignant gliomas, Parkinson's disease, known single gene disorders and cerebral ischaemia. However, the technical problems which will need to be overcome are formidable and will not be easily solved. The problems include the efficient delivery of the vector to target cells, the maintenance and control of foreign gene expression, and the control of unwanted host immune responses.

Gene therapy of cancer

Gene therapy of cancer

The roles of herpes simplex virus in neuroscience.

Herpes simplex virus (HSV) has been a focus of research in many laboratories during the last 30-35 years, with the majority centered on the virus' replication, molecular biology and pathogenesis. Recently, HSV has begun to receive considerable attention in the field of neuroscience, where scientists have begun to use the virus as a tool or model for several areas of investigation. These areas include the construction and development of HSV-based vectors for gene therapy and the use of HSV as a neuronal tracer, as a model for demyelinating disease and to study interactions between the nervous, immune and endocrine systems. The goal of this paper is to review these different roles for HSV in the broad field of neuroscience.