CNS gene transfer could provide new approaches to the modelling of neurodegenerative diseases and devising potential therapies. One such disorder is Parkinson's disease (PD), in which dysfunction of several different metabolic processes has been implicated. Here we review the literature on gene transfer systems based on herpes simplex virus type 1 (HSV-1) and non-viral polyethyleneimine (PEI) and calcium phosphate nanoparticle methods. We also assess the usefulness of various CNS gene delivery methods and present some of our own data to exemplify such usefulness. Our data result from vectors stereotaxically introduced to the substantia nigra (SN) of adult rats and evaluated 1 week and/or 1 month post injection using histochemical methods to assess recombinant ss-galactosidase enzyme activity. Gene transfer using PEI or calcium phosphate-mediated transfections was observed for both methods and PEI was comparable to that of HSV-1 amplicon. Our data show that the amplicon delivery was markedly increased when packaged with a helper virus and was similar to the expression profile achieved with a full-size replication-defective HSV-1 recombinant (8117/43). We also examine whether PEI or HSV-1 amplicon-mediated gene transfer could facilitate assessment of the biological effects induced by a dominant negative FGF receptor-1 mutant to model the reduced FGF signalling thought to occur in Parkinson's disease.
A universal vaccine that generally prevents influenza virus infection and/or illness remains elusive. We have been exploring a novel approach to vaccination involving replication-competent controlled herpesviruses (RCCVs) that can be deliberately activated to replicate efficiently but only transiently in an administration site in the skin of a subject. The RCCVs are derived from a virulent wild-type herpesvirus strain that has been engineered to contain a heat shock promoter-based gene switch that controls the expression of, typically, two replication-essential viral genes. Additional safety against inadvertent replication is provided by an appropriate secondary mechanism. Our first-generation RCCVs can be activated at the administration site by a mild local heat treatment in the presence of an antiprogestin. Here, we report that epidermal vaccination with such RCCVs expressing a hemagglutinin or neuraminidase of an H1N1 influenza virus strain protected mice against lethal challenges by H1N1 virus strains representing 75 years of evolution. Moreover, immunization with an RCCV expressing a subtype H1 hemagglutinin afforded full protection against a lethal challenge by an H3N2 influenza strain, and an RCCV expressing a subtype H3 hemagglutinin protected against a lethal challenge by an H1N1 strain. Vaccinated animals continued to gain weight normally after the challenge. Protective effects were even observed in a lethal influenza B virus challenge. The RCCV-based vaccines induced robust titers of in-group, cross-group and even cross-type neutralizing antibodies. Passive immunization suggested that observed vaccine effects were at least partially antibody-mediated. In summary, RCCVs expressing a hemagglutinin induce robust and very broad cross-protective immunity against influenza.
Diseased corneas are potential targets for viral-based gene therapy to normalize (stimulate or inhibit) the expression of specific proteins. The choice of viral vectors is important to achieve optimal effect. The purpose of this study was to compare the tropism to different corneal cells of recombinant adenovirus (rAV) and recombinant adeno-associated virus (rAAV) constructs using live rabbit and organ-cultured human corneas.rAV constructs harbored the green fluorescent protein (GFP) gene under the control of major immediate early cytomegalovirus (CMV) promoter. rAAV constructs from virus serotypes 1, 2 5, 7, and 8 had GFP under the chicken beta-actin promoter and CMV enhancer. For organ culture, 16 healthy and diabetic postmortem human corneas were used. Five or fifteen microl rAV at 10(7) plaque forming units per 1 microl were added for 2 days to culture medium of uninjured corneas that were further cultured for 5-32 days. rAAV were added at 1.2-7.8x10(10) vector genomes per cornea for 3 days to each cornea; the culture then continued for another 14-23 days. Corneal cryostat sections were examined by immunohistochemistry. Live rabbit corneas were used following excimer laser ablation of the corneal epithelium with preservation of the basal cell layer. Equal numbers of rAAV particles (2x10(11) vector genomes) were applied to the cornea for 10 min. After seven days to allow for corneal healing and gene expression the animals were euthanized, the corneas were excised, and sections analyzed by immunohistochemistry.By direct fluorescence microscopy of live organ-cultured human corneas GFP signal after rAV transduction was strong in the epithelium with dose-dependent intensity. On corneal sections, GFP was seen in all epithelial layers and some endothelial cells but most keratocytes were negative. In rAAV-transduced organ-cultured human corneas GFP signal could only be detected with anti-GFP antibody immunohistochemistry. GFP was observed in the epithelium, keratocytes, and endothelium, with more pronounced basal epithelial cell staining with rAAV1 than with other serotypes. No difference in the GFP expression patterns or levels between normal and diabetic corneas was noted. The rabbit corneas showed very similar patterns of GFP distribution to human corneas. With all rAAV serotype vectors, GFP staining in the epithelium was significantly (p=0.007) higher than the background staining in non-transduced corneas, with a trend for rAAV1 and rAAV8 to produce higher staining intensities than for rAAV2, rAAV5 (p=0.03; rAAV5 versus rAAV1), and rAAV7. rAAV serotype vectors also transduced stromal and endothelial cells in rabbit corneas to a different extent.rAAV appears to reach many more corneal cells than rAV, especially keratocytes, although GFP expression levels were lower compared to rAV. rAV may be more useful than rAAV for gene therapy applications requiring high protein expression levels, but rAAV may be superior for keratocyte targeting.
Vaccines to prevent important infections involving, e.g. influenza viruses, severe acute respiratory syndrome-causing coronaviruses (e.g. SARS-CoV-2), respiratory syncytial viruses (RSV), and human immunodeficiency viruses (HIV) have remained insufficiently effective or are not available at all. Regulated microbes constitute novel vaccine platforms that may be employed for the development of more potent and/or more broadly effective vaccines. We review the development and characterization of the vaccine potential of replication-competent controlled herpesviruses (RCCVs) which represent the first examples of regulated microbes used as vaccines. The RCCVs developed to date are suitable for application to the skin and can be activated deliberately to replicate efficiently, but only transiently, in the administration site. Without activation, the RCCVs are incapable of replicating in the nervous system and elsewhere. The RCCVs were found to induce potent anti-herpetic immune responses in mice. Vaccination with RCCVs expressing an influenza virus hemagglutinin broadly protected animals against lethal influenza virus challenges. This protection appeared to be at least in part antibody-mediated. These findings support a rational expectation that RCCVs may be developed into universal, non-seasonal vaccines against influenza and, possibly, against other rapidly evolving pathogens.
To analyze the rabbit host global gene expression patterns in uninfected and herpes simplex virus type 1 (HSV-1) latent trigeminal ganglia (TG) for identification of host response-initiated transcriptional changes during the maintenance of viral latency.The corneas of eight-week-old New Zealand White rabbits were scarified and inoculated with HSV-1 strain McKrae, 5x10(5) plaque forming units/eye. Corneal infection was verified by slit-lamp examination. Prior to sacrifice at 30 days post infection, ocular swabs confirmed no infectious virus was present. TG were aseptically removed from rabbits and placed in RNA stabilization solution. Host RNA was isolated from two groups of TG, uninfected and HSV-1 latent infected, and used to create labeled cRNA. Labeled cRNA was hybridized to two new and novel custom oligonucleotide rabbit arrays, containing a total of 3,123 probes for rabbit genes.The rabbit TG expressed approximately 80% of genes out of a total of 3,123. A one-way ANOVA performed on the log2 transformed signal ratios showed 611 genes were significantly altered (p< or =0.05) in HSV-1 latent TG. These genes, if annotated, were separated by biologic process categories. Five broad categories were most heavily represented: protein processing, carbohydrate processing, cell adhesion, apoptosis, and host defense and immune response. Sixty of the significantly altered genes were found to be altered by more than 2 fold, and five were altered by more than 4 fold. The genes altered by more than 4 fold were all upregulated and related to host defense and immune response. Viral latency had a large effect on protein processing. Of the differentially expressed genes with an assigned biologic process, 90/349 (25.7%) were associated with protein processing. The next most populated categories were carbohydrate metabolism 39/349 (11.1%) and host defense and immune response 17/349 (4.9%).The results of this microarray study demonstrate that host gene expression is altered in the HSV-1 latent rabbit TG. The shift in molecular processes at a pathway level reveals the presence of potential therapeutic significance inherent in the maintenance of HSV-1 latency. This is the first large-scale rabbit gene expression study, using microarray analysis, that documents the involvement of host immunity in maintaining HSV-1 latency.
A number of aspects of the natural biology of herpes simplex virus (HSV) make it an attractive candidate for a vector to express foreign genes within the nervous system. Some of the advantages of an HSV vector are
