Herpes simplex virus vector-mediated expression of Bcl-2 prevents 6-hydroxydopamine-induced degeneration of neurons in the substantia nigra in vivo

6-Hydroxydopamine (6-OHDA) is widely used to selectively lesion dopaminergic neurons of the substantia nigra (SN) in the creation of animal models of Parkinson’s disease. In vitro, the death of PC-12 cells caused by exposure to 6-OHDA occurs with characteristics consistent with an apoptotic mechanism of cell death. To test the hypothesis that apoptotic pathways are involved in the death of dopaminergic neurons of the SN caused by 6-OHDA, we created a replication-defective genomic herpes simplex virus-based vector containing the coding sequence for the antiapoptotic peptide Bcl-2 under the transcriptional control of the simian cytomegalovirus immediate early promoter. Transfection of primary cortical neurons in culture with the Bcl-2-producing vector protected those cells from naturally occurring cell death over 3 weeks. Injection of the Bcl-2-expressing vector into SN of rats 1 week before injection of 6-OHDA into the ipsilateral striatum increased the survival of neurons in the SN, detected either by retrograde labeling of those cells with fluorogold or by tyrosine hydroxylase immunocytochemistry, by 50%. These results, demonstrating that death of nigral neurons induced by 6-OHDA lesioning may be blocked by the expression of Bcl-2, are consistent with the notion that cell death in this model system is at least in part apoptotic in nature and suggest that a Bcl-2-expressing vector may have therapeutic potential in the treatment of Parkinson’s disease.

Severe genital herpes infections in HIV-infected individuals with impaired herpes simplex virus-specific CD8+ cytotoxic T lymphocyte responses

The specific mechanisms underlying the varied susceptibility of HIV-infected (HIV+) individuals to opportunistic infections (OI) are still incompletely understood. One hypothesis is that quantitative differences in specific T cell responses to a colonizing organism determine the development of an AIDS-defining OI. We evaluated this hypothesis for herpes simplex virus (HSV) infection, a common OI in HIV+ patients. Using limiting dilution analyses, the frequency of HSV-specific CD8+ cytotoxic T lymphocyte precursors (pCTL) and proliferative precursors were quantitated in peripheral blood mononuclear cells from 20 patients coinfected with HIV and HSV-2. The frequency of HSV-specific CD8+ pCTL in HSV+HIV+ individuals was significantly lower than in HSV+HIV− individuals (1 in 77,000 vs. 1 in 6,000, P = .0005) and was not different than in HSV-HIV− individuals (1 in 100,000, P = .24). HIV+ patients who suffered more severe genital herpes recurrences had significantly lower HSV-specific CD8+ pCTL frequencies than those patients with mild recurrences (1 in 170,000 vs. 1 in 26,000, P = .03). In contrast, no significant difference was seen in proliferative precursor frequencies between those patients with mild vs. severe genital herpes (1 in 3,800 vs. 1 in 6,600, P > .5). Quantitative differences in pCTL frequency to HSV appear to be the most important host factor influencing the frequency and severity of HSV reactivation in HIV+ patients. Studies to reconstitute such immunity, especially in people with acyclovir-resistant HSV, appear warranted.

An initial ATP-independent step in the unwinding of a herpes simplex virus type I origin of replication by a complex of the viral origin-binding protein and single-strand DNA-binding protein

Using a spectrophotometric assay that measures the hyperchromicity that accompanies the unwinding of a DNA duplex, we have identified an ATP-independent step in the unwinding of a herpes simplex virus type 1 (HSV-1) origin of replication, Oris, by a complex of the HSV-1 origin binding protein (UL9 protein) and the HSV-1 single-strand DNA binding protein (ICP8). The sequence unwound is the 18-bp A + T-rich segment that links the two high-affinity UL9 protein binding sites, boxes I and II of Oris. P1 nuclease sensitivity of Oris and single-strand DNA-dependent ATPase measurements of the UL9 protein indicate that, at 37°C, the A + T-rich segment is sufficiently single stranded to permit the binding of ICP8. Binding of the UL9 protein to boxes I and II then results in the formation of the UL9 protein–ICP8 complex, that can, in the absence of ATP, promote unwinding of the A + T-rich segment. On addition of ATP, the helicase activity of the UL9 protein–ICP8 complex can unwind boxes I and II, permitting access of the replication machinery to the Oris sequences.

Herpes simplex virus DNA packaging sequences adopt novel structures that are specifically recognized by a component of the cleavage and packaging machinery

The product of the herpes simplex virus type 1 UL28 gene is essential for cleavage of concatemeric viral DNA into genome-length units and packaging of this DNA into viral procapsids. To address the role of UL28 in this process, purified UL28 protein was assayed for the ability to recognize conserved herpesvirus DNA packaging sequences. We report that DNA fragments containing the pac1 DNA packaging motif can be induced by heat treatment to adopt novel DNA conformations that migrate faster than the corresponding duplex in nondenaturing gels. Surprisingly, these novel DNA structures are high-affinity substrates for UL28 protein binding, whereas double-stranded DNA of identical sequence composition is not recognized by UL28 protein. We demonstrate that only one strand of the pac1 motif is responsible for the formation of novel DNA structures that are bound tightly and specifically by UL28 protein. To determine the relevance of the observed UL28 protein–pac1 interaction to the cleavage and packaging process, we have analyzed the binding affinity of UL28 protein for pac1 mutants previously shown to be deficient in cleavage and packaging in vivo. Each of the pac1 mutants exhibited a decrease in DNA binding by UL28 protein that correlated directly with the reported reduction in cleavage and packaging efficiency, thereby supporting a role for the UL28 protein–pac1 interaction in vivo. These data therefore suggest that the formation of novel DNA structures by the pac1 motif confers added specificity on recognition of DNA packaging sequences by the UL28-encoded component of the herpesvirus cleavage and packaging machinery.

Oncolytic herpes simplex virus vector with enhanced MHC class I presentation and tumor cell killing

Oncolytic herpes simplex virus type 1 (HSV-1) vectors are promising therapeutic agents for cancer. Their efficacy depends on the extent of both intratumoral viral replication and induction of a host antitumor immune response. To enhance these properties while employing ample safeguards, two conditionally replicating HSV-1 vectors, termed G47Δ and R47Δ, have been constructed by deleting the α47 gene and the promoter region of US11 from γ34.5-deficient HSV-1 vectors, G207 and R3616, respectively. Because the α47 gene product is responsible for inhibiting the transporter associated with antigen presentation (TAP), its absence led to increased MHC class I expression in infected human cells. Moreover, some G47Δ-infected human melanoma cells exhibited enhanced stimulation of matched antitumor T cell activity. The deletion also places the late US11 gene under control of the immediate-early α47 promoter, which suppresses the reduced growth properties of γ34.5-deficient mutants. G47Δ and R47Δ showed enhanced viral growth in a variety of cell lines, leading to higher virus yields and enhanced cytopathic effect in tumor cells. G47Δ was significantly more efficacious in vivo than its parent G207 at inhibiting tumor growth in both immune-competent and immune-deficient animal models. Yet, when inoculated into the brains of HSV-1-sensitive A/J mice at 2 × 106 plaque forming units, G47Δ was as safe as G207. These results suggest that G47Δ may have enhanced antitumor activity in humans.

Immunization with DNA vaccines encoding glycoprotein D or glycoprotein B, alone or in combination, induces protective immunity in animal models of herpes simplex virus-2 disease.

DNA vaccines expressing herpes simplex virus type 2 (HSV-2) full-length glycoprotein D (gD), or a truncated form of HSV-2 glycoprotein B (gB) were evaluated for protective efficacy in two experimental models of HSV-2 infection. Intramuscular (i.m.) injection of mice showed that each construction induced neutralizing serum antibodies and protected the mice from lethal HSV-2 infection. Dose-titration studies showed that low doses (< or = 1 microgram) of either DNA construction induced protective immunity, and that a single immunization with the gD construction was effective. The two DNAs were then tested in a low-dosage combination in guinea pigs. Immune sera from DNA-injected animals had antibodies to both gD and gB, and virus neutralizing activity. When challenged by vaginal infection with HSV-2, the DNA-immunized animals were significantly protected from primary genital disease.

Herpes simplex virus vectors overexpressing the glucose transporter gene protect against seizure-induced neuron loss.

We have generated herpes simplex virus (HSV) vectors vIE1GT and v alpha 4GT bearing the GLUT-1 isoform of the rat brain glucose transporter (GT) under the control of the human cytomegalovirus ie1 and HSV alpha 4 promoters, respectively. We previously reported that such vectors enhance glucose uptake in hippocampal cultures and the hippocampus. In this study we demonstrate that such vectors can maintain neuronal metabolism and reduce the extent of neuron loss in cultures after a period of hypoglycemia. Microinfusion of GT vectors into the rat hippocampus also reduces kainic acid-induced seizure damage in the CA3 cell field. Furthermore, delivery of the vector even after onset of the seizure is protective, suggesting that HSV-mediated gene transfer for neuroprotection need not be carried out in anticipation of neurologic crises. Using the bicistronic vector v alpha 22 beta gal alpha 4GT, which coexpresses both GT and the Escherichia coli lacZ marker gene, we further demonstrate an inverse correlation between the extent of vector expression in the dentate and the amount of CA3 damage resulting from the simultaneous delivery of kainic acid.

Directed selection of recombinant human monoclonal antibodies to herpes simplex virus glycoproteins from phage display libraries.

Human monoclonal antibodies have considerable potential in the prophylaxis and treatment of viral disease. However, only a few such antibodies suitable for clinical use have been produced to date. We have previously shown that large panels of human recombinant monoclonal antibodies against a plethora of infectious agents, including herpes simplex virus types 1 and 2, can be established from phage display libraries. Here we demonstrate that facile cloning of recombinant Fab fragments against specific viral proteins in their native conformation can be accomplished by panning phage display libraries against viral glycoproteins "captured" from infected cell extracts by specific monoclonal antibodies immobilized on ELISA plates. We have tested this strategy by isolating six neutralizing recombinant antibodies specific for herpes simplex glycoprotein gD or gB, some of which are against conformationally sensitive epitopes. By using defined monoclonal antibodies for the antigen-capture step, this method can be used for the isolation of antibodies to specific regions and epitopes within the target viral protein. For instance, monoclonal antibodies to a nonneutralizing epitope can be used in the capture step to clone antibodies to neutralizing epitopes, or antibodies to a neutralizing epitope can be used to clone antibodies to a different neutralizing epitope. Furthermore, by using capturing antibodies to more immunodominant epitopes, one can direct the cloning to less immunogenic ones. This method should be of value in generating antibodies to be used both in the prophylaxis and treatment of viral infections and in the characterization of the mechanisms of antibody protective actions at the molecular level.

High prevalence of herpes simplex virus (HSV)- type 2 co-infection among HIV-positive women in Ukraine, but no increased HIV mother-to-child transmission risk.

BACKGROUND Over 3500 HIV-positive women give birth annually in Ukraine, a setting with high prevalence of sexually transmitted infections. Herpes simplex virus Type 2 (HSV-2) co-infection may increase HIV mother-to-child transmission (MTCT) risk. We explored factors associated with HSV-2 seropositivity among HIV-positive women in Ukraine, and its impact on HIV MTCT. METHODS Data on 1513 HIV-positive women enrolled in the Ukraine European Collaborative Study from 2007 to 2012 were analysed. Poisson and logistic regression models respectively were fit to investigate factors associated with HSV-2 seropositivity and HIV MTCT. RESULTS Median maternal age was 27 years (IQR 24-31), 53% (796/1513) had been diagnosed with HIV during their most recent pregnancy and 20% had a history of injecting drugs. Median antenatal CD4 count was 430 cells/mm(3) (IQR 290-580). Ninety-six percent had received antiretroviral therapy antenatally. HSV-2 seroprevalence was 68% (1026/1513). In adjusted analyses, factors associated with HSV-2 antibodies were history of pregnancy termination (APR 1.30 (95% CI 1.18-1.43) for ≥ 2 vs. 0), having an HIV-positive partner (APR 1.15 (95% CI 1.05-1.26) vs partner's HIV status unknown) and HCV seropositivity (APR 1.23 (95 % CI 1.13-1.35)). The overall HIV MTCT rate was 2.80% (95% CI 1.98-3.84); no increased HIV MTCT risk was detected among HSV-2 seropositive women after adjusting for known risk factors (AOR 1.43 (95% CI 0.54-3.77). CONCLUSION No increased risk of HIV MTCT was detected among the 68% of HIV-positive women with antibodies to HSV-2, in this population with an overall HIV MTCT rate of 2.8%. Markers of ongoing sexual risk among HIV-positive HSV-2 seronegative women indicate the importance of interventions to prevent primary HSV-2 infection during pregnancy in this high-risk group.

The production, harvest and adsorptive recovery of an infectious herpes simplex virus vaccine

At present there is not a reliable vaccine against herpes virus. Viral protein vaccines as yet have proved unsuccessful to meet the challenge of raising an appropriate immune response. Cantab Pharmaceuticals has produced a virus vaccine that can undergo one round of replication in the recipient in order to produce a more specific immune reaction. This virus is called Disabled Infectious Single Cycle Herpes Simplex Virus (DISC HSV) which has been derived by deleting the essential gH gene from a type 2 herpes virus. This vaccine has been proven to be effective in animal studies. Existing methods for the purification of viruses rely on laboratory techniques and for vaccine production would be on a far too small a scale. There is therefore a need for new virus purification methods to be developed in order to meet these large scale needs. An integrated process for the manufacture of a purified recombinant DISC HSV is described. The process involves culture of complementing Vero (CR2) cells, virus infection and manufacture, virus harvesting and subsequent downstream processing. The identification of suitable growth parameters for the complementing cell line and optimal limes for both infection and harvest are addressed. Various traditional harvest methods were investigated and found not to be suitable for a scaled up process. A method of harvesting, that exploits the elution of cell associated viruses by the competitive binding of exogenous heparin to virus envelope gC proteins, is described and is shown to yield significantly less contaminated process streams than sonication or osmotic approaches that involve cell rupture (with> 10-fold less complementing cell protein). High concentrations of salt (>0.8M NaCl) exhibit the same effect, although the high osmotic strength ruptures cells and increase the contamination of the process stream. This same heparin-gC protein affinity interaction is also shown to provide an efficient adsorptive purification procedure for herpes viruses which avoids the need to pre-treat the harvest material, apart from clarification, prior to chromatography. Subsequent column eluates provide product fractions with a 100-fold increase in virus titre and low levels of complementing cell protein and DNA (0.05 pg protein/pfu and 1.2 x 104 pg DNA/pfu respectively).