Michaela Nygårdas

Low copy number detection of herpes simplex virus type 1 mRNA and mouse Th1 type cytokine mRNAs by Light Cycler quantitative real-time PCR.

The real-time principle of reverse transcriptase polymerase chain reaction (RT-PCR) provides a quantitative and reproducible method to detect low copy number transcripts. The quantitative detection of cytokines from tissue samples is complicated by the low expression rates and the short half-lives of the cytokine proteins. The methods have been insensitive and labor-intensive. The LightCycler technique provides a 30-min PCR system with continuous fluorescent detection, analysis of the melting points of the products and user-friendly software for the analysis of the unknown samples. External copy number standards enable the measurement of amounts of the desired targets. We demonstrate the dynamic range of the RT-PCR system from a 100 to 10(7) mRNA copies of the mouse Th1 cytokines interleukin- (IL-) 12p35, 12p40 and IL-23p19 as well as gamma interferon (IFN-gamma) and the housekeeping gene beta-actin, with the usage of fluorescent hybridization probes. The cytokine quantitation was exemplified in murine nervous system samples. A viral transcript, mRNA of alpha trans-inducing factor (alphaTIF), or VP16 gene, of herpes simplex virus (HSV) type 1 was used to quantitate the viral replication in infected cells and in murine nervous system samples. For this viral transcript the linear dynamic range spanned from ten copies to one million copies (highest tested). For all tested cytokine transcripts, the detection level with the dsDNA binding dye SYBR Green I was one log lower than with the hybridizing fluorescent probes. The viral transcript was detected even with the SYBR Green I system at the level of ten copies. The specificity of the PCR was reached with the use of TaqStart antibody, by careful design of primers and probes, by melting temperature analysis and comparison with the gel electrophoresis and Southern blot analysis.

Spread and Replication of and Immune Response to γ134.5-Negative Herpes Simplex Virus Type 1 Vectors in BALB/c Mice

ABSTRACT We have previously shown that intracranial infection of herpes simplex virus type 1 (HSV-1) vector R8306 expressing interleukin-4 (IL-4) can abolish symptoms of experimental autoimmune encephalomyelitis, which is used as a model for human multiple sclerosis (Broberg et al., Gene Ther. 8:769-777, 2001). The aim of the current study was to search for means other than intracranial injection to deliver HSV-derived vectors to the central nervous system of mice. We also aimed to study the replication efficiency of these vectors in nervous system tissues and to elucidate the effects of the viruses on the immune response. We studied the spread and replication of the following viruses with deletions in neurovirulence gene γ134.5: R3616, R849 (lacZ transgene), R3659 (alpha-tk), R8306 (murine IL-4 transgene), and R8308 (murine IL-10 transgene). The samples were taken from trigeminal ganglia and brains of BALB/c mice after corneal, intralabial, and intranasal infection, and the viral load was examined by viral culture, HSV DNA PCR, and VP16 reverse transcription (RT)-PCR. The results show that (i) intranasal infection was the most efficient means of spread to the central nervous system (CNS) besides intracranial injection; (ii) the viruses did not grow in the culture from the brain samples, but the viral DNA persisted even until day 21 postinfection; (iii) viral replication, as observed by VP16 mRNA RT-PCR, occurred mainly on days 4 and 7 postinfection in trigeminal ganglia and to a low extent in brain; (iv) R3659, R8306, and R8308 showed reactivation from the trigeminal ganglia in explant cultures; (v) in the brain, the vectors spread to the midbrain more efficiently than to other brain areas; and (vi) the deletions in the R3659 genome significantly limited the ability of this virus to replicate in the nervous system. The immunological studies show that (i) the only recombinant to induce IL-4 mRNA expression in the brain was R8306, the gamma interferon response was very low in the brain for R3659 and R8306, and the IL-23p19 response to R8306 decreased by day 21 postinfection, unlike for the other viruses; (ii) Δγ134.5 HSV vectors modulated the subsets of the splenocytes differently depending on the transgene; (iii) R3659 infection of the nervous system induces expression and production of cytokines from the stimulated splenocytes; and (iv) HSV vectors expressing IL-4 or IL-10 induce expression and production of both of the Th2-type cytokines from splenocytes. We conclude that the intranasal route of infection is a possible means of delivery of Δγ134.5 HSV vectors to the CNS in addition to intracranial infection, although replication in the CNS remains minimal. The DNA of the HSV vectors is able to reside in the brain for at least 3 weeks. The features of the immune response to the vectors must be considered and may be exploited in gene therapy experiments with these vectors.

Treatment of experimental autoimmune encephalomyelitis in SJL/J mice with a replicative HSV-1 vector expressing interleukin-5.

Experimental autoimmune encephalomyelitis (EAE) is an autoimmune inflammation of the central nervous system and is used as the experimental model of multiple sclerosis (MS). The exact mechanism behind the disease is still unknown, but interleukin (IL)-17 expressing T cells are thought to mediate the disease. Toll-like receptors (TLRs) are known to have a role in the innate immune response against pathogens, and several TLRs have also a role in the disease course of EAE. Here, we show that treatment with a herpes simplex virus type 1 vector expressing the Th2 cytokine IL-5 ameliorates EAE and decreases the numbers of infiltrating lymphocytes in the brain. The effect involves downregulation of TLR 2, 3 and 9 mRNA expression and upregulation of type I interferons (IFNs) in brains during onset of disease. The elevated expression of type I IFNs was also observed during recovery.

A Herpes Simplex Virus-Derived Replicative Vector Expressing LIF Limits Experimental Demyelinating Disease and Modulates Autoimmunity

Herpes simplex virus type 1 (HSV-1) has properties that can be exploited for the development of gene therapy vectors. The neurotropism of HSV enables delivery of therapeutic genes to the nervous system. Using a bacterial artificial chromosome (BAC), we constructed an HSV-1(17+)-based replicative vector deleted of the neurovirulence gene γ134.5, and expressing leukemia inhibitory factor (LIF) as a transgene for treatment of experimental autoimmune encephalomyelitis (EAE). EAE is an inducible T-cell mediated autoimmune disease of the central nervous system (CNS) and is used as an animal model for multiple sclerosis. Demyelination and inflammation are hallmarks of both diseases. LIF is a cytokine that has the potential to limit demyelination and oligodendrocyte loss in CNS autoimmune diseases and to affect the T-cell mediated autoimmune response. In this study SJL/J mice, induced for EAE, were treated with a HSV-LIF vector intracranially and the subsequent changes in disease parameters and immune responses during the acute disease were investigated. Replicating HSV-LIF and its DNA were detected in the CNS during the acute infection, and the vector spread to the spinal cord but was non-virulent. The HSV-LIF significantly ameliorated the EAE and contributed to a higher number of oligodendrocytes in the brains when compared to untreated mice. The HSV-LIF therapy also induced favorable changes in the expression of immunoregulatory cytokines and T-cell population markers in the CNS during the acute disease. These data suggest that BAC-derived HSV vectors are suitable for gene therapy of CNS disease and can be used to test the therapeutic potential of immunomodulatory factors for treatment of EAE.

Enzymatically produced pools of canonical and Dicer-substrate siRNA molecules display comparable gene silencing and antiviral activities against herpes simplex virus.

RNA interference (RNAi)-based sequence-specific gene silencing is applied to identify gene function and also possesses great potential for inhibiting virus replication both in animals and plants. Small interfering RNA (siRNA) molecules are the inducers of gene silencing in the RNAi pathway but may also display immunostimulatory activities and promote apoptosis. Canonical siRNAs are 21 nucleotides (nt) in length and are loaded to the RNA Induced Silencing Complex when introduced into the cells, while longer siRNA molecules are first processed by endogenous Dicer and thus termed Dicer-substrate siRNA (DsiRNA). We have applied RNA polymerases from bacteriophages T7 and phi6 to make high-quality double-stranded RNA molecules that are specific for the UL29 gene of herpes simplex virus (HSV). The 653 nt long double-stranded RNA molecules were converted to siRNA and DsiRNA pools using Dicer enzymes originating from human or Giardia intestinalis, producing siRNAs of approximately 21 and 27 nt in length, respectively. Chemically synthesised 21 and 27 nt single-site siRNA targeting the UL29 were used as references. The impact of these siRNAs on cell viability, inflammatory responses, gene silencing, and anti-HSV activity were assayed in cells derived from human nervous system and skin. Both pools and the canonical single-site siRNAs displayed substantial antiviral activity resulting in four orders of magnitude reduction in virus titer. Notably, the pool of DsiRNAs caused lower immunostimulation than the pool of canonical siRNAs, whereas the immunostimulation effect was in relation to the length with the single-site siRNAs. Our results also propose differences in the processivity of the two Dicers.

Inhibition of coxsackievirus B3 and related enteroviruses by antiviral short interfering RNA pools produced using φ6 RNA-dependent RNA polymerase

Coxsackievirus B3 (CBV3) is a member of the human enterovirus B species and a common human pathogen. Even though much is known about the enteroviral life cycle, no specific drugs are available to treat enterovirus infections. RNA interference (RNAi) has evolved to be an important tool for antiviral experimental therapies and gene function studies. We describe here a novel approach for RNAi against CBVs by using a short interfering (siRNA) pool covering 3.5 kb of CBV3 genomic sequence. The RNA-dependent RNA polymerase (RdRP) of bacteriophage phi6 was used to synthesize long double-stranded RNA (dsRNA) from a cloned region (nt 3837-7399) of the CBV3 genome. The dsRNA was cleaved using Dicer, purified and introduced to cells by transfection. The siRNA pool synthesized using the phi6 RdRP (phi6-siRNAs) was considerably more effective than single-site siRNAs. The phi6-siRNA pool also inhibited replication of other enterovirus B species, such as coxsackievirus B4 and coxsackievirus A9.

Inhibition of clinical pathogenic herpes simplex virus 1 strains with enzymatically created siRNA pools

Herpes simplex virus (HSV) is a common human pathogen causing severe diseases such as encephalitis, keratitis, and neonatal herpes. There is no vaccine against HSV and the current antiviral chemotherapy fails to treat certain forms of the disease. Here, we evaluated the antiviral activity of enzymatically created small interfering (si)RNA pools against various pathogenic HSV strains as potential candidates for antiviral therapies. Pools of siRNA targeting 0.5–0.8 kbp of essential HSV genes UL54, UL29, or UL27 were enzymatically synthesized. Efficacy of inhibition of each siRNA pool was evaluated against multiple clinical isolates and laboratory wild type HSV‐1 strains using three cell lines representing host tissues that support HSV‐1 replication: epithelial, ocular, and cells that originated from the nervous system. The siRNA pools targeting UL54, UL29, and UL27, as well as their equimolar mixture, inhibited HSV replication, with the pool targeting UL29 having the most prominent antiviral effect. In contrast, the non‐specific control siRNA pool did not have such an effect. Moreover, the UL29 pool elicited only a minimal innate immune response in the HSV‐infected cells, thus evidencing the safety of its potential clinical use. These results are promising for the development of a topical RNA interference approach for clinical treatment of HSV infection. J. Med. Virol. 88:2196–2205, 2016.

Innate responses to small interfering RNA pools inhibiting herpes simplex virus infection in astrocytoid and epithelial cells

Herpes simplex virus (HSV) is a human pathogen that can cause severe diseases such as encephalitis, keratitis and neonatal herpes. Control of HSV infection may be achieved by using small interfering (si)RNAs. We have designed and enzymatically produced pools of siRNAs targeting HSV. In addition to the target-specific effects, such siRNAs may induce innate immunity responses that may contribute to antiviral effects. HSV has versatile ways of modulating innate immunity, and it remains unclear whether HSV-specific antiviral treatment would benefit from the potential immunostimulatory effects of siRNAs. To address this, cell lines derived from epithelium and nervous system were studied for innate immunity reactions to HSV infection, to siRNA treatment, and to a combination of treatment and infection. In addition, the outcome of HSV infection was quantitated. We show that innate immunity reactions vary drastically between the cell lines. Moreover, our findings indicate only a minimal relation between the antiviral effect and the treatment-induced innate immunity responses. Thus, the antiviral effect is mainly sequence specific and the inhibition of HSV infection is not ascribed to the slight innate immunity induction.

The ERK-1 function is required for HSV-1-mediated G1/S progression in HEP-2 cells and contributes to virus growth

The herpes simplex virus 1 is able to readdress different cellular pathways including cell cycle to facilitate its replication and spread. During infection, the progression of the cell cycle from G1 to S phase makes the cellular replication machinery accessible to viral DNA replication. In this work we established that HSV-1, in asynchronized HEp-2 cells, strictly controls cell cycle progression increasing S-phase population from 9 hours post infection until the end of HSV-1 replication. The G1/S phases progression depends on two important proteins, cyclin E and CDK2. We demonstrate that their phosphorylated status and then their activity during the infection is strongly correlated to viral replication events. In addition, HSV-1 is able to recruit and distribute ERK1/2 proteins in a spatio-temporal fashion, highlighting its downstream regulatory effects on cellular processes. According with this data, using chemical inhibitor U0126 and ERK dominant negative cells we found that the lack of ERK1 activity affects cyclin E protein accumulation, viral gene transcription and percentage of the cells in S phase, during the viral replication. These data suggested a complex interaction between ERK, cell cycle progression and HSV-1 replication.

Topical treatment of herpes simplex virus infection with enzymatically created siRNA swarm

BACKGROUND Herpes simplex virus (HSV) is a common human pathogen. Despite current antivirals, it causes a significant medical burden. Drug resistant strains exist and they are especially prevalent in immunocompromised patients and in HSV eye infections. New treatment modalities are needed. METHODS BALB/c mice were corneally infected with HSV and subsequently treated with a swarm of enzymatically created, Dicer-substrate small interfering RNA (siRNA) molecules that targeted the HSV gene UL29. Two infection models were used, one in which the infection was predominantly peripheral and another in which it spread to the central nervous system. Mouse survival, as well as viral spread, load, latency and peripheral shedding, was studied. RESULTS The anti-HSV-UL29 siRNA swarm alleviated HSV infection symptoms, inhibited viral shedding and replication and had a favourable effect on mouse survival. CONCLUSIONS Treatment with anti-HSV-UL29 siRNA swarm reduced symptoms and viral spread in HSV infection of mice and also inhibited local viral replication in mouse corneas.