Henrik Paavilainen

Obatoclax, saliphenylhalamide and gemcitabine inhibit influenza A virus infection

Background: Novel options should be developed for treatment of IAV infections. Results: Obatoclax, saliphenylhalamide, and gemcitabine target host factors and inhibit IAV and several other viruses infections. Conclusion: These compounds represent potent antiviral agents. Significance: These compounds could be exploited in treatment of severe viral infections. Influenza A viruses (IAVs) infect humans and cause significant morbidity and mortality. Different treatment options have been developed; however, these were insufficient during recent IAV outbreaks. Here, we conducted a targeted chemical screen in human nonmalignant cells to validate known and search for novel host-directed antivirals. The screen validated saliphenylhalamide (SaliPhe) and identified two novel anti-IAV agents, obatoclax and gemcitabine. Further experiments demonstrated that Mcl-1 (target of obatoclax) provides a novel host target for IAV treatment. Moreover, we showed that obatoclax and SaliPhe inhibited IAV uptake and gemcitabine suppressed viral RNA transcription and replication. These compounds possess broad spectrum antiviral activity, although their antiviral efficacies were virus-, cell type-, and species-specific. Altogether, our results suggest that phase II obatoclax, investigational SaliPhe, and FDA/EMEA-approved gemcitabine represent potent antiviral agents.

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.

Anticancer compound ABT-263 accelerates apoptosis in virus-infected cells and imbalances cytokine production and lowers survival rates of infected mice

ABT-263 and its structural analogues ABT-199 and ABT-737 inhibit B-cell lymphoma 2 (Bcl-2), BCL2L1 long isoform (Bcl-xL) and BCL2L2 (Bcl-w) proteins and promote cancer cell death. Here, we show that at non-cytotoxic concentrations, these small molecules accelerate the deaths of non-cancerous cells infected with influenza A virus (IAV) or other viruses. In particular, we demonstrate that ABT-263 altered Bcl-xL interactions with Bcl-2 antagonist of cell death (Bad), Bcl-2-associated X protein (Bax), uveal autoantigen with coiled-coil domains and ankyrin repeats protein (UACA). ABT-263 thereby activated the caspase-9-mediated mitochondria-initiated apoptosis pathway, which, together with the IAV-initiated caspase-8-mediated apoptosis pathway, triggered the deaths of IAV-infected cells. Our results also indicate that Bcl-xL, Bcl-2 and Bcl-w interact with pattern recognition receptors (PRRs) that sense virus constituents to regulate cellular apoptosis. Importantly, premature killing of IAV-infected cells by ABT-263 attenuated the production of key pro-inflammatory and antiviral cytokines. The imbalance in cytokine production was also observed in ABT-263-treated IAV-infected mice, which resulted in an inability of the immune system to clear the virus and eventually lowered the survival rates of infected animals. Thus, the results suggest that the chemical inhibition of Bcl-xL, Bcl-2 and Bcl-w could potentially be hazardous for cancer patients with viral infections.

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.

Regulation of kynurenine biosynthesis during influenza virus infection

Influenza A viruses (IAVs) remain serious threats to public health because of the shortage of effective means of control. Developing more effective virus control modalities requires better understanding of virus–host interactions. It has previously been shown that IAV induces the production of kynurenine, which suppresses T‐cell responses, enhances pain hypersensitivity and disturbs behaviour in infected animals. However, the regulation of kynurenine biosynthesis during IAV infection remains elusive. Here we showed that IAV infection induced expression of interferons (IFNs), which upregulated production of indoleamine‐2,3‐dioxygenase (IDO1), which catalysed the kynurenine biosynthesis. Furthermore, IAV attenuated the IDO1 expression and the production of kynurenine through its NS1 protein. Interestingly, inhibition of viral replication prior to IFN induction limited IDO1 expression, while inhibition after did not. Finally, we showed that kynurenine biosynthesis was activated in macrophages in response to other stimuli, such as influenza B virus, herpes simplex virus 1 and 2 as well as bacterial lipopolysaccharides. Thus, the tight regulation of the kynurenine biosynthesis by host cell and, perhaps, pathogen might be a basic signature of a wide range of host–pathogen interactions, which should be taken into account during development of novel antiviral and antibacterial drugs.

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.

An investigation of herpes simplex virus type 1 latency in a novel mouse dorsal root ganglion model suggests a role for ICP34.5 in reactivation.

After a primary lytic infection at the epithelia, herpes simplex virus type 1 (HSV-1) enters the innervating sensory neurons and translocates to the nucleus, where it establishes a quiescent latent infection. Periodically, the virus can reactivate and the progeny viruses spread back to the epithelium. Here, we introduce an embryonic mouse dorsal root ganglion (DRG) culture system, which can be used to study the mechanisms that control the establishment, maintenance and reactivation from latency. Use of acyclovir is not necessary in our model. We examined different phases of the HSV-1 life cycle in DRG neurons, and showed that WT HSV-1 could establish both lytic and latent form of infection in the cells. After reactivating stimulus, the WT viruses showed all markers of true reactivation. In addition, we showed that deletion of the γ(1)34.5 gene rendered the virus incapable of reactivation, even though the virus was clearly able to replicate and persist in a quiescent form in the DRG neurons.

Antiviral Properties of Chemical Inhibitors of Cellular Anti-Apoptotic Bcl-2 Proteins

Viral diseases remain serious threats to public health because of the shortage of effective means of control. To combat the surge of viral diseases, new treatments are urgently needed. Here we show that small-molecules, which inhibit cellular anti-apoptotic Bcl-2 proteins (Bcl-2i), induced the premature death of cells infected with different RNA or DNA viruses, whereas, at the same concentrations, no toxicity was observed in mock-infected cells. Moreover, these compounds limited viral replication and spread. Surprisingly, Bcl-2i also induced the premature apoptosis of cells transfected with viral RNA or plasmid DNA but not of mock-transfected cells. These results suggest that Bcl-2i sensitizes cells containing foreign RNA or DNA to apoptosis. A comparison of the toxicity, antiviral activity, and side effects of six Bcl-2i allowed us to select A-1155463 as an antiviral lead candidate. Thus, our results pave the way for the further development of Bcl-2i for the prevention and treatment of viral diseases.

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.