Herpes simplex virus type 1 (HSV-1) pathogenesis and HSV gene therapy of experimental autoimmune encephalomyelitis

The aim of this thesis was to develop new herpes simplex virus (HSV) vectors for gene therapy of experimental autoimmune encephalomyelitis (EAE), the principal model of multiple sclerosis (MS), and to study the pathogenesis of wild-type HSV-1 and HSV-1 vectors in vivo. By introducing potential immunomodulatory factors into mice with EAE we strived to develop therapies and possibly find molecules improving recovery from EAE. We aimed at altering the immune response by inducing favorable Th2-type cytokines, thus shifting the immune response from a Th1- or a Th17-response. Our HSV vector expressing interleukin (IL)-5 modulated the cytokine responses, decreased inflammation and alleviated EAE. The use of a novel method, bacterial artificial chromosome (BAC), for engineering recombinant HSV facilitated the construction of a new vector expressing leukemia inhibitory factor (LIF). LIF is a neurotropic cytokine with broad functions in the central nervous system (CNS). LIF promotes oligodendrocyte maturation and decreases demyelination and oligodendrocyte loss. The BAC-derived HSV-LIF vector alleviated the clinical symptoms, induced a higher number of oligodendrocytes and modulated T cell responses. By administering HSV via different infection routes, e.g. peripherally via the nose or eye, or intracranially to the brain, the effect of the immune response on HSV spread at different points of the natural infection route was studied. The intranasal infection was an effective delivery route of HSV to the trigeminal ganglion and CNS, whereas corneal infection displayed limited spread. The corneal and intranasal infections induced different peripheral immune responses, which might explain the observed differences in viral spread.Herpes simplex virus tyyppi 1 (HSV-1) infektion patogeneesi ja HSV-välitteinen geeniterapia kokeellisessa autoimmuuni-enkefalomyeliitissä. Väitöskirjatutkimuksen tarkoituksena oli kehittää uusia herpes simplex virus (HSV)-pohjaisia geenikuljettimia (vektoreita) kokeellisen autoimmuuni-enkefalomyeliitin (EAE) hoitoon, ja tutkia HSV-1 luonnonkannan ja vektoreiden aiheuttamia immuunivasteita in vivo. EAE toimii multippeliskleroosin (MS-taudin) ensisijaisena koemallina. Tavoitteemme oli kehittää uusia hoitomuotoja, jotka perustuivat virusvälitteiseen geeninsiirtoon. Pyrimme muokkaamaan aivoja kohtaan suuntautuneita immuunivasteita tuomalla hermostoon viruksen avulla suotuisia Th2-tyypin sytokiineja, ja siten muuttamaan immuunivasteen tasapainoa pois Th17-tyypin vasteesta. Interleukiinia (IL)-5 tuottava HSV-vektori lievensi EAE:ta, kykeni vähentämään tulehdusta ja muokkaamaan taudille tyypillistä immunologista tilaa. Kehitin bacterial artificial chromosome (BAC)-teknologialla uuden HSV-vektorin, joka ilmentää LIF-sytokiinia (leukemia inhibitory factor). LIF on neurotrooppinen sytokiini, jolla on useita vaikutusmekanismeja. LIF edistää oligodendrosyyttien kypsymistä ja estää demyelinaatiota ja oligodendrosyyttien tuhoa. HSV-LIF-vektorilla pystyimme lieventämään EAE-oireita ja lisäämään oligodendrosyyttien määrää aivoissa. Myös tautiin liittyvissä T-soluvasteissa tapahtui myönteisiä muutoksia. Havaitsin työssäni, että autoimmuunitaudin hoitotutkimuksissa yleisesti käytetyllä SJL/J-hiirikannalla saavutettiin HSV:n tehokas kulkeutuminen aivoihin annostelemalla virus nenän kautta, mikä edesauttaa geenihoidon toteutusmahdollisuuksia. Viruksen annostelu muita reittejä ei ollut yhtä tehokasta tällä hiirikannalla. Eroavaisuudet immuunivasteessa voivat selittää vaihtelun viruksen leviämisessä.Siirretty Doriast

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

Peer reviewe

Spread and Replication of and Immune Response to γ(1)34.5-Negative Herpes Simplex Virus Type 1 Vectors in BALB/c Mice

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 γ(1)34.5: 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) Δγ(1)34.5 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 Δγ(1)34.5 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

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

Innate immunity responses to siRNA pools and single-site siRNA molecules.

<p>HaCaT and U373MG cells were transfected with either 1 (light grey) or 10 (dark grey) pmol of the indicated siRNA molecules; 1 pmol of 88-bp dsRNA (black bar) or water (control, grey bar); or left untreated (control, white bar). The expression levels of <i>IFN-β</i> (<i>A</i>), <i>IFN-λ1</i> (<i>B</i>) or <i>ISG54</i> (<i>C</i>) were assessed by qRT-PCR 8 h, 24 h or 48 h post transfection. Values were normalized to the <i>GAPDH</i> housekeeping gene and shown on a logarithmic scale. The mean values+S.D. of at least two independent experiments, each with a minimum of three biological replicates, are shown. Data were compared by Mann-Whitney U-test. The statistical significance is indicated as (×) p < 0.01 or (¤) p < 0.05 against the controls; (**) p < 0.01 or (*) p < 0.05 against a group of comparison; (#) p < 0.01 against all other transfections. UL29GD, GD-digested anti-<i>UL29</i> siRNA pool; UL29HD, HD-digested anti-<i>UL29</i> siRNA pool; UL29L, 27-nt chemically synthesized anti-<i>UL29</i> siRNA; UL29S, 21-nt chemically synthesized single anti-<i>UL29</i> siRNA. No significant signal was detected for IFN-λ1 mRNA in U373MG cell culture (B, right panel) if no dsRNA was applied (non- and mock-transfected cells).</p

Inhibition of HSV-1 replication by <i>UL29</i>-specific siRNA molecules.

<p>HaCaT and U373MG cells on 96-well plates were transfected with 10 pmol of indicated siRNA molecules or water. After 4 h the cells were infected with 1000 pfu of HSV-1 and incubated for 44 h. (<i>A</i>) Dilutions of the HaCaT and U373MG supernatant, collected 48 h after transfection (44 h post infection) were assayed for released virus by plaque formation on Vero cell culture. (<i>B</i>) The relative expression of the target HSV-1 gene <i>UL29</i> was measured by qRT-PCR from samples of the infected cultures. Values were normalized to the <i>GAPDH</i> housekeeping gene and shown on a logarithmic scale. The mean values+S.D. are shown for six replicates. Data were compared by Mann-Whitney U-test. The statistical significance is indicated as (×) p < 0.01 and (¤) p<0.05 against the controls; (*) p < 0.05 against a group of comparison; (#) p < 0.01 against all other siRNA transfections. NT, no transfection; mock, transfection with water; eGFPGD, anti-eGFP siRNA pool; eGFPS, single-site anti-eGFP siRNA; UL29GD, GD-digested anti-UL29 siRNA pool; UL29HD, HD-digested anti-UL29 siRNA pool; UL29L, 27-nt single-site anti-UL29 siRNA; UL29S, 21-nt single-site anti-UL29 siRNA.</p