Gene Therapy (2011) 18, 646–655 & 2011 Macmillan Publishers Limited All rights reserved 0969-7128/11 www.nature.com/gt

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

M Nyga˚rdas1, C Aspelin1, H Paavilainen1,MRo¨ytta¨2,MWaris1 and V Hukkanen1,3

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. Gene Therapy (2011) 18, 646–655; doi:10.1038/gt.2011.4; published online 17 February 2011

Keywords: experimental autoimmune encephalomyelitis (EAE); Herpes simplex virus (HSV); interleukin; interferon; Toll-like receptor

INTRODUCTION but helpful in understanding the role of the different cytokines. Multiple sclerosis (MS) is a demyelinating autoimmune disease of the Th2 cytokines have, for example, been used for therapy of EAE, either central nervous system (CNS), characterized by infiltration of inflam- directly administered or expressed from viral vectors. Administration matory cells into the CNS. Experimental autoimmune encephalo- of IL-4 by viral vectors has shown beneficial effects in EAE myelitis (EAE), a common animal model for MS, can be induced recovery.25–28 Along with others, we have previously used intrathecally by immunization with myelin-derived antigens in adjuvants1–5 or by or intracranially delivered Th2 cytokine-expressing HSV vectors for adoptive transfer of myelin-activated T cells.6 Cytokines are an treatment of EAE.26–30 Still, mice lacking the IL-4 gene have shown important part of the host’s immune response against pathogens variable susceptibility to EAE, depending on mouse strain.31,32 Studies and an imbalance in the cytokine network has a role in the initiation with IL-10-deficient mice have, on the other hand, shown that IL-10 is of autoimmune diseases.7,8 CD4+ T helper 1 (Th1) cells, characterized critical in the regulation of EAE.33 IL-5 is also a Th2 cytokine and by secretion of interferon (IFN)-g, tumor necrosis factor and inter- induces maturation and differentiation of eosinophils.34,35 Treatments leukin- (IL-) 2, were until some years ago believed to be the essential in use for MS, such as glatiramer acetate, seem to involve the upregu- T-cell population involved in the pathology and onset of the disease. lation of IL-5 together with IL-13.36–38 Still, certain IL-5-deficient Treatment with antibodies against IL-12, a cytokine that induces Th1 mice do develop EAE, which indicates that the role of IL-5 in the responses, was shown to ameliorate EAE.9,10 Later it was shown that development of EAE is still controversial.39 IL-23 and not IL-12 is the crucial cytokine in CNS inflammation.11–13 Important factors in innate immune response are the pathogen- IL-23 also drives the expression of IL-17,14 a relatively recently identified associated pattern recognition molecules, such as toll-like receptors cytokine with a role in inflammation and autoimmune disease.14–17 The (TLRs). TLRs are important molecules also in the initiation of the T cells producing IL-17 make a separate Th17 T-cell population, distinct adaptive immune responses. In all, 11 human TLRs and 13 mouse 15,18 + from Th1 and Th2. Both the CD4 Th1 cells and Th17 T cells are TLRs have been identified so far. Typical for all TLRs are that they are probably important during disease onset. Regulatory CD4+CD25+ ligand-specific40 and that they activate intracellular signaling pathways T cells (T-regs) are important in maintaining the balance between that lead to the induction of inflammatory cytokines, such as IFN-a, tolerance and immunity,19 suppressing pathogenic autoreactive IL-6 and IL-12 (Th1 responses). Several groups have studied the role Tcells.20,21 CD8+ T cells may also have a regulatory role in EAE.22 of TLRs in autoimmune disease. TLR agonists have been shown Evidence suggests that Th2 type cytokines, such as IL-4 and IL-10 to induce autoimmune diseases, for example, EAE and diabetes.41,42 are associated with the recovery phase of EAE and also of MS,23 On the other hand, TLR stimulation can also suppress autoimmune whereas certain cytokines such as IFN-g, tumor necrosis factor-a pathogenesis, showing a more complex role for TLR in autoimmune and tumor necrosis factor-b have a disease-promoting role.24 The diseases.43,44 Th1/Th2 paradigm in autoimmune disease has been targeted for Vectors based on herpes simplex virus type 1 (HSV-1) have several potential treatments of EAE. Results have been contradicting features making them a good candidate for gene therapy of CNS

1Department of Virology, University of Turku, Turku, Finland; 2Department of Pathology, University of Turku, Turku, Finland and 3Department of Microbiology, University of Oulu, Oulu, Finland Correspondence: Dr M Nyga˚rdas, Department of Virology, University of Turku, Kiinamyllynkatu 13, 20520 Turku, Finland. E-mail: michaela.nygardas@utu.fi Received 31 January 2010; revised 10 January 2011; accepted 10 January 2011; published online 17 February 2011 HSV-1 expressing IL-5 ameliorates EAE MNyga˚rdas et al 647 disease. HSV-1 is naturally neurotropic, thus after primary infection of the mice infected with R8316 (IL-5) after day 15 post induction epithelial cells, the virus is transported along nerves to the sensory (Figure 1). There was no EAE mortality, either, in the R8316 (IL-5) ganglia, where it can establish a latent infection.45,46 In addition, the treatment group (Table 1). The EAE cumulative index was lowest in large DNA genome contains several non-essential genes, which can be the HSV-1–IL-5-treated group (Table 1). Since IL-10 has a role during removed or replaced by transgenes. The neurovirulence gene g134.5 the recovery phase of EAE, we also studied the effect of an HSV-1 can also be removed from the vectors.47,48 HSV vectors can be expressing IL-10. Treatment with R8308 (IL-10) caused no significant administered into CNS by intracranial injection, intrathecally or alleviation of EAE in comparison with untreated mice. The control through peripheral organs, such as the eye and nose.49 Intranasal virus R3659 also alleviated the EAE scores during the recovery phase administration of wild-type HSV-1 yields an effective means of starting from day 18 (Figure 1), indicating a beneficial effect of the delivery in SJL/J mice.50 We have previously studied the spread and backbone HSV vector during the late phase of the EAE. replication of wild-type HSV-1 and vectors deleted of g134.5 after corneal, intralabial and intranasal infection. We also found that the Microscopical analysis of brain samples intranasal route was effective for delivery of HSV-1 and g134.5- Hematoxylin–eosin-stained brain paraffin sections were analyzed for deletion viruses into the CNS.51 amount of lymphocyte infiltrates and pathological changes typical for As a beneficial role of both IL-5 and IL-10 has been indicated in EAE (Figures 2 and 3). At day 14 post induction, a strong inflamma- EAE and MS, we wanted to investigate their therapeutic effect in SJL/J tion could be seen in the brain of untreated EAE mice, with numerous mice when expressed from HSV vectors. We have here studied the infiltrating lymphocytes. Also in the HSV-treated groups inflamma- treatment with replicative HSV-1 vectors expressing IL-5 or IL-10 on tion could be seen during the acute disease (Figure 2). By day 21, the EAE after peripheral administration and also studied the cytokine and inflammation had decreased in the brains and there was a significant TLR profiles during the treatments. We saw a significant decrease in disease score with the HSV-1 expressing IL-5, but not with IL-10-HSV. The IFN-b mRNA expression was upregulated in the CNS by the Th2 5.0 cytokine-expressing HSV vectors. We also observed a trend of decreas- ing IL-17 mRNA expression during the onset of disease after treatment 4.0 with the cytokine-expressing HSV-1 vectors. TLR2, TLR3 and TLR9 expression were also modulated after administration of the replicative 3.0 IL-5–HSV and they may be involved in the recovery of the animals. To our knowledge, this is the first study where IL-5 expressed from a 2.0

viral vector has been used in gene therapy of EAE. Disease grade 1.0 RESULTS Replicative HSV-1 vector expressing IL-5 precludes 0.0 EAE in SJL/J mice 0 5 10 15 20 25 ***** * We studied the effect of intranasal administration of recombinant ## # HSV-based vectors, expressing Th2 type cytokines, to the disease Days post induction course of EAE in SJL/J mice. The experiment included four groups EAE, no virus EAE, IL-10 virus (R8308) of mice, all induced for EAE: (1) no treatment, (2) infection with an EAE, IL-5 virus (R8316) EAE, R3659 HSV-1 deleted of the g 34.5 genes and expressing IL-10 (R8308), 1 Figure 1 Clinical scores of EAE with or without treatment of HSV vectors. (3) infection with an HSV-1 deleted of the g134.5 genes and expressing The blue diamonds indicate EAE without virus treatment, the green squares IL-5 (R8316) and (4) infection with an HSV-1 deleted of the g134.5 indicate EAE treated with the R3659 virus, the purple dots indicate genes but not expressing any cytokine (R3659). The mice were treatment with IL-10 virus and the red triangles indicate treatment with the observed daily for disease grade. All untreated mice developed IL-5 virus. The IL-5-expressing HSV R8316 ameliorates the EAE disease symptoms, showing that the induction of EAE was successful. The course, with significantly lower disease scores from day 15 on (*Po0.05, time of disease onset did not differ significantly between the groups. indicates statistically significant difference when compared with uninfected mice with EAE). The virus R3659 reduced EAE scores from day 18 on Most mice developed symptoms starting on days 11–12 post induc- (#Po0.05, indicates statistically significant difference when compared with tion (Table 1). The EAE disease peak also occurred approximately uninfected mice with EAE). The IL-10-expressing HSV R8308 did not have at the same time for all groups, but disease scores were highest in the a significant therapeutic effect. The color reproduction of this figure is untreated group and a significant decrease (Po0.05) could be seen in available on the html full text version of the manuscript.

Table 1 EAE parameters in the different treatment groups

Group No. of mice Mean max EAE Mean day of onset Incidence Cumulative Mortality (day 10) score (s.d.) (mean grade 41) (%) index d19 (s.d.) (EAE %)

EAE, no virus 15 2.23 (1.39) 11 12/15 (80) 21.7 (7.8) 20% EAE, backbone (R3659) 13 1.77 (1.36) 11 9/13 (69) 14.19 (9.6)* 0% EAE, IL-5 virus (R8316) 14 1.57 (1.4) 11 10/15 (67) 10.75 (7.0)** 0% EAE, IL-10 virus (R8308) 14 2.05 (1.63) 12 10/15 (67) 14.3 (4.9)* 14.3%

Abbreviations: EAE, experimental autoimmune encephalomyelitis; IL, interleukin. *P¼0.04, **P¼0.01.

Gene Therapy HSV-1 expressing IL-5 ameliorates EAE MNyga˚rdas et al 648

3.5 upregulated all studied TLRs throughout the disease. Significant 3 differences were found between the Th2 cytokine-expressing vectors 2.5 and R3659. R8316 (IL-5) slightly upregulated the mRNA expression of TLR2, TLR9 and MyD88 at day 14 post induction in comparison with 2 untreated EAE, but not to the extent that R3659 did. Treatment 1.5 with R8308 (IL-10) showed a similar tendency, but the upregulation * * 1 was not significant. 0.5 The type 1 IFN mRNA expression followed a common pattern 0 (Figure 5). R8316 (IL-5) infection significantly (Po0.05) upregulated Inflammation (arbitrary units) day 10 day 14 day 21 the IFN-a4, IFN-a6andIFN-b mRNA expression at day 10 and Days post EAE induction day 21 post EAE induction in brains. R8308 (IL-10) infection EAE, no virus EAE, IL-10 virus (R8308) downregulated the type 1 IFN mRNA expression during day 10, but EAE, R3659 EAE, IL-5 virus (R8316) thereafter significantly upregulated the mRNA expression on both day 14 and day 21. Also the R3659 virus upregulated the IFN responses at Figure 2 Inflammation in brain sections based on hematoxylin–eosin day 14. Still, both R8308 (IL-10) and R8316 (IL-5) showed higher type staining. The scoring was: 0, no infiltration; 1, perivascular infiltration; I IFN mRNA expression than R3659 during recovery. 2, perivascular inflammatory cuffs; and 3, inflammation of the brain substance. Each group at each time point consisted of 4–5 mice. *Po0.05, indicates statistically significant difference when compared with uninfected HSV expressing IL-5 or IL-10 modulates the cytokine response in mice with EAE. the nervous system Expression of several cytokines was studied from brain and TG decrease (Po0.05) in immunological cell infiltrates in brains of mice samples, including IL-4, IL-5, IL-10, IFN-g, IL-17, IL-23, IL-12p35 infected with IL-5–HSV and the control virus R3659 (Figure 2). and IL-12p40 mRNA. Infiltrates were composed of leukocytes (CD45), including CD4+ In brains of SJL/J mice induced for EAE and treated with R8316 and CD8+ T-lymphocytes (Figure 3). There was a trend of lower (IL-5) or with R8308 (IL-10), we could see a trend of a down- CD4+ to CD8+ ratio in HSV-treated mice. Antigen-presenting cells regulation of the pro-inflammatory cytokine IL-17 during onset of (F4/80) were also found in the infiltrates. There was, as well, a trend disease (day 10) (Figure 6). A significant reduction was seen in IL-23 toward fewer F4/80-positive cells in HSV-treated mice than in mRNA expression after R3659 infection on day 10 and 21 in untreated EAE (Figure 3). The IL-5–HSV treatment was not associated comparison with both untreated EAE and treatment with the Th2 with significant increase in eosinophilic leukocytes in the CNS cytokine-expressing vectors. R8308 (IL-10) downregulated IL-12p40 (Supplementary Figure 1). during the acute EAE phase (day 14). Both R8308 and R8316 showed In spinal cords of mice recovered from severe disease (grade 3), lower IL-12p40 mRNA expression than R3659, which upregulated inflammatory infiltrates were still found at day 21 post induction IL-12p40 throughout the disease. R8316 downregulated IL-12p35 in both untreated and treated EAE mice. However, less infiltrates during recovery (day 21). No significant changes were seen in the were seen in the IL-5–HSV treatment group (Figure 4). There were no IFN-g mRNA expression in comparison with untreated mice, significant differences between the treatment and no treatment groups although the Th2 cytokine-expressing HSV vectors had lower IFN-g in infiltrate composition, although there was a trend toward higher levels than R3659. The HSV-1s expressing IL-5 or IL-10 did not proportion of CD8+ cells in HSV-treated groups. increase the IL-5 expression in the brains at day 4 post infection, but later during the treatment IL-5 was observed (Figure 6). The virus Viral transcripts in the nervous system R3659 upregulated IL-5 on day 14 and day 21. The IL-10 mRNA As the viruses were administered intranasally, we studied the tri- expression was upregulated after R8316 (IL-5) at day 14 and day 21 geminal ganglion (TG) samples for the viral DNA (PCR for the HSV-1 and at day 21 after R8308 (IL-10)-treatment. Both Th2 cytokine- gD target). The majority of all TGs from infected animals were positive expressing vectors induced higher IL-10 mRNA expression than R3659 for HSV-1 DNA (Supplementary Table 2A). In addition, we studied the at the recovery phase. TG with a transgene-specific PCR. IL-5 transgene copies could be found The phenomena in the TG may affect the outcome of EAE, in a few TG samples from R8316-infected mice at day 10 post induction providing a contact of the immune system with HSV vectors expres- (day 4 post infection) and in almost all TGs later in the disease sing cytokines. In the TG, we found a significant upregulation of the (Supplementary Table 2B). TG samples from R8308 (IL-10)-infected IL-5 expression in SJL/EAE mice infected with R8316 (IL-5) at day 14 mice were positive for the IL-10 transgene. As expected, TGs of mice and day 21 post induction (day 8 and 15 post infection), compared infected with R3659 were negative for the tested transgenes. with both untreated EAE mice and mice infected with R3659 (Figure 7). Mice infected with the IL-5 virus also showed a significant TLR and type I IFN expression in nervous system of SJL/J mice upregulation of the IL-10 mRNA expression in the TG at day 14, but a with EAE downregulation during the recovery phase (Figure 7). This indicates We studied the effect on the TLR expression in the CNS of the that the R8316 (IL-5) induced a Th2-type response in the TG. replicative HSV-1 vectors expressing IL-5 or IL-10 and the R3659. The A temporary upregulation of IL-17 was seen in TG, but not in mRNA expression of TLR2, TLR3 and TLR9 were analyzed from brain brain, at day 14 post EAE induction in mice infected with R8316 samples. In addition, expression of MyD88, and the expression of (IL-5). R8308 (IL-10) also induced similar patterns but not a sig- type 1 IFNs IFN-a4, IFN-a6 and IFN-b were analyzed. nificant response. Neither of the cytokine-expressing viruses signifi- All of the studied TLRs were significantly (Po0.05) downregulated cantly affected the IL-12p40 or IFNg mRNA expression in the TG. in brains of the mice infected with R8316 (IL-5) during the onset of R3659 upregulated the IL-12p40 expression also in TGs. Only single the EAE disease as compared with untreated mice (Figure 5). Infection TG samples were positive for IL-12p35 or IL-4 and therefore no with R8308 (IL-10) showed a similar tendency. Infection with R3659 significant changes were seen (data not shown).

Gene Therapy HSV-1 expressing IL-5 ameliorates EAE MNyga˚rdas et al 649

Figure 3 Cellular infiltrates in the hematoxylin–eosin (HE) and immunohistochemically stained paraffin brain sections of mice with EAE during acute disease, with or without HSV vector treatment. Figure 3 depicts vertically the different treatment groups, including EAE no treatment, infection with R3659, R8316 (IL-5) or R8308 (IL-10). Parallel paraffin brain sections were stained with HE, or reacted with antibodies against leukocytes (CD45), CD4+ and CD8+ T lymphocytes and antigen-presenting cells (F4/80). The arrows indicate cells with positive staining in the lymphocyte infiltrates.

DISCUSSION tendency toward a higher proportion of CD8+ cells in the CNS of In this study, we found that an HSV-1 vector expressing IL-5 HSV-treated mice. This is in accordance with the proposed regulatory significantly ameliorates EAE. We observed marked improvement of role of CD8+ cells in EAE.22 Eosinophilic leukocytes were observed in the disease score, the cumulative disease index and the EAE mortality infiltrates of R8316 (IL-5)-treated EAE mice, but were also observed rate in the animals treated with the virus R8316 (IL-5). The effect of in the other EAE groups in accordance with observations in other EAE the HSV vector involves an upregulation of the type 1 IFN response in models52 (Supplementary Figure 1). the CNS, especially IFN-b. Overall, contradicting views exists regarding the roles of different When treating the mice with the control virus R3659, we also saw a cytokines and their effects on the disease course of EAE and MS. reduction in EAE scores during the late phase of EAE, as expected Several studies have indicated the importance of IFN-b in the recovery based on earlier reports.28,29 Treatments with all of the HSV-1 vectors from EAE. Mice depleted of the type I IFN receptor (IFNAR)53,54 or used in this study showed a significantly lower cumulative disease negative for the IFN-b gene showed exacerbated EAE.55 Guo et al.54 index than for the untreated EAE mice. This confirms earlier findings suggested that the type I IFNs constrain IL-17, whereas a study by 53 that replicative g134.5-negative HSV as such also have beneficial effects Prinz et al. shows that IFN-b suppresses chemokine expression and on EAE. No clear differences in CNS inflammatory infiltrate composi- upregulation of major histocompatibility complex class II molecules tion were seen between the different treatment groups, but there was a on macrophages and microglia, as also discussed by Axtell and

Gene Therapy HSV-1 expressing IL-5 ameliorates EAE MNyga˚rdas et al 650

Figure 4 Hematoxylin–eosin (HE) and immunohistochemically stained spinal cord sections during recovery. Parallel spinal cord paraffin sections of EAE mice without treatment or treated with R3659 or R8316 (IL-5) were stained with HE or reacted with antibodies against leukocytes, CD4+, and CD8+ lymphocytes and antigen-presenting cells (F4/80). The arrows in HE-staining indicate the areas of higher magnification.

Steinman.56 Studies have also shown that type 1 IFNs enhance IL-4, IL-5 or IL-10 in combination with ConA stimulation. IL-5 also production of IL-4 and IL-1057–59 and diminish the production of downregulated the TLR3 expression during polyinosinic:polycytidylic the pro-inflammatory Th1 cytokines IFN-g and tumor necrosis acid stimulation (data not shown). Mueller et al.61 have also shown factor-a in MS.60 In this study, we saw a significantly higher upregula- that the Th2 cytokines IL-4 and IL-13 downregulated TLR3 and TLR4, tion in IFN-b mRNA expression with R8316 (IL-5) in comparison while IFN-g enhanced their expression in human intestinal epithelial with both R3659 and untreated mice. cells. The group also studied the effect of IL-5, but did not see the Touil et al.44 have shown that TLR3 stimulation with the TLR3 same effect as with IL-4 and IL-13, probably due to the lack of IL-5 agonist polyinosinic:polycytidylic acid (double-stranded RNA analog) receptor in the used cell line. The TLR3 changes that we saw might suppressed demyelization in SJL/J mice with EAE by inducing IFN-b. therefore be a response to the expressed Th2 cytokines. This is The IFN-b upregulation that we obtained is most likely dependent on also supported by the fact that there were significant differences in signaling from other sensors than TLR3 since we obtained a decrease TLR3 mRNA expression between the backbone virus R3659 and the in TLR3 mRNA expression after treatment with HSV-1 expressing Th2 cytokine-expressing HSV vectors. IL-5 or IL-10. In addition, our unpublished observations indicate that The treatment with HSV vectors expressing IL-5 or IL-10 in SJL/J TLR3 is downregulated in spleen cells treated with the Th2 cytokines mice with EAE also influences other TLR responses, as we, in addition,

Gene Therapy HSV-1 expressing IL-5 ameliorates EAE MNyga˚rdas et al 651

MyD88 TLR2 TLR3 TLR9 1000000 10000000 10000000 100000000 * ** ** * # # #### # * ** * ## # # # ## * # # # # ## # # 1000000 * 1000000 * 10000000 100000 * # ## * # # * * * * 1000000 * 100000 100000 * 10000 * 100000 10000 * 10000 * 10000 1000

-actin x 10^8 -actin x 10^8 1000 -actin x 10^8 -actin x 10^8

 1000    1000 100 100 100 100 10 10 Copies/ 10 Copies/ Copies/ 10 Copies/ 1 1 1 1 day 10 day 14 day 21 day 10 day 14 day 21 day 10 day 14 day 21 day 10 day 14 day 21 day 4 p.inf day 8 p.inf day 15 p.inf day 4 p.inf day 8 p.inf day 15 p.inf day 4 p.inf day 8 p.inf day 15 p.inf day 4 p.inf day 8 p.inf day 15 p.inf Days post induction/infection Days post induction/infection Days post induction/infection Days post induction/infection

α IFN-α4 IFN- 6 IFN-β # # ## # # # 1000000 10000000 10000000 * * * * * * 1000000 1000000 * * 100000 # * * * * * * * # 100000 100000 10000 10000 10000 1000 -actin x 10^8

-actin x 10^8 1000 -actin x 10^8 1000    100 100 100 10

10 10 Copies/ Copies/ Copies/ 1 1 1 day 10 day 14 day 21 day 10 day 14 day 21 day 10 day 14 day 21 day 4 p.inf day 8 p.inf day 15 p.inf day 4 p.inf day 8 p.inf day 15 p.inf day 4 p.inf day 8 p.inf day 15 p.inf Days post induction/infection Days post induction/infection Days post induction/infection

EAE, no virus EAE, R3659 virus EAE, IL-10 virus (R8308) EAE, IL-5 virus (R8316)

Figure 5 TLR and IFN mRNA expression in brains of mice with EAE, with or without HSV vector treatment. Light gray bar indicates EAE without treatment, dark gray bar R3659 treatment, black bar treatment with HSV-1 expressing IL-10 (R8308) and white bar treatment with HSV-1 expressing IL-5 (R8316). (a) MyD88, (b)TLR2,(c) TLR3, (d)TLR9,(e)IFN-a4, (f)IFN-a6and(g)IFN-b. Significant changes are indicated with * and ** (Po0.05 and Po0.01, respectively) in comparison with untreated mice at different time points. Similarly, # and ## indicate significant changes in comparison with R3659. The copy number values are shown on a logarithmic scale.

IL-17 ## IL-23 IL-12 p40 IL-12 p35 1000000 # # 100000 * * 100000 # * 10000000 * # * 100000 * 1000000 # ## * * 10000 # # 10000 10000 100000 ** * # # 1000 1000 1000 10000 * -actin x 10^8 -actin x 10^8 -actin x 10^8 -actin x 10^8   1000 100 100 100   100 10 10 10 10

Copies/ Copies/ # Copies/ Copies/ 1 1 1 1 day 10 day 14 day 21 day 10 day 14 day 21 day 10 day 14 day 21 day 10 day 14 day 21 day 4 p.inf day 8 p.inf day 15 p.inf day 4 p.inf day 8 p.inf day 15 p.inf day 4 p.inf day 8 p.inf day 15 p.inf day 4 p.inf day 8 p.inf day 15 p.inf Days post induction/infection Days post induction/infection Days post induction/infection Days post induction/infection

IFN-γ IL-5 IL-10 ## 1000000 10000 1000000 * # ## ## ### * * * * 100000 100000 1000 * * * 10000 10000

1000 100 1000 -actin x 10^8 -actin x 10^8 -actin x 10^8    100 100 10 10 10 Copies/ Copies/ Copies/ 1 1 1 day 10 day 14 day 21 day 10 day 14 day 21 day 10 day 14 day 21 day 4 p.inf day 8 p.inf day 15 p.inf day 4 p.inf day 8 p.inf day 15 p.inf day 4 p.inf day 8 p.inf day 15 p.inf Days post induction/infection Days post induction/infection Days post induction/infection

EAE, no virus EAE, R3659 virus EAE, IL-10 virus (R8308) EAE, IL-5 virus (R8316)

Figure 6 Cytokine mRNA expression in brains of mice with EAE, with or without HSV vector treatment. (a)IL-17,(b)IL-23,(c) IL-12p40, (d) IL-12p35, (e)IFN-g,(f) IL-5 and (g) IL-10. Light gray bar indicates EAE without treatment, dark gray bar R3659 treatment, black bar treatment with HSV-1 expressing IL-10 (R8308) and white bar treatment with HSV-1 expressing IL-5 (R8316). * and ** indicate significant changes (Po0.05 and Po0.01, respectively) in comparison with untreated mice at different time points. Similarly, # and ## indicate significant changes in comparison with R3659. The copy number values are shown on a logarithmic scale. observed a decrease in the TLR2, TLR9 and MyD88 mRNA expression expression during the recovery phase (day 21 post induction). during the onset of EAE and an increase during the acute phase when For all studied TLRs the Th2 cytokine-expressing vectors induced treating with the HSV-1 vectors (in comparison with plain EAE). significantly lower mRNA expression than R3659. Several reports have There was also a trend of downregulation of the TLR mRNA shown that TLRs have a role in the pathogenesis of EAE. MyD88 is

Gene Therapy HSV-1 expressing IL-5 ameliorates EAE MNyga˚rdas et al 652

IL-17 # # IL-23 IL-12 p40 IFN-γ 10000000 * 1000000 100000 10000000 1000000 * 1000000 100000 10000 ** # #### # # 100000 * 100000 10000 10000 1000 10000 1000 -actin x 10^8 1000 -actin x 10^8 -actin x 10^8 -actin x 10^8 1000    100  100 100 100 10 10 10

Copies/ 10 Copies/ Copies/ Copies/ 1 1 1 1 day 10 day 14 day 21 day 10 day 14 day 21 day 10 day 14 day 21 day 10 day 14 day 21 day 4 p.inf day 8 p.inf day 15 p.inf day 4 p.inf day 8 p.inf day 15 p.inf day 4 p.inf day 8 p.inf day 15 p.inf day 4 p.inf day 8 p.inf day 15 p.inf Days post induction/infection Days post induction/infection Days post induction/infection Days post induction/infection

IL-5 IL-10 # # # 1000000 1000000 * ** * 100000 100000 # ** 10000 10000

1000 1000 -actin x 10^8 -actin x 10^8   100 100

10 10 Copies/ Copies/ 1 1 day 10 day 14 day 21 day 10 day 14 day 21 day 4 p.inf day 8 p.inf day 15 p.inf day 4 p.inf day 8 p.inf day 15 p.inf Days post induction/infection Days post induction/infection

EAE, no virus EAE, R3659 virus EAE, IL-10 virus (R8308) EAE, IL-5 virus (R8316)

Figure 7 Cytokine mRNA expression in TG of mice with EAE, with or without HSV vector treatment. (a)IL-17,(b)IL-23,(c) IL-12p40, (d)IFN-g,(e)IL-5 and (f) IL-10. Light gray bar indicates EAE without treatment, dark gray bar R3659 treatment, black bar treatment with HSV-1 expressing IL-10 (R8308) and white bar treatment with HSV-1 expressing IL-5 (R8316). * and ** indicate significant changes (Po0.05 and Po0.01, respectively) in comparison with untreated mice at different time points. Similarly, # and ## indicate significant changes in comparison with R3659. The copy number values are shown on a logarithmic scale.

believed to be crucial for the induction of EAE since MyD88À/À mice Treatment with R8316 (IL-5) upregulated the Th2 cytokine IL-10, are resistant to EAE.62 T cells from MyD88-negative mice do not both in brains and TG, during the acute phase. Both cytokine- express IL-17.63 This is accordance with our findings that MyD88 is expressing HSV vectors upregulated IL-10 mRNA expression during downregulated early during the treatment with HSV vectors expres- recovery in brains, also in comparison with R3659. We also saw an sing Th2 cytokines, and thus might explain why these HSV vectors upregulation of the IL-5 expression in the TG after R8316 (IL-5) ameliorated the EAE disease course. infection, indicating that the virus expressed IL-5 properly. This IL-10 has been presented as a critical Th2 cytokine in recovery and was further supported by our results from a transgene PCR, regulation of EAE.23,33 We therefore also studied the effect of IL-10 where transgene transcripts were found in most TGs (Supplementary expressed from a HSV-1 vector. We did not see a significant effect on Table 2). Our results also indicate that the empty vector R3659 and the the EAE disease course when the IL-10–HSV was administered cytokine-expressing HSV vectors cause different changes in cytokine intranasally as we did for the IL-5-expressing HSV. This is in profiles and TLR expression. accordance with an previous study of ours, where IL-10, when The changes observed in the CNS and TG are not similar in expressed from an HSV-1 vector, did not have a significant alleviating all respects. The cytokine profiles and changes observed in the CNS effect on the disease course.29 Still, both HSV-1 vectors expressing Th2 during treatment are likely influenced by the pathogenic and repair cytokines had similar effect on many of the studied cytokines. We have processes occurring in EAE, meanwhile the cytokine changes in the earlier observed upregulation of IL-23 in nervous system of mice TG are likely less affected by the EAE pathogenesis, reflecting 64 infected with wild-type HSV-1. However, the g134.5 deletion viruses more directly the effects of the HSV vectors and of the innate used in this study did not upregulate IL-23 in the TG, except for antiviral responses. The vectors also reach the CNS later than they R8316 at day 21. R3659 even downregulated IL-23 in the brain during reach the TG. onset of disease. In addition, R3659 upregulated IL-12p40 both in The cytokine IL-5 has not been widely studied in MS or in EAE. brains and TGs. The IL-10 virus also affected IL-12p40 in the brain, Our study indicates that IL-5 has a role in the control of the disease, whereas the IL-5 virus downregulated IL-12p35. This indicates that and might involve regulation of IL-17 and IFN-b. The HSV vectors IL-10 and IL-5 might limit the inflammation through downregulation expressing IL-5 or IL-10 also affected TLR responses, which we believe of these cytokines. Studies by Becher and Cua11–13 has shown that are also important factors in the pathogenesis of the disease. The IL-23 and IL-12 are involved in the inflammation in EAE, although therapeutic outcome of R8316 is likely a combination of the beneficial IL-17 is now believed to be the main disease-promoting cytokine in effect of the HSV-1 backbone and the IL-5. As observed earlier, R3659 CNS inflammation.65 Although it is known that HSV infection activates is able to induce Th2 responses by itself, explaining the effects IFN-g expression,66,67 no significant changes were seen in mRNA of the HSV backbone.28,51,68 This study also supports previous 26,27,29,69 expression of IFN-g in the brain or in the TG with our g134.5 deletion studies, indicating that HSV vectors are good candidates for viruses. There was, however, a trend of decreased IFN-g expression after gene therapy of CNS diseases. Still, it is important to study the safety treatment with R8316 (IL-5) and a significant lower IFN-g expression of these vectors and also optimize the expression efficiency in the was seen for the IL-5–HSV and the IL-10–HSV than for R3659. target tissue.

Gene Therapy HSV-1 expressing IL-5 ameliorates EAE MNyga˚rdas et al 653

MATERIALS AND METHODS Heat-induced epitope retrieval was done in sodium citrate buffer, pH 6.0 Viral vectors (10 mM). Samples were stained for leukocytes (CD45, AbDSerotec, Oxford, The vectors used in this study are based on the HSV-1 virus strain F, and are UK, 1:2000), T lymphocytes (CD4, Abcam, Cambridge, UK, 1:1000; CD8, derived from the virus R3659.70 The virus R3659 carries the thymidine kinase AbDSerotec, 1:100), and antigen-presenting cells (F4/80, AbDSerotec 1:80). gene under the a27 gene promoter in the deletion of g134.5 locus. As a Sections were incubated with the primary antibody overnight at +4 1C. Primary consequence, the viruses do not replicate in nondividing cells such as neurons. antibodies were detected using Vectastain ABC kit (Vector Laboratories, Both the R8308 (IL-10) and the R8316 (IL-5) virus have an insertion of the Burlingame, CA, USA) according to the manufacturer’s instructions and mouse Th2 cytokine in the deleted regions of the g134.5 gene. The viruses were stained with diaminobenzidine. All antibodies were diluted in Tris-buffered thymidine kinase-positive. The virus R3659 and genomic structure of the saline containing 1% bovine serum albumin. cytokine-expressing HSV vectors have been described before.29,68,71 Cytokine expression from the HSV vectors expressing IL-10 and IL-5 was Viral culture tested from supernatants with EIA (eBioscience, San Diego, CA, USA). Shortly, Brain and TG samples were homogenized by shaking the samples in tubes with CSM rat neuronal cells were infected with R8308 and R8316 and supernatants glass beads (Assistant Ø 1 mm, VWR International Oy, Espoo, Finland) at –1 were collected. IL-10 was produced at the level of 850 pg ml at 24 h after +4 1C for 2Â45 s with Mikro-dismembrator II device (Medical Braun Ab, –1 R8308 infection. The R8316 virus produced 5.1 pg ml of IL-5 at 24 h post Espoo, Finland). Thereafter the samples were centrifuged for 30 s. The TG infection. Both viruses were a kind gift from Dr Bernard Roizman, the samples were diluted 1:2 and the brain samples 1:10 in RPMI medium University of Chicago, Chicago, IL, USA. containing 0.1% bovine serum albumin before overlaying cells in six-well culture dishes with 0.5 ml virus dilution. Cells were incubated for 1–2 h at room Mice temperature before changing the medium to Eagle’s minimal essential medium Specific pathogen-free 4- to 6-week-old female SJL/J mice were obtained from containing 7% fetal calf serum and 20 mgml–1 human immunoglobulin G. Cells the Central Animal Laboratory, University of Turku, Turku, Finland. The mice were then incubated for 3 days at 37 1C and 5% CO2,fixedinmethanolfor were maintained at the animal facility of the Microbiological Institute, 10 min and stained with 0.1% crystal violet in 2% ethanol. Samples taken on University of Turku, permit no. 1513/05 of the Ethical Committee for Animal day 21 post induction were incubated in culture medium for 5 days before Experiment of the University of Turku. The recombinant viruses were used homogenization and plaque titration to reactivate the possible latent virus.51,72 under the notifications no. 4/P/99 and 19/M/07 of the Board of Gene Technology, Finland. DNA and RNA extraction DNA was extracted from brain and TG samples with EZNA tissue extraction kit EAE induction (OMEGA, Bio-tek, Norcross, GA, USA) according to the manufacturer’s EAE was induced by immunization in both footpads with 50 mlof50mg manual. TG samples were processed from viral culture tubes. proteolipid protein peptide139À151 (NeoMPS, Strasbourg, France) in Freund’s RNA was extracted with Trizol Reagent (Invitrogen Life Technologies, incomplete adjuvant supplemented with 200 mg Mycobacterium tuberculosis Carlsbad, CA, USA) according to the manufacturer’s instructions. In all, 1 ml (strain H37RA; Difco Laboratories, Detroit, MA, USA) and 12.5 mg Myco- of Trizol was used for brain samples and 500 mlwasusedforTGsamples.The bacterium butyricum (Difco Laboratories). Mice received 200 ng Pertussis RNA was diluted in 20 ml of water treated with diethylpyrocarbonate. Samples toxin (Alexis Biochemicals, Enzo Life Sciences, Plymouth Meeting, PA, USA) were then stored at À70 1C until complementary DNA (cDNA) transcription. in 250 ml of phosphate-buffered saline intraperitoneally at days 0 and 1 after induction. Clinical scores (0, healthy; 1, fur ruffling; 2, tail atonia; 3, hind limb Reverse transcription paralysis; 4, tetraparalysis or moribund; 5, dead) were recorded daily. RNA was transcribed into cDNA as follows: 0.023 U pd(T)12À18 (0.7 ml of 33 Uml–1) (Amersham Biosciences/GE Healthcare Bio Science, Pittsburgh, Treatment of EAE PA, USA) and 13.5 ml diethylpyrocarbonate-water were mixed and 5 mlRNA In all, 1Â107 p.f.u. of HSV-1 viral vectors R3659, R8308 (IL-10) and R8316 sample was added. The sample mixture was incubated at 70 1C for 5 min and (IL-5) were diluted in 10 ml of phosphate-buffered saline. In total, 5 mlvirus then put on ice. Moloney murine leukemia virus RT 5Âbuffer, 5 mM dNTP, dilution was administered in anesthetized mice in each of the nostrils on day 6 moloney murine leukemia virus reverse transcriptase Rnase H Minus enzyme post EAE induction (day 0 of infection). Control EAE mice were left untreated. (40 units) and Rnase inhibitor (8 units), all from Promega (Madison, WI, All groups consisted of 15–20 mice. USA) was then mixed in a PCR clean room and 5.7 ml mixture was added to the samples. The reverse transcriptase-reaction was carried out in PerkinElmer PCR Dissection and processing of tissues machine (Waltham, MA, USA) under the following conditions; at +37 1Cfor Five mice from each group were killed under CO2 anesthesia at 10, 14 and 21 60 min followed by +90 1C for 5 min whereafter the samples were cooled to days after EAE induction. Mice were perfused with phosphate-buffered saline +4 1C. The cDNA was stored at À70 1C and used for quantitative real-time via the left chamber of the heart after lethal CO2 anesthesia. Samples were taken PCR. from the midbrain level for virus culture and PCR. The rest of the brain samples were fixed in 4% phosphate-buffered formaldehyde for microscopy. Quantitation of HSV-1 gD gene, cytokine and TLR cDNA Spinal cords were taken on day 21 and fixed in 4% phosphate-buffered with real-time PCR formaldehyde. In all, 4 mm paraffin sections were made and transferred onto Quantitative real-time PCR was performed with Rotor-Gene 6000 real-time glass microscope slides. Samples from the TG were taken for viral culture and instrument (Corbett Research, Mortlake, Victoria, Australia). Each reaction was for DNA and RNA extraction. carried out in a total volume of 20 ml containing 10 ml Quantitect SybrGreen PCR enzyme buffer mixture (Qiagen, Hilden, Germany), 10–20 pmol of each Neuropathological examinations primer, 7 mlwaterand2ml cDNA samples. The amount of viral genome in the Formaldehyde-fixed 4 mm paraffin brain and spinal cord sections were stained samples was studied by quantifying HSV-1 gD gene target.64 This PCR mixture with hematoxylin and eosin. The specialist of neuropathology scored contained 10 ml SybrGreen, 3.75 pmol of each primer, 6 ml PCR-grade water and the severity of inflammatory infiltrates in the CNS. The scoring was: 0, no 4 ml DNA. The enzyme mixture was pipetted in a separate PCR clean room, infiltration; 1, perivascular infiltration; 2, perivascular inflammatory cuffs; and 3, where no PCR products were handled. The reaction was carried out using the inflammation of the brain substance. following conditions: initial denaturation at 95 1C for 15 min, 45 cycles at 95 1C Immunohistochemistry was performed to analyze the cell composition of for 15 s, 55 or 60 1C for 30 s, 72 1C for 45 s, followed by generation of melting infiltrates in brains and spinal cords. Fixed samples were deparaffinized and curve from 72 to 95 1C. Primer sequences for IL-4, IL-23p19, IL-12p35, rehydrated by incubating slides overnight at +60 1C and washed in Xylene IL-12p40, B-actin, IFN-g,64 HSV-1 gD73 and TLR962 have been published (2Â5 min), and subsequently in 100%, 96% and 70% ethanol (2Â2 min each). earlier. For additional primer sequences and annealing temperatures,

Gene Therapy HSV-1 expressing IL-5 ameliorates EAE MNyga˚rdas et al 654

see Supplementary Table 1. The sensitivity of the quantitative reverse tran- 17 Hofstetter H, Ibrahim S, Koczan D, Kruse N, Weishaupt A, Toyka K et al. Therapeutic scriptase-PCR tests was between 1 and 10 copies of target per sample. efficacy of IL-17 neutralization in murine experimental autoimmune encephalomyelitis. Cell Immunol 2005; 237: 123–130. 18 Harrington L, Hatton R, Mangan P, Turner H, Murphy T, Murphy K et al. Interleukin Statistical analysis 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type Clinical disease scores were compared for the statistically significant difference 1and2lineages.Nat Immunol 2005; 6: 1123–1132. between mouse groups and different timepoints. Cytokine and TLR mRNA 19 Sakaguchi S. Naturally arising CD4+ regulatory t cells for immunologic self-tolerance and negative control of immune responses. Annu Rev Immunol 2004; 22: expression after viral infection in SJL/J mice with EAE were compared 531–562. with uninfected EAE mice. In addition, changes in mRNA expression were 20 Miyara M, Sakaguchi S. Natural regulatory T cells: mechanisms of suppression. Trends calculated between R3659 (backbone virus) and Th2-expressing HSV vectors. Mol Med 2007; 13: 108–116. We used Statview software (Abacus Concepts, Berkeley, CA, USA) to calculate 21 Sakaguchi S. Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in immunological tolerance to self and non-self. Nat Immunol 2005; 6: 345–352. the Mann–Whitney U-test results. 22 Fletcher J, Lalor S, Sweeney C, Tubridy N, Mills K. T cells in multiple sclerosis and experimental autoimmune encephalomyelitis. Clin Exp Immunol 2010; 162:1–11. 23 Kennedy M, Torrance D, Picha K, Mohler K. Analysis of cytokine mRNA expression in CONFLICT OF INTEREST the central nervous system of mice with experimental autoimmune encephalomyelitis The authors declare no conflict of interest. reveals that IL-10 mRNA expression correlates with recovery. JImmunol1992; 149: 2496–2505. 24 Issazadeh S, Mustafa M, Ljungdahl A, Ho¨jeberg B, Dagerlind A, Elde R et al. Interferon ACKNOWLEDGEMENTS gamma, interleukin 4 and transforming growth factor beta in experimental autoimmune This study was financially supported by the Academy of Finland, Grant nos. encephalomyelitis in Lewis rats: dynamics of cellular mRNA expression in the central 118366 and 128915, the Swedish Cultural Foundation in Finland, the nervous system and lymphoid cells. JNeurosciRes1995; 40: 579–590. Finnish Cultural Foundation, the Research and Science foundation of Farmos, 25 Butti E, Bergami A, Recchia A, Brambilla E, Del Carro U, Amadio S et al. IL4 gene delivery to the CNS recruits regulatory T cells and induces clinical recovery in mouse the Paulo Foundation and Turku Graduate School of Biomedical Sciences. models of multiple sclerosis. Gene Therapy 2008; 15: 504–515. We are grateful for the technical assistance by Terhi Helander and Leena 26 Furlan R, Poliani P, Galbiati F, Bergami A, Grimaldi L, Comi G et al. Central nervous Marjatta Ja¨rvi and animal care by Seija Lindqvist. We also thank Dr Bernard system delivery of interleukin 4 by a nonreplicative herpes simplex type 1 viral vector Roizman for the recombinant HSV-1 vectors. ameliorates autoimmune demyelination. Hum Gene Ther 1998; 9: 2605–2617. 27 Furlan R, Poliani P, Marconi P, Bergami A, Ruffini F, Adorini L et al. Central nervous system gene therapy with interleukin-4 inhibits progression of ongoing relapsing- remitting autoimmune encephalomyelitis in Biozzi AB/H mice. Gene Therapy 2001; 8:13–19. 28 Broberg E, Salmi A, Hukkanen V. IL-4 is the key regulator in herpes simplex virus-based 1 Amor S, Groome N, Linington C, Morris M, Dornmair K, Gardinier M et al. Identification gene therapy of BALB/c experimental autoimmune encephalomyelitis. Neurosci Lett of epitopes of myelin oligodendrocyte glycoprotein for the induction of experimental 2004; 364: 173–178. allergic encephalomyelitis in SJL and Biozzi AB/H mice. JImmunol1994; 153: 29 Broberg E, Seta¨la¨ N, Ro¨ytta¨ M, Salmi A, Era¨linnaJ,HeBet al. Expression of 4349–4356. interleukin-4 but not of interleukin-10 from a replicative herpes simplex virus type 1 2 Linington C, Berger T, Perry L, Weerth S, Hinze-Selch D, Zhang Y et al. T cells specific viral vector precludes experimental allergic encephalomyelitis. Gene Therapy 2001; 8: for the myelin oligodendrocyte glycoprotein mediate an unusual autoimmune inflam- 769–777. matory response in the central nervous system. Eur J Immunol 1993; 23: 1364–1372. 30 Martino G, Furlan R, Galbiati F, Poliani P, Bergami A, Grimaldi L et al. Agenetherapy 3 Mendel I, Kerlero de Rosbo N, Ben-Nun A. A myelin oligodendrocyte glycoprotein approach to treat demyelinating diseases using non-replicative herpetic vectors peptide induces typical chronic experimental autoimmune encephalomyelitis in H-2b engineered to produce cytokines. Mult Scler 1998; 4: 222–227. mice: fine specificity and T cell receptor V beta expression of encephalitogenic T cells. 31 Liblau R, Steinman L, Brocke S. Experimental autoimmune encephalomyelitis in Eur J Immunol 1995; 25: 1951–1959. IL-4-deficient mice. Int Immunol 1997; 9: 799–803. 4 Zamvil S, Steinman L. The T lymphocyte in experimental allergic encephalomyelitis. 32 Falcone M, Rajan A, Bloom B, Brosnan C. A critical role for IL-4 in regulating disease Annu Rev Immunol 1990; 8: 579–621. severity in experimental allergic encephalomyelitis as demonstrated in IL-4-deficient 5 Tuohy V, Lu Z, Sobel R, Laursen R, Lees M. A synthetic peptide from myelin proteolipid C57BL/6 mice and BALB/c mice. JImmunol1998; 160: 4822–4830. protein induces experimental allergic encephalomyelitis. J Immunol 1988; 141: 33 Bettelli E, Das M, Howard E, Weiner H, Sobel R, Kuchroo V. IL-10 is critical in the 1126–1130. regulation of autoimmune encephalomyelitis as demonstrated by studies of IL-10- and 6 Pettinelli C, McFarlin D. Adoptive transfer of experimental allergic encephalomyelitis in IL-4-deficient and transgenic mice. JImmunol1998; 161: 3299–3306. SJL/J mice after in vitro activation of lymph node cells by myelin basic protein: 34 Sanderson C. Interleukin-5: an eosinophil growth and activation factor. Dev Biol Stand requirement for Lyt 1+ 2- T lymphocytes. JImmunol1981; 127: 1420–1423. 1988; 69:23–29. 7 Liblau R, Singer S, McDevitt H. Th1 and Th2 CD4+ T cells in the pathogenesis of organ- 35 Sanderson C. Interleukin-5, eosinophils, and disease. Blood 1992; 79: 3101–3109. specific autoimmune diseases. Immunol Today 1995; 16:34–38. 36 Wiesemann E, Klatt J, So¨nmez D, Blasczyk R, Heidenreich F, Windhagen A. Glatiramer 8 Issazadeh S, Ljungdahl A, Ho¨jeberg B, Mustafa M, Olsson T. Cytokine production in the acetate (GA) induces IL-13/IL-5 secretion in naive T cells. J Neuroimmunol 2001; 119: central nervous system of Lewis rats with experimental autoimmune encephalomyelitis: 137–144. dynamics of mRNA expression for interleukin-10, interleukin-12, cytolysin, tumor 37 Wiesemann E, Klatt J, Wenzel C, Heidenreich F, Windhagen A. Correlation of serum necrosis factor alpha and tumor necrosis factor beta. J Neuroimmunol 1995; 61: IL-13 and IL-5 levels with clinical response to Glatiramer acetate in patients with 205–212. multiple sclerosis. Clin Exp Immunol 2003; 133:454–460. 9 Ichikawa M, Koh C, Inoue A, Tsuyusaki J, Yamazaki M, Inaba Y et al. Anti-IL-12 38 Sanna A, Fois M, Arru G, Huang Y, Link H, Pugliatti M et al. Glatiramer acetate reduces antibody prevents the development and progression of multiple sclerosis-like relapsing– lymphocyte proliferation and enhances IL-5 and IL-13 production through modulation remitting demyelinating disease in NOD mice induced with myelin oligodendrocyte of monocyte-derived dendritic cells in multiple sclerosis. Clin Exp Immunol 2006; glycoprotein peptide. J Neuroimmunol 2000; 102:56–66. 143:357–362. 10 Leonard J, Waldburger K, Goldman S. Prevention of experimental autoimmune 39 Weir C, Bernard C, Ba¨ckstro¨m B. IL-5-deficient mice are susceptible to experimental encephalomyelitis by antibodies against interleukin 12. JExpMed1995; 181: autoimmune encephalomyelitis. Int Immunol 2003; 15: 1283–1289. 381–386. 40 Akira S. TLR signaling. Curr Top Microbiol Immunol 2006; 311:1–16. 11 Becher B, Durell B, Noelle R. IL-23 produced by CNS-resident cells controls 41 Lang K, Recher M, Junt T, Navarini A, Harris N, Freigang S et al. Toll-like receptor T cell encephalitogenicity during the effector phase of experimental autoimmune engagement converts T-cell autoreactivity into overt autoimmune disease. Nat Med encephalomyelitis. JClinInvest2003; 112: 1186–1191. 2005; 11: 138–145. 12 Cua D, Sherlock J, Chen Y, Murphy C, Joyce B, Seymour B et al. Interleukin-23 rather 42 Segal B, Chang J, Shevach E. CpG oligonucleotides are potent adjuvants for the than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain. activation of autoreactive encephalitogenic T cells in vivo. JImmunol2000; 164: Nature 2003; 421: 744–748. 5683–5688. 13 Becher B, Durell B, Noelle R. Experimental autoimmune encephalitis and inflammation 43 Buenafe A, Bourdette D. Lipopolysaccharide pretreatment modulates the disease in the absence of interleukin-12. JClinInvest2002; 110: 493–497. course in experimental autoimmune encephalomyelitis. J Neuroimmunol 2007; 182: 14 Langrish C, Chen Y, Blumenschein W, Mattson J, Basham B, Sedgwick J et al. IL-23 32–40. drives a pathogenic T cell population that induces autoimmune inflammation. 44 Touil T, Fitzgerald D, Zhang G, Rostami A, Gran B. Cutting Edge: TLR3 stimulation JExpMed2005; 201: 233–240. suppresses experimental autoimmune encephalomyelitis by inducing endogenous 15 Park H, Li Z, Yang X, Chang S, Nurieva R, Wang Y et al. A distinct lineage of CD4 T cells IFN-beta. JImmunol2006; 177: 7505–7509. regulates tissue inflammation by producing interleukin 17. Nat Immunol 2005; 6: 45 Stevens J, Cook M. Latent herpes simplex virus in spinal ganglia of mice. Science 1133–1141. 1971; 173: 843–845. 16 Agrawal S, Yong V. Immunopathogenesis of multiple sclerosis. Int Rev Neurobiol 2007; 46 Stevens J, Cook M. Latent infections induced by herpes simplex viruses. Cancer Res 79: 99–126. 1973; 33: 1399–1401.

Gene Therapy HSV-1 expressing IL-5 ameliorates EAE MNyga˚rdas et al 655

47 Chou J, Kern E, Whitley R, Roizman B. Mapping of herpes simplex virus-1 neuroviru- 61 Mueller T, Terada T, Rosenberg I, Shibolet O, Podolsky D. Th2 cytokines down-regulate lence to gamma 134.5, a gene nonessential for growth in culture. Science 1990; 250: TLR expression and function in human intestinal epithelial cells. JImmunol2006; 1262–1266. 176: 5805–5814. 48 Markovitz N, Baunoch D, Roizman B. The range and distribution of murine central 62 Prinz M, Garbe F, Schmidt H, Mildner A, Gutcher I, Wolter K et al. Innate immunity nervous system cells infected with the gamma(1)34.5- mutant of herpes simplex virus mediated by TLR9 modulates pathogenicity in an animal model of multiple sclerosis. 1. JVirol1997; 71: 5560–5569. JClinInvest2006; 116: 456–464. 49 Frampton AJ, Goins W, Nakano K, Burton E, Glorioso J. HSV trafficking and develop- 63 Marta M, Andersson A, Isaksson M, Ka¨mpe O, Lobell A. Unexpected regulatory roles of ment of gene therapy vectors with applications in the nervous system. Gene Therapy TLR4 and TLR9 in experimental autoimmune encephalomyelitis. Eur J Immunol 2008; 2005; 12:891–901. 38: 565–575. 50 Hudson S, Dix R, Streilein J. Induction of encephalitis in SJL mice by intranasal 64 Broberg E, Seta¨la¨ N, Era¨linna J, Salmi A, Ro¨ytta¨ M, Hukkanen V. Herpes simplex virus infection with herpes simplex virus type 1: a possible model of herpes simplex type 1 infection induces upregulation of interleukin-23 (p19) mRNA expression in encephalitis in humans. JInfectDis1991; 163:720–727. trigeminal ganglia of BALB/c mice. J Interferon Cytokine Res 2002; 22:641–651. 51 Broberg E, Peltoniemi J, Nyga˚rdas M, Vahlberg T, Ro¨ytta¨ M, Hukkanen V. Spread and 65 Steinman L. A brief history of T(H)17, the first major revision in the T(H)1/T(H)2 replication of and immune response to gamma134.5-negative herpes simplex virus hypothesis of T cell-mediated tissue damage. Nat Med 2007; 13: 139–145. type 1 vectors in BALB/c mice. JVirol2004; 78: 13139–13152. 66 Smith P, Wolcott R, Chervenak R, Jennings S. Control of acute cutaneous herpes 52 Stefferl A, Brehm U, Storch M, Lambracht-Washington D, Bourquin C, Wonigeit K et al. simplex virus infection: T cell-mediated viral clearance is dependent upon interferon- Myelin oligodendrocyte glycoprotein induces experimental autoimmune encephalomye- gamma (IFN-gamma). Virology 1994; 202:76–88. litis in the ‘‘resistant’’ Brown Norway rat: disease susceptibility is determined by MHC 67 Cantin E, Hinton D, Chen J, Openshaw H. Gamma interferon expression during acute and MHC-linked effects on the B cell response. JImmunol1999; 163:40–49. and latent nervous system infection by herpes simplex virus type 1. JVirol1995; 69: 53 Prinz M, Schmidt H, Mildner A, Knobeloch K, Hanisch U, Raasch J et al. Distinct and 4898–4905. nonredundant in vivo functions of IFNAR on myeloid cells limit autoimmunity in the 68 Peltoniemi J, Broberg E, Nyga˚rdas M, Era¨linna J, Waris M, Hukkanen V. Enhancement central nervous system. Immunity 2008; 28:675–686. of Th2 responses to replicative herpes simplex virus type 1 vectors by immunomodu- 54 Guo B, Chang E, Cheng G. The type I IFN induction pathway constrains Th17-mediated lative chemotherapy. Int Immunopharmacol 2006; 6: 817–829. autoimmune inflammation in mice. JClinInvest2008; 118: 1680–1690. 69 Furlan R, Bergami A, Brambilla E, Butti E, De Simoni M, Campagnoli M et al. HSV-1- 55 Teige I, Treschow A, Teige A, Mattsson R, Navikas V, Leanderson T et al. IFN-beta gene mediated IL-1 receptor antagonist gene therapy ameliorates MOG(35-55)-induced deletion leads to augmented and chronic demyelinating experimental autoimmune experimental autoimmune encephalomyelitis in C57BL/6 mice. Gene Therapy 2007; encephalomyelitis. JImmunol2003; 170: 4776–4784. 14:93–98. 56 Axtell R, Steinman L. Type 1 interferons cool the inflamed brain. Immunity 2008; 28: 70 Lagunoff M, Roizman B. The regulation of synthesis and properties of the protein 600–602. product of open reading frame P of the herpes simplex virus 1 genome. JVirol1995; 57 Rudick R, Ransohoff R, Peppler R, VanderBrug Medendorp S, Lehmann P, Alam J. 69: 3615–3623. Interferon beta induces interleukin-10 expression: relevance to multiple sclerosis. 71 Andreansky S, He B, van Cott J, McGhee J, Markert J, Gillespie G et al. Treatment of Ann Neurol 1996; 40: 618–627. intracranial gliomas in immunocompetent mice using herpes simplex viruses that 58 Rudick R, Ransohoff R, Lee J, Peppler R, Yu M, Mathisen P et al. In vivo effects of express murine interleukins. Gene Therapy 1998; 5:121–130. interferon beta-1a on immunosuppressive cytokines in multiple sclerosis. Neurology 72 Peltoniemi J, Broberg E, Halenius A, Setala N, Eralinna J, Salmi A et al. Immunomo- 1998; 50: 1294–1300. dulation by roquinimex decreases the expression of IL-23 (p19) mRNA in the brains of 59 Kozovska M, Hong J, Zang Y, Li S, Rivera V, Killian J et al. Interferon beta induces herpes simplex virus type 1 infected BALB/c mice. Clin Exp Immunol 2004; 137: T-helper 2 immune deviation in MS. Neurology 1999; 53: 1692–1697. 305–312. 60 Noronha A, Toscas A, Jensen M. Interferon beta decreases T cell activation 73 Hukkanen V, Rehn T, Kajander R, Sjo¨roos M, Waris M. Time-resolved fluorometry PCR and interferon gamma production in multiple sclerosis. J Neuroimmunol 1993; 46: assay for rapid detection of herpes simplex virus in cerebrospinal fluid. JClinMicrobiol 145–153. 2000; 38: 3214–3218.

Supplementary Information accompanies the paper on Gene Therapy website (http://www.nature.com/gt)

Gene Therapy