Gene Therapy (2001) 8, 769–777 2001 Nature Publishing Group All rights reserved 0969-7128/01 $15.00 www.nature.com/gt RESEARCH ARTICLE Expression of interleukin-4 but not of interleukin-10 from a replicative herpes simplex virus type 1 viral vector precludes experimental allergic encephalomyelitis E Broberg1,2,3, N Seta¨la¨ 1,3,MRo¨ytta¨4, A Salmi1, J-P Era¨linna1,5,BHe6, B Roizman6 and V Hukkanen1,2 Departments of 1Virology, 4Pathology, 5Neurology and 2MediCity Research Laboratory, University of Turku, Turku; 3Turku Graduate School of Biomedical Sciences, Turku, Finland; and 6The Marjorie B Kovler Viral Oncology Laboratories, University of Chicago, Chicago, IL, USA We have used interleukin (IL)-4 and -10-producing HSV-1 any infection, mice infected with backbone virus R3659 and ␥ 134.5 deletion viruses in gene therapy of a BALB/c model mock-infected mice. Weights and symptoms of the mice of experimental allergic encephalomyelitis (EAE), a T cell- were recorded daily and the tissue specimens were col- mediated demyelinating disease of the central nervous sys- lected at specific time-points. The results indicate that the tem. It is known that in EAE of mice the Th2-type cytokines intracranial infection with IL-4-producing virus (1) precludes are down-regulated and the Th1-type cytokines up-regulated EAE symptoms, (2) protects the spinal cord from massive during the onset and relapse of the disease. Therefore, we leukocyte infiltrations and (3) prevents demyelination and tested two HSV-1 recombinants expressing the Th2-type axonal loss. The IL-10-expressing virus R8308 did not have cytokines IL-4 and IL-10. The recombinant viruses were a similar favorable effect on the recovery of the mice as did injected intracranially (i.c.) in BALB/c mice 6 days after the IL-4 virus R8306. Gene Therapy (2001) 8, 769–777. induction of EAE. As control groups we used mice without Keywords: EAE; cytokines; gene therapy; herpes simplex virus vectors Introduction tions when planning gene therapy for a CNS disease. Administration of cytokine proteins intranasally requires Experimental allergic encephalomyelitis (EAE) is an ani- daily administration.14 Intramuscular plasmid injections15 1 mal model for human multiple sclerosis (MS). It is a T and cytokine antibody injections16 require several admin- cell-mediated demyelinating disease of the central ner- istrations to prevent development of EAE. On the other 2 vous system (CNS). There is strong evidence for a dis- hand, viral vectors often require only one time-point ␥ ease-promoting role of certain cytokines, such as IFN- , delivery. However, there are several different virus vec- ␣ ␤ ␤ TNF- and TNF- (Th1 type) while IL-10 and TGF- (Th2 tor types with their own particular properties. HSV-based 3–8 type) may limit the disease. Similar changes in the vectors have the ability to infect neurons, to infect the Th1/Th2 balance have also been observed in cerebro- target for a lifetime, to remain latent and to express lat- 9 spinal fluid samples of MS patients. The critical role ency-associated genes.17 The genome of the virus is not especially of IL-4 has been suggested in recovery from integrated into the genome of the infected cell, and large 10 EAE. The mechanisms underlying the impact of IL-4 on parts of the viral genome can be changed or deleted for 8 EAE have been ascribed to the IL-12 suppression. IL-12 safe gene delivery purposes.18 The virus is not recognized has been shown to have a significant role in the patho- by the immune system during latency and the latently 11 genesis of autoimmune demyelination. infected cells may remain undamaged.19 Therefore, there In gene therapy of EAE different methods of cytokine is ongoing interest to develop HSV vectors further for or cytokine antibody delivery have already been stud- expression of inserted genes during latency.20 The ability 12–16 ied. The inhibition of Th1-type signaling or stimu- of wild-type HSV-1 to cause a cytopathic effect in lation of Th2-type cytokine expression has been the key infected cells has been one of the problems with HSV- in inhibition or amelioration of disease symptoms. The based vectors. In the vectors used in this study, the neu- route of delivery and length of expression are key ques- ␥ rovirulence gene 134.5 has been deleted and replaced by a cytokine-encoding gene.21 HSV vectors with the ␥ deletion of the 134.5 gene have proved safe in phase I 22,23 Correspondence: E Broberg, Department of Virology, University of Turku, studies on therapy of brain tumors. We have studied Kiinamyllynkatu 13, FIN-20520 Turku, Finland the effect of intracranially delivered HSV vectors enco- Received 11 October 2000; accepted 15 March 2001 ding immunosuppressive cytokines on the disease course Heterologous IL-4 expression precludes EAE E Broberg et al 770 of EAE. We have chosen the replication-competent cyto- The disease incidence (score у2) on day 18 varied in kine-expressing vectors R8306 and R830821 for expression mouse groups between 0% in the EAE/IL-4 virus group of the murine interleukins 4 and 10, respectively. and 100% in the EAE/IL-10 virus group. The EAE/PBS group developed normal EAE with typical hind limb par- Results alysis. The average disease grade of these mice was even higher than that of the EAE/no virus group during the EAE peak period (days 15–20). The mice in the EAE/IL- Clinical course of EAE 10 virus group developed typical EAE signs earlier than The main objective of this study was to examine the effect in other groups but their average disease grade stabilized of intracranial (i.c.) infections of recombinant Th2-type around grade 2 (tail atonia). The group with the back- cytokine-expressing HSV vectors to the disease course of bone virus R3659 infection did not develop the typical EAE. The experiment included five different groups of hind limb paralysis and moribund state of EAE but their mice, all with EAE induction: (1) no treatment, (2) average grade stabilized at 1.5 (between fur ruffling and PBS/glucose injection, (3) infection with IL-4-expressing tail atonia). There were more unexplained deaths in this virus R8306, (4) infection with IL-10-expressing virus group than in others. The volume of injection (10 ␮l) was R8308 and (5) infection with empty backbone virus vector probably not the cause of the deaths, because three times R3659. The mice were followed daily for scoring of the larger volumes have been used without any conse- EAE disease grade. The mice that did not receive infec- quences.24 tion developed the expected signs of BALB/c EAE by day The main result of the clinical EAE scoring was that 15 (Figure 1). Mortality in this group was 11% (Table 1), the IL-4 virus infected mice did not develop signs of EAE while in other groups there were no deaths due to EAE. (disease score у2, Table 1) except for one mouse that The EAE disease peak in the untreated group was developed hind limb paralysis. The mean day of EAE observed on days 15–20 after EAE induction (Figure 1). onset (grade Ͼ1) was day 11 for all groups except for the The IL-4 virus infected EAE-induced mice did not EAE/IL-10 virus group (day 10). develop typical EAE (disease score у2) at all (Figure 1). Overall mortality varied between 0 and 22% (Table 1). Only in the EAE/no virus group were there deaths due to EAE symptoms. Those deaths were seen on days 13 and 20 after EAE induction after exacerbation of EAE symptoms. In virus infected mice there were one sudden death on day 12 in the EAE/IL-10 virus group, one sud- den death on each of days 9 and 10 in the EAE/IL-4 virus group and three sudden deaths in the EAE/backbone virus group on days 10, 11 and 17. One mouse died dur- ing the intracranial infection procedure in the EAE/backbone virus group. The statistical analysis by Mann–Whitney U test con- firmed that the IL-4 group differed statistically in clinical status from all the other groups beginning at least on day 15 (P р 0.045) and from the backbone virus group on days 16–21 (P р 0.010). The backbone virus alone gave a statistically significant effect when compared with PBS/glucose injection during days 15–21 (P р 0.025) and during days 16–19 when compared with EAE/no virus (P р 0.046). The EAE/no virus and the EAE/PBS groups did not differ significantly during days 9–21. Virus culture Brain samples taken from the infection site were cultured on VERO cells. The results are summarized in Table 2. Figure 1 Interleukin (IL)-4 expressing herpes simplex virus vector pre- There were only seven positive virus cultures of all stud- cluded the signs of EAE. Figure shows scores of experimental allergic ied brain samples. Five of them were in the EAE/IL-10 encephalomyelitis (EAE) disease and standard deviations in different virus group, one in the EAE/IL-4 virus group and one mouse groups. The disease score of each mouse was recorded daily based in EAE/backbone virus group. All positive virus cultures on the following: 0, healthy; 1, fur ruffling; 2, tail atonia; 3, hind limb were grown to 100% cytopathic effect and the viral DNA paralysis; 4, tetraparalysis or moribund; 5, dead. The infections were made was collected and studied further. These viral DNAs in a volume of 10 ␮l of 1% glucose in PBS and 106 p.f.u.