NZ620196B2 - Polyoma virus jc peptides and proteins in vaccination and diagnostic applications - Google Patents

Abstract

Discloses use of a protein or peptide for the manufacture of a medicament for treating, preventing, or diagnosing progressive multifocal leukoencephalopathy (PML) or progressive multifocal leukoencephalopathy-immune reconstitution inflammatory syndrome (PML-IRIS) in a human subject, wherein said protein or peptide comprises at least one CD4+ epitope derived from polyoma virus JC (JCV), wherein the protein or peptide is present in the form of a virus like particle (VLP), and wherein the protein or peptide comprises VP1 . Also discloses related uses of kits and adjuvants. tein or peptide comprises at least one CD4+ epitope derived from polyoma virus JC (JCV), wherein the protein or peptide is present in the form of a virus like particle (VLP), and wherein the protein or peptide comprises VP1 . Also discloses related uses of kits and adjuvants.

Description

Polyoma virus JC peptides and proteins in vaccination and diagnostic applications The present invention relates to the field of vaccination or zation, in particular thera- peutic vaccination, and diagnosis. Pharmaceutical itions and kits capable of eliciting a protective immune se against polyoma virus JC (JCV) are sed, which may be used e.g., for therapy or for prevention of progressive multifoeal leukoencephalopathy (PML) and/or ssive ocal leukoencephalopathy—immune reconstitution inflammatory syn- drome RIS). Individuals in danger of such PML or PML-IRIS may, c.g., be immuno- compromised or immunosuppressed patients or patients having an autoimmune disease eligible for immunosuppressive treatment. The invention also relates to compositions comprising at least one CD4+ epitopc of a JCV protein and to therapeutic. prophylactic and diagnostic uses thereof.

Progressive multifocal leukoencephalopathy and progressive multifocal leukoencephalopathy- immunc reconstitution atory syndrome are caused by infection of the central nervous system with the polyoma virus JC‘ (JCV). Both have recently emerged as complications of monoclonal antibody therapy in multiple sclerosis and other autoimmune diseases.

Progressive multifocal ncephalopathy (PML) was first described in I958 . In 197] the polyoma virus JC (JCV) was identified as ive agent of PML. PML is an opportunis- tic and often fatal infection that occurs in states of immunocompromisc such as HIV infection, cancer. organ transplantation, immtmodeftciencies. or rarely during autoimmune es. In AIDS patients, PML was one of the most serious complications, although its incidence de- creased afier introduction of antiretroviral y (C inque ct al.. 200] ). Infection with JCV is highly prevalent in y adults. and 60% or more of the population carn'cs a latent/persistent infection (Egli et al., 2009). In recent years PML has emerged as an increasingly common seri- ous adverse event in monoclonal antibody therapy of autoimmune diseases, in particular of multiple sclerosis (MS) and treatment with natalizumab (anti-VLA-4) (Kleinschmidt- DeMasters and Tyler, 2005) (Langer-Gould et al., 2005) (Jilek et al.) (Anonymous, 20] I).

Other monoclonal antibodies such as rituximab (anti-CD20), mab (anti-tumor necrosis factor (TNF)-alpha) and the lgGl-TNF receptor 2 fusion protein ctancrccpt that are used to treat toid arthritis (RA) have also been associated with PML. Efalizumab (anti- leukocyte function-associated antigen-l) had to be withdrawn from the market already (Pugashetti and K00. 2009). With > 120 PML cases reported in MS patients receiving natali- zumab, the PML incidence is between 1:500 and 1:1.000 and jeopardizes the use of this highly effective treatment (Anonymous. 201 l).

The pathogenesis of PML is characterized by a lytic infection of -forming oligodendro- cytes and abortive infection of astrocytes in the absence of a notable immune reaction. How- ever, other CNS cells such as cerebellar granule s can also be infected by JCV (Du Pas- quier et al., 2003a). Although the mechanisms of controlling JCV infection are as yet in- completely understood, latency of JCV infection is probably controlled by effective humoral and/or cellular immune responses in healthy duals (Du Pasquier et al., 2001) (Du Pas- quier et al., 2004a) (Weber et al., 1997). Accordingly, the presence of JCV-specific CD8+ cy- totoxic T cells has been linked to the recovery from PML, while these cells were absent in PML cases with fatal outcome (Du Pasquier et al., 2004a; Du Pasquier et al., 2006; Koralnik et al., 2002). Also, PML occurs preferentially in situations of decreased CD4+ T cell numbers or compromised CD4+ cell functions such as AIDS and idiopathic CD4+ lymphopenia (Stoner et al., 1986) (Gillespie et al., 1991; Zonios et al., 2008). Comparable to the role of CD8+ JCV- c T cells, the tion of PML follows the restoration of CD4+ number and fianction, indicating that both CD4+ and CD8+ virus-specific T cells are l for host protection.

In contrast to the profound irnmunosuppression in AIDS, in Non-Hodgkin lymphoma and leu- kemias, monoclonal antibody-based ies inhibit specific immune fianctions such as cell migration across endothelial rs in LA-4/natalizumab therapy, or eliminate certain immune cells such as CD20-expressing B cells in the case of rituximab (Lutterotti and Martin, 2008). In the context of anti-VLA-4 therapy current hypotheses assume that PML results from compromised immune surveillance of the CNS, since activated T cells and CD209+ im- mature dendritic cells cannot cross the brain-barrier (BBB) and access the brain (del Pilar Martin et al., 2008; Stuve et al., 2006; Yednock et al., 1992). As a result, local antigen tation in the CNS is compromised (del Pilar Martin et al., 2008).

Alternatively, it has been considered that the inhibition of VLA-4/vascular cell adhesion molecule-1 (VCAM-l) interactions, which serve as a retention signal for hematopoietic precursor cells in the bone marrow, leads to release of JCV from one of its natural niches (Tan et al., 2009a), increased viral replication and ence of JCV variants with tropism for CNS cells (Houff et al., 1988; off, 2005).

Cessation of therapy with these monoclonal antibodies in PML reestablishes logical surveillance for JCV-infected cells in the CNS and leads to clinically apparent inflammatory responses in this compartment. Inflammation can be visualized by contrast-enhancing lesions on magnetic resonance imaging (MRI) due to opening of the BBB and influx of T cells and monocytes/macrophages. The latter manifestation of PML has been termed immune PML- reconstitution inflammatory syndrome (PML-IRIS) (Koralnik, 2006; Tan et al., 2009b). PML- IRIS can lead to rapid deterioration of the patient’s clinical state and death in about 30% to 50% of cases (Tan et al., 2009b). Its cellular and molecular pathogenesis, i.e. which T cell sub- types, dies or cytokines are involved, is currently poorly understood.

Progressive multifocal ncephalopathy-immune reconstitution inflammatory syndrome may obscure the diagnosis of progressive multifocal leukoencephalopathy and lead to marked immunopathogenesis with severe clinical disability and possibly death. Different from progres- sive multifocal leukoencephalopathy, in which demyelination results from oligodendrocyte lysis by JC virus in the absence of an immune response at the site of infection, tissue destruction in progressive multifocal ncephalopathy - immune reconstitution inflammatory syndrome is caused by a vigorous immune response against JC virus-infected oligodendrocytes and astro- cytes and inflammatory swelling of the brain. PML-IRIS starts when irnmunocompetence is re- ished, e.g. in AIDS patients treated with highly active retroviral therapy or in MS patients treated with the anti-VLA-4 monoclonal antibody natalizumab and after g out the anti- body. During PML-IRIS, immune cells enter the brain and eliminate JCV-infected astrocytes and oligodendrocytes. The cells and mediators that are involved in progressive multifocal leu- koencephalopathy - immune reconstitution atory syndrome are poorly understood in the state of the art. sing PML and PML-IRIS as early as possible and identifying effective therapies based on the underlying disease mechanisms are important goals not only in MS, but also in a number of other autoimmune diseases, during acquired immunodeficiencies, during malignancies and in transplant medicine. Methods for diagnosis of JCV are known. For example, it is possible to detect the virus by PCR. An alternative approach is detection of antibodies to JCV, e.g., by ELISA. Exemplary methods for diagnosing JCV ion, e.g., using an ELISA to the JCV core protein VPl are taught in Goldmann et al., 1999, or in DE 195 43 553. s of treatment of the disease have been ched less so far. Goldmann et al., 1999, or DE 195 43 553 suggest vaccination with VPl protein, which may be led to virus like particles. It is taught that vaccination with Freund’s Complete Adjuvant (FCA) induces an im- mune response, while vaccination without adjuvant is not genic. However, Freund’s Complete nt may not be used in humans due to its toxicity.

In light of the dangers of a pathogenic immune se, as prevalent in PML-IRIS, it is a par- ticular challenge to develop a ation which allows for treatment of PML and prevention ofPML and PML-IRIS.

This problem was solved by the invention, in particular, by the subject matter of the claims. (followed by 4A) The present invention provides in a first aspect a protein or peptide comprising at least one CD4+ epitope derived from JCV, n the epitope is selected from the group comprising SEQ ID NO: 1 – 92. In a second aspect, the invention provides a pharmaceutical kit comprising a protein or peptide comprising at least one CD4+ e derived from JCV, n the epitope is ed from the group comprising SEQ ID NO: 1 - 92, and an nt. Preferably, the adjuvant is ed from the group comprising a TLR-7 agonist and/or TLR-8 agonist.

In a particular aspect the present invention provides the use of a protein or peptide for the manufacture of a medicament for treating or preventing progressive multifocal ncephalopathy (PML) or progressive multifocal leukoencephalopathy-immune reconstitution inflammatory me (PML-IRIS) in a human subject, wherein said protein or peptide comprises at least one CD4+ epitope derived from polyoma virus JC (JCV), wherein the protein or peptide is t in the form of a virus like particle (VLP), and wherein the protein or peptide comprises VP1 .

In another aspect the t invention particularly provides the use of a pharmaceutical kit for the manufacture of a medicament for ng or preventing PML or PML-IRIS in a subject, wherein said kit comprises a protein or peptide comprising at least one CD4+ epitope derived from JCV, wherein the protein or peptide comprises VP1 and is present in the form of a virus like particle, and wherein said kit comprises an adjuvant.

In a still further aspect, the present invention particularly provides for the use of an adjuvant, in the manufacture of a ment, for treating or preventing PML or PML-IRIS in a subject, wherein the treating or ting comprises administration of the adjuvant in combination with a n or peptide comprising at least one CD4+ epitope derived from JCV, wherein the protein or peptide comprises VP1 and is present in the form of a virus like particle.

The inventors have surprisingly shown the biological relevance of the CD4+ response in controlling JCV infection and preventing PML. It was previously believed that the main role in controlling JCV was played by CD8+ cells and the cellular immune response. (followed by 5) The inventors identified a number of CD4+ epitopes, in particular from the VP1 protein of JVC which are suitable for being used in therapeutic and prophylactic vaccines. For e, the peptides or proteins for use in these vaccines can comprise one or more of the epitope sequence from one or more of the JCV proteins disclosed herein. The protein or peptide of the invention can, for example, comprise the amino acid sequence of the VP1 protein or a n having at least 70% amino acid identity with the VP1 protein. According to a preferred aspect, the invention refers to vaccination with the VP1 protein or a protein having at least 70% amino acid identity with VP1. The VP1 protein or its variant can be present in the vaccine as a pentamer, i.e. in the form of a JCV capsomer. Preferably, however, the VP1 protein or its variant is present in the form of a virus-like le (VLP). The VLP can consist of VP1 or it may also se other JCV ns, such VP2 and/or VP3. The pharmaceutical kit of the invention may thus comprise, as one component, an antigen, i.e., a protein or e comprising at least one CD4+ epitope derived from JCV comprises VP1 or a protein having at least 70% amino acid identity with VP1.

Protein and peptide are used largely in exchange for each other in the context of this application. Typically, ns are longer than peptides, and, e.g., se more than 100 amino acids, while peptides have between 5 and 100 amino acids.

A CD4+ epitope is a peptide e of being recognized by a CD4+ T cell’s T cell receptor in the context of a MHC class II molecule. The inventors have identified CD4+ T cell epitopes, which are recognized by CD4+ T cells of y controls and patients having PML/PML-IRIS. These peptides are disclosed in Table 1 below (SEQ ID NO: 1-92). Several of these epitopes could not be identified by classical methods using peripheral T cells, but were only identified as reactive with T cells isolated from the brain biopsy of a patient with PML-IRIS. Since this patient showed low to absent JCV viral load in the brain and CSF, the JCV-specific intracerebral CD4+-mediated immune response s to have cleared or almost cleared the viral infection from the brain, and therefore the experiments performed ensure the high biologi- cal relevance of the identified CD4+ T cell epitopes, in particular those identified as recognized by brain-derived T cells. T cell epitopes that have been fied by brain-derived T cells are depicted in SEQ ID NO:l-3, 7-9, ll, 23, 37-38, 43-45, and 69-71 and peptides and proteins comprising these epitopes are particularly preferred for the prophylactic and therapeutic vacci- nation approaches described . In a preferred embodiment, the protein or peptide of the invention comprises at least one CD4+ epitope selected from the group comprising SEQ ID NO:l-3, 7-9, ll, 23, 37-38, 43-45, and 69-71.

VPl is the major capsid protein of JCV, and comprises a high proportion of the identified im- munodominant epitopes. The sequence of wild type VPl is disclosed, e.g., in DE 195 43 553.

VPl can, however, also be mutated, e.g., in positions 55, 269 and others. It has been shown that more than 50% of the VP1 mutations ing in vivo are in those positions. Epitopes corresponding to peptides from mutated VPl proteins may also be employed in the context of the ion. Some of the peptides in Table 1 correspond to mutated VPl fragments. The proteins or peptides comprising a CD4+ epitope of the ion consist of the es of SEQ ID NO:l-92, or they may be longer, e.g., having a length of l2, l3, l4, l5, l6, 17, 18, 19, 20, 21, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or more, 40 or more, 50 or more, 75 or more, 100 or more, 125 or more, 150 or more or 200 or more amino acids.

They may consist of amino acids of wild type or naturally occurring mutated VPl or other ICV proteins or comprise different sequences, such as sequences not originating from the same vi- rus n or not in their natural arrangement. E. g., they may be fusion proteins comprising 2, 3, 4, 5, 6, 7, 8, 9, 10 or more epitopes selected from the group comprising SEQ ID NO: 1-92.

In one embodiment, the protein has at least 70% amino acid identity with VPl. Preferably, it comprises at least one epitope selected from the group comprising SEQ ID NO: 1-92. The n having at least 70% amino acid ty with VPl may be a fiJsion protein fiarther com- prising at least one epitope selected from the group comprising SEQ ID NO: I and SEQ ID NO: 46 — 76. Preferably, the protein comprises at least one of the epitopes depicted in SEQ ID NO:l-3, 7-9, 11, 23, 37-38, 43-45, and 69-71 In one ment, the protein comprising at least one CD4+ epitope derived from JCV is present in the form of a virus like particle. VPl protein or derivatives thereof, e.g., mutants or fragments comprising at least 70% amino acid ce identity to wild type VPl as described in DE 195 43 553, or fiJsion proteins thereof, can assemble into virus like les, which is also described in DE 195 43 553.

Of course, the kit of the invention may also comprise two or more es or proteins com- prising at least one CD4+ epitope derived from JCV selected from the group of SEQ ID NO: 1- 92, preferably, in one composition. Preferably, the kit comprises two or more peptides or pro- teins having CD4+ epitopes from JCV selected from the group of SEQ ID NO: 1-3, 7-9, ll, 23, 37-38, 43-45, and 69-71. As both CD4+ es and CD8+ epitopes appear to be relevant for an effective immune response against JCV, it is particularly advantageous if the e or protein of the invention comprises both at least one CD4+ epitope and one CD8+ epitope.

As demonstrated in the Examples of the present invention, the peptides and proteins of the invention are useful for being stered to a subject who is afflicted with PML to induce or enhance a specific intracerebral CD4+-mediated immune response against JCV. Thus, accord- ing to one aspect of the invention, the protein or peptide comprising the at least one CD4+ epitope derived from JCV is used in a method of treating PML in a subject. The treatment of a subject who already suffers from PML and/or PML-IRIS is referred to herein as therapeutic ation. In a red embodiment, a protein comprising the amino acid sequence of the VP1 n or the amino acid sequence of a protein having at least 70% amino acid identity with VPl is used in the treatment of PML. In another preferred embodiment, the peptide or protein used in the treatment of PML comprises or consists of an epitope selected from the group ofSEQ ID NO:l-3, 7-9, ll, 23, 37-38, 43-45, and 69-71.

When treating subjects who developed a PML, it has been found that treatment based on the administration of the es or proteins of the invention can be further ed by admini- stration of a cytokine capable of expanding and maintaining T cells. It has been found in the course of the invention that the co-administration of such a cytokine provides a stimulus for reconstitution of important immune functions. Several cytokines can be used, e.g., IL-7, IL-2, IL-lS and IL-Zl. The use of IL-7 or derivatives of IL-7 is particularly preferred.

In a still fiarther embodiment, PML treatment by therapeutic vaccination also comprises the administration of an adjuvant. The adjuvant can be any adjuvant which is suitable for being administered to a human subject and results in T cell tion and/or antigen presentation at the site of administration. For example, the adjuvant to be used in the methods and kits of the present invention may be selected from the group of MF59, aluminium hydroxide, m ate gel, lipopolysaccharides, imidazo-quinolines (e.g. imiquimod, S-28463), u- cleotide ces with CpG motifs, stearyl tyrosine, DTP-GDP, DTP-DPP, threonyl-MDP, 7- allyloxoguanosine, glycolipid bay R1005, antigen peptide system, polymerized hap- tenic peptides, bacterial extracts, TLR-7 agonists, TLR—8 agonists, vit-A, and the like. ably, the adjuvant f to be used in practising the invention is a TLR—7 agonist or a TLR—8 agonist. Several TLR—7 agonists are known and commercially available, e.g., from Invivogen, San Diego. Examples are the adenine analog CL264, the guanosine analogue Loxoribine, or, preferably, imidazoquinoline compounds such as Resiquimod, GardiquimodTM or Imiquirnod (4-amino-l-isobutyl-lH-imidazol[4,5-c]chinolin). TLR—8 agonists are known to have similar biological effects as TLR—7 agonists and can thus also, or alternatively, be used. Examples of TLR—8 agonists are stranded RNAs or E.coli RNA. ary TLR-7/8 Ligands are the thiazoloquinoline compound CL075, the imidazoquinoline compound R848, or the water- soluble R848 imidazoquinoline compound CL097, thymidine homopolymer phosphorothioate ODN (Poly(dT).

The preferred adjuvant used in the invention is imiquimod. More than one adjuvant, preferably selected from the group comprising a TLR—7 t and/or TLR—8 agonist, can be used in the context of the invention, and if required, additional means to stimulate an immune se can be employed, e.g., as described below.

According to a preferred ment, therapeutic vaccination ses the administration of VPl (or a protein having at least 70% amino acid identity with VPl) in combination with an adjuvant, such as imiquimod, and a cytokine, such as IL-7. The VPl protein or its variant can be present in this combination as a pentamer, i.e. in the form of a JCV capsomer. Preferably, however, the VP1 protein or its variant is t in the form of a virus-like particle (VLP).

The VLP can consist of VPl or it may also comprise other JCV proteins, such VP2 and/or VP3. It is particularly preferred that a VLP ting of VPl or a protein having at least 70% amino acid identity with VPl is stered in ation with an adjuvant and a cytokine.

Most preferably, the VLP consisting of VPl is administered in combination with imiquimod and IL-7 for therapeutic vaccination.

In one ment, e.g., the kit may fiarther comprise IL-7. IL-7 is preferably used in cases where a subject suffering from PML is treated (i.e. in a therapeutic vaccination) and no suffi- cient immune response is ed without fiarther stimulation, e.g., if the patient is immunode- f1cient. It was shown by the inventors that administration of IL-7 with the other components of the kit was able to induce a protective immune response in an individual with a congenital im- mune defect. IL-7 may also be employed in subjects receiving imrnunosuppressive medication or in patients compromised due to HIV infection.

Preferably, the protein or peptide of the invention comprising at least one CD4+ epitope de- rived from JCV is to be administered subcutaneously. Other modes of administration may also be chosen, e. g., dermal, intramuscular, enous, pulmonary or oral administration.

The adjuvant is preferably to be stered to the subject in a way suitable for inducing an immune response to the protein or e of the invention. For example, the adjuvant may be administered in the same way and at the time of administration of the protein or e of the invention, and both may be in one composition, e.g., contained in one vial. In one embodiment, both the n or peptide comprising the CD4+ epitope and the adjuvant are for subcutane- ous administration. Alternatively, they are administered in a way which allows stimulation of an immune response to the e, e.g., the antigen is administered subcutaneously and the adju- vant is administered topically or ly, in particular, it may be administered at the site of the ion of the antigen, e.g., in a form or a cream or . Ways of dermal application of adjuvants such as imiquimod are known in the state of the art. For example, a cream comprising an effective concentration of the adjuvant may be administered to the skin in the vicinity of the subcutaneous inj ection over an area of about 5cm x 5cm.

The adjuvant and the antigen are preferably to be administered simultaneously, or consecu- tively within a short time span. For example, an imiquimod cream may be dermally adminis- tered directly after subcutaneous injection. The cream may be covered to prevent fithher spreading, and wiped off after about 4-12 hours, e.g., 8 hours.

After the first administration of the protein or peptide comprising at least one of the CD4+ epitopes and the nt, fiarther courses of administration may be carried out for boosting the immune response, e.g., two, three or four courses of administration. The time between courses may be about 1 to about 4 weeks, preferably, about 2 to about 3 weeks, e.g., 10 days.

In one embodiment, the first administration is followed by a booster immunisation after 2 weeks and another after 6 weeks. In an immunodeficient or immunocompromised t, it is advantageous to administrate both antigen and nt for boosting. In a subject who is not immunodeficient or immunocompromised, it is also le to only use adjuvant for the first, or for the first and second immunisation, i.e. to use the antigen only for later immunisations.

Apart from being used in the treatment of subjects which have ped PML, the protein or peptide of the invention can also effectively be used for preventing PML and/or PML-IRIS in a subject who has not yet developed PML, but is at risk of developing this disease. This approach is referred to herein as prophylactic vaccination. The subject to be treated by prophy- lactic vaccination can be a subject who is not yet infected with JVC, which means that prophy- lactic vaccination is used to prevent infection of the subject. Preferably, however, the subject to be treated by lactic vaccination is a subject who is already infected with JCV. Prophy- lactic vaccination does not need to include the administration of an adjuvant. It is red that prophylactic vaccination neither includes the administration of an adjuvant nor the admini- WO 14134 stration of a cytokine such as IL-7. Prophylactic vaccination approaches can, however, also include the administration of these compounds to the respective subject.

According to a red aspect, prophylactic vaccination includes the administration of the VP1 protein or a protein having at least 70% amino acid identity with VPl. The VPl protein or its variant can be present in the prophylactic e as a pentamer, i.e. in the form of a JCV capsomer. Preferably, however, the VP1 protein or its variant is present in the prophylactic vaccine in the form of a virus-like particle (VLP). The VLP can consist of VPl or it may also comprise other JCV proteins, such VP2 and/or VP3.

The t to whom the protein or e of the invention is administered, either in a thera- peutic or prophylactic vaccination regimen, has an inherited or ed immunodeficiency, which means that several aspects of adaptive or innate immune filnction are dysfilnctional or impaired. Said immunodeficiency may result from an inherited dysfilnction such as idiopathic CD4+ lymphopenia or Hyper-IgE-Syndrome, or due to an acquired immunodeficiency result- ing from a disease or pathological ion, such as AIDS, leukemia, lymphoma, multiple myeloma or infection with hepatitis virus B or C. The subject may also be immunocompro- mised as a result from a therapeutic intervention. For e, cancer treatment often involves chemotherapeutic or radiation courses that lead to certain dysfilnctions of the immune system.

Also, immunosuppressive treatments which are commonly used in transplantation medicine and also in the treatment of autoimmune diseases may be responsible for the immunodeficiency of the subject to be treated ing to the invention. ing to a preferred embodiment, the subject to be treated by the peptides or proteins of the invention, either by prophylactic or therapeutic treatment, is undergoing an immunosup- pressive treatment or will undergo an immunosuppressive treatment. This means that once it has been d by the ing ian that a patient is to be treated by the administration of an immunosuppressive agent, it will be le to ster to said patient one or more of the peptides or proteins of the present invention in order to prevent the development of PML and/or PML/IRIS. This is particularly usefill, e.g., for patients which will receive organ trans- plantation. atively, the subject undergoing or being eligible for immunosuppressive treatment can be patients who suffer from an autoimmune disease, preferably an autoimmune disease which is characterized in that T cells play a pathogenetic role or are the target of immunosuppression.

As used herein, autoimmune diseases comprise, for example, acute disseminated encephalo- myelitis , ankylosing spondylitis, antiphospholipid syndrome, autoimmune - myopathy, autoimmune cardiomyopathy, autoimmune hepatitis, autoimmune inner ear disease, autoimmune proliferative syndrome, autoimmune eral neuropathy, autoimmune pancreatitis, autoimmune polyendocrine syndrome, autoimmune progesterone dermatitis, auto- immune thrombocytopenic purpura, autoimmune urticaria, autoimmune uveitis, Behcet's dis- ease, celiac disease, Crohn's disease, dermatomyositis, diabetes mellitus type 1, eosinophilic fasciitis, gastrointestinal pemphigoid, Goodpasture's syndrome, Graves' disease, Guillain—Barré syndrome (GBS), Hashimoto's alopathy, Hashimoto's thyroiditis, Lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious a, polymyositis, primary biliary cirrhosis, psoriasis, psoriatic arthritis, relapsing polychondritis, rheumatoid ar- thritis, Sjogren's syndrome, transverse myelitis, ulcerative colitis, vasculitis, and Wegener's omatosis. Preferably, the autoimmune disease is multiple sis. Among autoimmune diseases other than MS those, in which The components of the kit of the invention may therefore be for administration to a t selected from the group consisting of a subject diagnosed with PML or a subject at risk of de- veloping PML.

Subjects at risk of ping PML are well known in the state of the art. These subjects can be treated prophylactically with the proteins and peptides of the present ion. As outlined above, examples are patients who are deficient or imrnunocompromised, e.g., due to an HIV infection or AIDS, or due to a tumor. Such patients may also have a congenital immu- nodeficiency, such as patients with idiopathic CD4+ lymphopenia or Hyper-IgE-Syndrome.

Alternatively, the immune system may be compromised due to immunosuppressive treatment which is presently taking place or which is planned. Patients may be eligible for immunosup- pressive treatment e.g., if they have an mune disease, e.g., multiple sclerosis, rheuma- toid arthritis, lupus erythematodes, Crohn’s disease or psoriasis, or if they are transplantation patients, i.e. patients having received a transplant or about to receive a transplant. Any patient with acquired or congenital state of reduced ty, in particular of compromised CD4+ T cell numbers and function, are potentially at risk to develop PML and subsequent IS, if the underlying immunocompromise were to be corrected.

In the context of the invention, immunosuppressive treatment may be treatment e.g., with cyc- losporin or FK506 g protein, with cytotoxic drugs (e.g. cyclophosphamide, ntron, busulphan and many others that are in standard use as single drug- or combination therapy in the treatment of hematologic or solid tumors) or with an immunosuppressive or immunomodu- latory antibody, e.g., a monoclonal antibody selected from the group comprising natalizumab, efalizumab, rituxirnab, ocrelizumab and zumab. Immunosuppressive treatment may also be ation or chemotherapy.

The immunosuppressive ent preferably comprises treatment of the subject with one or more immunosuppressive antibodies, more preferably one or more monoclonal antibodies or other biologic, cell therapy or small molecule-based treatments. It has been shown that immu- nosuppressive treatment, e.g., with several major monoclonal antibodies that are in use in can- cer and mune diseases including natalizumab (anti-VLA-4), efalizumab (anti-leukocyte fianction—associated antigen-l) y withdrawn from the market), rituximab (anti-CD20), ocrelizumab CD20), alemtuzumab CD52) or infliximab (anti-tumor necrosis factor (TNF)-alpha) or with the IgGl-TNF or 2 fusion protein etanercept may lead to PML and/or PML-IRIS. This risk may be prevented or reduced by means of the invention. It is an- ticipated that other biologicals or small les, e.g. the sphingosine—phosphate receptor 1 agonist, fingolimod, that compromise certain immune fianctions such as ion of activated immune cells into the CNS g CNS immune surveillance), as is the case for natalizumab, or trap cells in secondary lymphoid organs, as is the case for fingolimod, and others with simi- lar effects may lead to increased risk to develop PML/PML-IRIS. Accordingly, it is preferred that the subject to be treated by the peptides and proteins of the invention, either by lac- tic or eutic treatment, is a subject that is currently treated by one or more monoclonal antibodies selected from the group of natalizumab, efalizumab, rituximab, ocrelizumab and alemtuzumab or another immunosuppressive agent (see above), or a subject for whom such treatment is planned. Preferably, the subject is treated with the antibody natalizumab or a de- rivative thereof In a preferred embodiment, the invention relates to prophylactic or therapeutic vaccination which includes the administration of the VP1 protein or a protein having at least 70% amino acid identity with VPl, for example in the form of a pentamer or in the form of a VLP com- g or consisting of VPl or a VPl variant, to a subject which receives one or more of the above immunosuppressive antibodies, preferably natalizumab.

Treatment of a subject with the kit of the invention may be especially advantageous for a sub- ject which has been diagnosed to be a carrier of JCV. r, there also is a significant risk of subjects being newly infected with JCV e.g. in the course of an immunosuppressive treat- ment. It is therefore also advantageous to immunize ts to JCV by means of the inventive kit if no sis of JCV infection is performed or if a test for JCV has been negative.

The components of the kit or the invention are preferably to be administered to a subject se- lected from the group of: a) immunocompromised or immunodeficient subjects, such as carriers of HIV, subjects having immunosuppressive treatment or congenital immunodeficient patients such as pa- tients with idiopathic CD4+ lymphopenia or Hyper-IgE-Syndrom; b) subjects eligible for immunosuppressive ent.

In one aspect, the present invention is directed to a pharmaceutical kit as described herein for use in treating PML, i.e. in treating a subject diagnosed with PML. As surprisingly shown by the ors, immunity to JCV can be induced by means of the ion, JCV can be elimi- nated fiom the brain and the symptoms ofPML may be healed (reduced or abolished).

The invention is also directed to a pharmaceutical kit as described herein for use in preventing PML and/or PML-IRIS in a subject selected fiom the group of: a) immunocompromised or immunodeficient subjects, such as carriers of HIV, subjects having immunosuppressive treatment or congenital immunodeficient patients such as pa- tients with idiopathic CD4+ lymphopenia or Hyper-IgE-Syndrom; and b) subjects eligible for immunosuppressive treatment.

Immunosuppressive treatment may, e.g., be treatment of a subject sed with an autoim- mune disease or a transplantation patient. The components of the kit may be administered to said patient before, after or during immunosuppressive treatment. In ular if start of the immunosuppressive treatment is not ng, it may be advantageous to start or to boost an immune response to JCV by means of the invention before suppressive treatment is started. However, the inventors have shown that it is also possible to e an immune re- sponse to JCV sufficient to treat PML in an immunocompromised individual. This is a situation similar to subjects oing immunosuppressive treatment.

Under some circumstances, it may be advantageous to reduce or upt immunosuppressive treatment for the first days or weeks (e. g., 2 days, 5 days, 7 days, 14 days or 28 days after the immunisation or the invention, or until clearance of JCV from the brain is shown or the symp- toms of PML healed. This may be d by the medical practitioner depending on the im- mune status of the patient and the risks of reducing or interrupting the immunosuppressive treatment in the patient. atively or additionally, an immune stimulatory treatment such as treatment with IL-7 can be administered to a subject.

The present invention is also directed to a method of treating PML and a method of preventing PML and/or PML-IRIS, wherein a) a protein or peptide sing at least one CD4+ epitope derived from JCV, wherein the epitope is selected from the group comprising SEQ ID NO: 1 - 92, is administered to a patient, b) optionally, an adjuvant selected from the group comprising a TLR-7 agonist and/or TLR—8 agonist is administered to a patient.

The ion is also directed to a protein or peptide comprising at least one CD4+ epitope derived from JCV, wherein the e is selected from the group sing SEQ ID NO: 1 — 92. Said protein or peptide is one component of le the kit of the invention as described herein, and thus suitable for preparing said kit, adding adjuvant.

The present inventors have shown the biological relevance of the CD4+ epitopes disclosed, and have first isolated the peptides consisting of these epitopes. In the context of the invention, the epitopes may be presented in the context of MHC II with or without further sing by the antigen presenting cell, i.e., the term epitope relates to the amino acid sequence as dis- closed in SEQ ID NO: l-92, which was shown by the ors to be able to induce a CD4+ T cell response, and not necessarily to the peptide which may be isolated from MHC II.

A protein or peptide comprising at least one CD4+ epitope derived from JCV, wherein the epitope is selected from the group comprising SEQ ID NO: 1 — 92, may be a fusion protein of VPl or a protein having at least 70% amino acid identity with VPl, further comprising at least one epitope selected from the group comprising SEQ ID NO: 1 and SEQ ID NO: 46 — 76. The protein or peptide of the invention may thus se epitopes from more than one native JCV protein, e. g., from two, three or four ICV proteins.

As described above, the protein or peptide comprising at least one CD4+ e derived from JCV, wherein the epitope is selected from the group comprising SEQ ID NO: 1 — 92 may be employed for preparing the kit of the invention, i.e., for immunisation of a subject to JCV, e.g., for treating or preventing PML or PML-IRIS. Alternatively, the protein or e comprising at least one CD4+ epitope derived from JCV, wherein the epitope is selected from the group comprising SEQ ID NO: 1 — 92 may be used for diagnosing infection with JCV. In particular, it may be used for diagnosing PML, preferably, in context with other methods such as is of symptoms and/or MRI or the brain.

The protein or peptides described herein are highly suitable for use in a method of sing an infection with JCV and/or for diagnosing PML. The present ion thus also s to a method for diagnosing infection with JCV or for diagnosing PML, comprising contacting a (followed by page 14A) sample from a subject with a protein or peptide comprising at least one CD4+ e derived from JCV, wherein the epitope is selected from the group comprising SEQ ID NO: 1 – 92.

In one embodiment, the method for diagnosing infection is d out under conditions suitable for binding of antibodies from the sample to said n or peptide. If g of antibodies to the sample is detected, e.g., by means of an ELISA, the subject is infected with JCV or has been infected with JCV.

Thus in a particular embodiment the present invention provides for the use of a protein or peptide for the manufacture of an agent for in vivo sing infection with JCV and/or for in vivo diagnosing PML, n said protein or peptide comprises at least one CD4+ epitope derived from polyoma virus JC (JCV), wherein the protein or peptide comprises VP1 and is present in the form of a virus like particle, and wherein the method of diagnosing comprises detecting a reaction of CD4+ T cells in a sample to a presence of the epitope.

In a preferred embodiment, the characterisation of the protein or peptide of the invention as comprising CD4+ epitopes is exploited. The method for diagnosing infection may be carried out under conditions suitable for detecting a on of CD4+ T cells in the sample to the presence of the epitope/epitopes. For example, a proliferation assay for T cells, which may e.g., measure incorporation of 3H-Thymidin, incorporation of bromo-2'-deoxyuridine (BrdU) or an assay for expression of tion markers such as CD25 or one or more nes may be used.

An ELISPOT assay may be used, but an ELISA assay, a scintillation assay or extracellular or intracellular FACS can also be suitable. An assay for CD4+ activation by the protein or peptide comprising the disclosed epitope/epitopes may e additional information with regard to a JCV infection or the immune status of the subject when combined with a PCR test and/or a test for the presence of antibodies to JCV in a sample from the patient, or it may be used instead of such a test previously known in the state of the art.

In one ment, CD4+ T cell activation is tested, and the phenotype of the CD4+ T cell is analysed, e.g., Th1, Th2 or Th1/2 phenotype is ined. This can be carried out based on expression of cytokines as known in the state of the art, and/or based on expression of other differentiation markers such as transcription factors. (followed by page 15) In the context of the invention, the sample to be analysed (and transformed by this analysis) ably is a blood sample, a brain tissue sample such as a sample from brain parenchyma or a sample of cerebrospinal fluid (CSF) e.g., from a brain biopsy or a puncture, or derived therefrom. For example, if T cell activity is to be analysed, PBMC or T cells may be isolated from the blood or CSF by methods known in the art. The inventors have however shown that it is preferable to analyse T cell activity of T cells from a brain tissue such as brain parenchyma.

If antibodies are to be ed, serum may be used.

In a preferred ment of the invention, the subject or patient is human. Alternatively, the subject or patient can also be humanized animal such as a humanized mouse susceptible to infection with JCV in an experimental system. As the subject/patient is human, proteins nced in this application, if not specifically mentioned otherwise, are also preferably human or of human . For example, IL-7 should be human IL-7 or a derivative f capable of binding to IL-7 receptor and mediating signalling thereof. It can be recombinant IL-7, e. g., in the form of a fusion protein. TLR are also human TLR.

The inventors, who recognized the ance of CD4+ responses in the immune response to JCV and in clearing the virus from the brain, mapped the genic epitopes of the polyoma virus JC. Immunodominant peptides fiom three open reading frames of JCV were identified. The peptides of a set of overlapping peptides spanning all open reading frames of JCV including important variants within the major capsid n were tested. The CD4+ T cell epitopes identified may be used for diagnostic examinations of JCV infectious status, but may also be used to vaccinate patients/controls, e.g. subjects who have weak immune response against JCV (constitutively or due to disease (e.g. AIDS, constitutive immunodeficiencies such as idiopathic CD4+ lymphopenia) or treatment (cancer y, monoclonal antibody y, e.g. natalizumab in MS, but others as well) and are at risk to develop or have already devel- oped PML.

The inventors have mapped the fine specificity of CD4+ T cell epitopes for all JCV proteins. T cell cultures and T cell clones from a brain biopsy of a patient suffering from PML and PML- immune reconstitution inflammatory syndrome RIS) were examined. Since this immune response is protective in the sense that it targets the most important epitopes of the virus and leads to its elimination and containing the ion, the mapping data from testing peripheral blood cytes of healthy controls and multiple sclerosis (MS) patients, but particularly the data from characterizing the antigen fine specificity of infiltrating T cells during PML- IRIS supports biological relevance and usefiJlness for diagnostic and vaccination/therapeutic purposes.

In the course of the invention, furthermore, an individual healing attempt was performed in a patient with idiopathic CD4+ lymphopenia, a rare constitutive deficiency, who devel- oped PML at the age of 64 years. In this patient, the inventors tested the circumstances if vac- cination with the entire major capsid protein VPl can under certain conditions boost the insuf- ficient immune response against JCV to the point that JCV can be eliminated from the brain.

The inventors vaccinated the patient by subcutaneous injection of recombinant VPl protein combined with a dermally applied TLR7 agonist (imiquimod, Aldara) and recombinant i.v. IL-7 (Cytheris). The patient not only showed an in vitro proliferative response against JCV VPl after only two vaccinations, but also reduced the serum JCV viral load to 0, began to show slight contrast enhancement by brain MRI imaging and ly elevated CSF cell counts, and finally is clinically improving, which all support that the vaccination worked in vivo.

The following examples are meant to illustrate the invention, but not to limit its scope. All pub- lications cited herein are th fially incorporated for all purposes.

Legends Fig. 1: a) FHA-expanded bulk mononuclear cell populations from the brain biopsy (left panel), CSF (second panel from left) and PBMC (third panel from left) as well as unmanipulated PBMC (right panel) were tested t JCV VPl/VLP protein and tetanus toxoid protein (TTX). Results show the mean SI i SEM. Note the different scales for the y-axis. b) EX-vivo quantification of Thl-, Th2-, Thl7- and Thl-2 cells in PHA-expanded CD4+ T cells from brain biopsy (left ), CSF (second panels from left), PBMCs (third panels from left) and in unmanipulated PBMCs (right panels). Num- bers represent the percentage of positive cells. Thl were identified as CD4+ IFN—gamma+ IL-17A' /IL- 4'; Thl7 cells as CD4+ IL-l7A+ IFN—gamma'; Th2 as CD4+ IL-4+ IFN—gamma', and Thl-2 as CD4+ IFN—gamma+ IL-4+.

Fig. 2: a) Proliferative response of brain-derived PHA-expanded cells against 204 pping lS-mer peptides spanning all open reading frames of JCV (covering Agno, VPl, VP2, VP3, Large-T, and T proteins) and organized in 41 pools of 5 peptides each. Results show the mean SI i SEM.

The different patterns of the bars pond to the different open reading frames. Schematic repre- sentation of the 5 open reading frames in the JCV genome (upper right hand figure). b) Proliferative response of brain-derived bulk mononuclear cell populations against individual JCV peptides. s show the mean SI i SEM. Note the different scales of the y-axes in panels a and b. c) Precursor fre- quency of T cells specific of the 5 JCV peptides inducing the est proliferative ses in PHA- expanded cells from brain biopsy. (1) tage of CD8+ T cells that bind HLA-A*02:Ol-VP136 tetramers (middle graph) and HLA-A*02:Ol-VP1100 tetramers (lower graph).

Fig. 3: a) ut representing the frequency of each individual TCC in the brain biopsy. b) Proliferative response of TCC against 64 individual VPl peptides. Results show the mean SI i SEM. 0) Schematic representation of the immunodominant peptides identified for the brain-derived bulk population (upper graph, show the mean SI i SEM) and for the different TCC (lower graph the dif- ferent ns correspond to the different TCC and each single cell growing culture is represented by a square).

Fig. 4: a) Representative flow try analysis of intracellular IFN—gamma and IL-4 produc- tion by a Thl-2 (upper plot) and a Thl (lower plot) VPl/VLP-specific CD4+ T cell clone. b) The dot- plot represents the percentage of VPl-specific, brain-derived single cell cultures with Thl-2 and Thl phenotype by intracellular cytokine staining. Each dot ponds to one of the 21 single cell cultures analyzed. The doughnut represents the filnctional phenotype of each TCC. c) ELISA detection of IFN—gamma and IL-4 production in culture supematants of Thl-2 TCC (n=5, black bars) and Thl TCC (n=6, white bars) 72 h after stimulation with FHA. Results show the mean i SEM. d) RT-PCR analysis for transcription factors Gata3 and t-bet of Thl-2 TCC (n=5, black bars) and Thl TCC (n=6, white bars). Values are relative expression compared to brain-derived PHA-expanded cells (calibrator =1). Results show the mean 1- SEM.

Fig. 5 Treatment scheme for immunisation of an immunocompromised subject with VP1 M=month, D=day, W=week, MRI= Magnetic Resonance Imaging. Adjuvant imod) was administered directly after administration ofVP 1.

Fig. 6 pment of VP1 immune response during treatment VLPl= virus like particlel composed of VP1 protein, TT= Tetanus toxoid Fig. 7 Course of ent Fig. 7A shows viral load and mean SI after VPl stimula- tion, and Fig. 7B shows leukocyte counts in the CSF. Time points of stration of IL-7 and VP1 are identical and correspond to the scheme shown in Fig. 5.

Fig. 8 Characterization of VP1-specific T-cells Only CD4+ cells proliferate after VP1 stimulus (6 weeks after immunization) Fig. 9 Proliferating CD4 cells are activated memory cells Fig. 10 CD4+ T cells activated by VP1 express a high background of IL-4 and produce IFN-gamma after VP1 stimulation. CD4+ T cells were stimulated with antigen on day 0. On day 6, they were restimulated with antigen, 1 hour later, secretion was blocked with din A and after 15 h, an intracellular cytokine staining was med and analysed by FACS.

Fig. 11 3H-thymidine oration assay At the time points indicated, peripheral blood mononuclear cells (PBMC) were obtained from the patients and freshly seeded (lxlOeS cells/well) with antigen (VP1/VLP). After 7 days of incubation, 3H-thymidine incorporation was measured. A stimulation index of >2 is considered a positive se. 81s are shown on the y-axis of the graph.

Example 1 — Identification of immunodominant CD4+ epitopes Material and Methods Patients HLA-class II types: DRBl*l3:Ol, l; DRB3*02:02; DRBS*02:O2; l:02, -*Ol:03; DQBl*05:02, -*06:03 (patient 1); and l:03, -*15:Ol; DRB3*02:02; DRBS*Ol:Ol; DQAl*Ol:02, -*05:XX (X indicating not typed to the exact subtype); DQBl*03:Ol, -*06:02 (patient Neuropathology Small tissue fragments of a total volume of approximately 0,1 ml, were obtained by open biopsy. Fol- lowing fixation in buffered formalin for 2 hours, tissue was embedded in paraffin. Microtome sections of 4 um were stained with hematoxiline-eosin (H&E), van Gieson's ome, PAS, Turnbull's stain for n and Luxol. Immunohistochemical staining was performed on an automated Ventana HX IHC system, benchmark (Ventana-Roche Medical systems, Tucson, AZ, USA) following the manufac- turer's instructions using the following antibodies: anti-CD45 / LCA (DAKO, Glostrup, Denmark; M701), anti-CD3 (DAKO; M1580), anti-CD45R0 (DAKO; M 0742), anti-CD20 (DAKO; M0755), anti-CD79a (DAKO; M7050), anti-CD68 (Immunotech / Beckmann-Coulter, Krefeld, Germany, 2164), anti-HLA-DR (DAKO; M775), anti-NF (Zymed / Invitrogen, Darmstadt, Germany, 80742971), anti-GFAP (DAKO; Z334) and 53 (DAKO; .

Brain Tissue Processing and Expansion of Brain-Derived, CSF-Derived and Peripheral Blood Mononuclear Cells A biopsy of approximately 0.033 ml was cut into small pieces and disrupted by incubation in a solution containing 1 mg/ml Collagenase A (Roche Diagnostics, Penzberg, Germany) and 0.1 mg/ml DNAse I (Roche) at 37°C in a water bath for 45 min. The resulting cell suspension was washed three times, and brain-derived mononuclear cells were separated using a Percoll density gradient centrifiagation (GE Healthcare, Munich, Germany). Cells were resuspended in a 30% Percoll solution and carfiJlly underlayered with a 78% Percoll solution. After centrifiagation brain-derived mononuclear cells were gathered from the interface of the gradient.

CSF-derived mononuclear cells were obtained directly from a diagnostic spinal tap, and peripheral blood clear cells were separated by Ficoll density gradient centrifiagation (PAA, Pasching, Austria).

, CSF- and peripheral blood-derived mononuclear cells were expanded in 96-well U-botton mi- crotiter plates by seeding 2000 cells per well together with 2 x 105 non-autologous, irradiated PBMC (3,000 rad) and 1 ug/ml of PHA-L (Sigma, St Louis, MO). Medium consisted of RPMI (PAA) con- taining 100 U/ml penicillin/streptomycin (PAA), 50 ug/ml gentamicin (BioWhittaker, Cambrex), 2 mM L-glutamine (GIBCO, Invitrogen) and 5% ecomplemented human serum (PAA). After 24 h, 20 U/ml of human recombinant IL-2 2, Tecin, Roche Diagnostics) were added and additional hrIL2 was added every 3-4 days. After two weeks cells were pooled and analyzed, eserved or ulated again with 1 ug/ml PHA, 20 U/ml hrIL-2 and allogeneic irradiated PBMC.

Flow Cytometry Analysis of Brain-Derived Mononuclear Cells Brain-derived clear cells ly from brain digestion were stained with the ing antibod- ies for surface markers: CD45 (AmCyan, 2D1, BD ngen, San Diego, USA), CD56 (Alexa 488, B159, BD Pharmingen), CD3 (PeCy7, UCHTl, ience, San Diego, USA), CD4 (APC, RPA-T4, eBioscience), CD8 (PB, DK25, Dako, Glostrup, Denmark), CD45RO (FITC, UCHLl, eBioscience), CD19 (FITC, HIBl9, BD Pharmingen), CD38 (APC, HIT2, BD Pharmingen), and CD27 (APC-Alexa 750, CLB-27/l, Invitrogen). Analysis was performed on a3 LSRII (BD Biosciences, Heidelberg, Germany) flow cytometer.

Proteins and Peptides For the identification of JCV-specific T cells, 204 (13-16 mer) peptides covering the entire JC viral proteome were applied. Peptides were synthesized and provided by pe (peptides and elephants GmbH, Potsdam, Germany). These 204 peptides overlap by 5 amino acids and e 35 common single amino acid ons. To account for amino acid variations, that occur among the ent ICV geno- types and strains, amino acid sequences of each JCV encoded protein including Agno, VPl, VP2, VP3, Large T antigen and small t antigen from all 479 JCV genomic sequences available in GenBank (by March 2008) were aligned and those polymorphisms, which were prevalent in more than 1% of the all retrieved sequences, were defined as common mutations.

In order to determine which individual peptides are recognized by CNS-derived T cells, a two- dimensional seeding scheme was applied. Peptides were arranged in a set of 82 pools, where each pool contains 5 ent es. By the combination of different peptides in each well according to a rec- tangular matrix and each individual peptide appearing in exactly two pools, in which the residual pep- tides differ, immunogenic candidate peptides could be identified at the intersections of the positive pools.

JCV VPl protein forms virus-like (VLP) particles, and VPl and VLP are ore used as inter- changeable terms. VPl protein forming VLP LP) was generated by the Life e Inkuba- tor, Bonn, Germany, as previously described (Goldmann et al., 1999). 20 mer myelin peptides with an overlap of 10 amino acids and covering MBP (16 peptides), MOG (25 peptides) and PLP (27 peptides ) were synthesized and provided by PEPScreen, Custom Peptide Libraries, SIGMA. Tetanus toxoid (TTX) (Novartis Behring, Marburg, y) was used as positive control.

Proliferative Assays Recognition of JCV Peptides, VPl/VLP and TTX was tested by seeding ates in 96-well U- botton microtiter plates 2-2.5 x 104 brain-derived, rived or eral blood-derived PHA- expanded cells per well and l x 105 autologous ated PBMC with or without peptides for 72 hours. Unmanipulated PBMC were tested at 2 x 105 cells/well in a 7-day primary proliferation. In addtion to TTX, PHA-L stimulation was added as positive control. All ICV peptides were either tested in pools or as individual peptides at a final concentration of 2 uM per peptide for peptides in pools and at a concentration of 10 uM for individual es. VPl/VLP was tested at 2 ug/ml, Tetanus toxoid (TTX) at 5 ug/ml and PHA at l ug/ml. Proliferation was measured by midine (Hartmann Ana- lytic, Braunschweig, Germany) incorporation in a scintillation beta counter (Wallac 1450, PerkinEl- mer, Rodgau-Jurgesheim, Germany). The stimulatory index (SI) was calculated as SI = Mean cpm (counts per minute)(peptide) / Mean cpm (background). Responses were considered as positive when SI > 3, cpm > 1000 and at least three standard deviations (SD) above average background cpm.

Myelin peptides were tested as individual peptides at 5 uM as described above. tion of Brain-Derived VPl/VLP-Specific T Cell Clones 2.5 X 104 brain-derived PHA-expanded cells were seeded in 96-well U-botton microtiter plates with 1 x 105 autologous irradiated PBMC with or without VPl/VLP protein. After 48 hours of culture, plates were split into mother and daughter plates. Proliferation was measured in daughter plates by methyl-3H-thymidine incorporation. VPl/VLP-responsive cultures were identified in mother , and IL-2 was added every 3-4 days until day 12. T cell clones (TCC) were established from positive cultures by seeding cells from VPl/VLP-responsive wells under ng on conditions at 0.3 and 1 cell/well in 96-well U-botton microtiter plates, and on of 2 x 105 allogeneic, ated PBMC and 1 ug/ml of PHA-L in complete RPMI. After 24 h, 20 U/ml of human recombinant IL-2 were added. P specificity was then confirmed seeding 2.5 X 104 cells from growing colonies with autologous irradiated PBMC with or without VPl/VLP protein for 72 h. Specific cultures were res- tirnulated every two weeks with 1 ug/ml PHA-L, 20 U/ml hrIL-2 and allogeneic irradiated PBMC, and hrIL2 was added every 3-4 days.

TCR Analysis TCR VB chain expression was ed in panded cells and T cell clones by 22 anti TCRBV monoclonal antibodies (Immunotech, Marseille, , (Muraro et al., 2000)) in combination with CD4 (APC, eBioscience) and CD8 (PB, PB, DakoCytomation, Denmark).

Determination of Precursors Frequency in CNS-Derived Mononuclear Cells Frequencies of VPl/VLP-specific cells were determined by ng dilution. 20, 200, 2.000 or 20.000 brain-derived PHA-expanded cells were seeded in quadruplicates in 96-well U-botton microtiter plates with 1 x 105 autologous ated PBMC with or without VPl/VLP protein. After 72 hours, prolif- eration was measured by methyl-3H-thymidine incorporation. Frequencies were ated as previ- ously described (Taswell, 1981). Observed data were: r, the number of negatively responding cultures or wells of each dose i; n, the total number of wells per dose i, and )L, the number of cells in the dose i.

Calculated data was: pi = ri / ni, the on of negatively responding cultures of each dose i. The fre- quency was calculated using the following formula: f = - (ln pi) / hi.

Cytokine Production For ellular cytokine staining, PHA-expanded cells and TCC were analyzed 12 days after last restirnulation. Cells were stimulated with PMA (50 ng/ml, Sigma) and ionomycin (l ug/ml, Sigma) in the presence of Brefeldin A (10 ug/ml, eBioscience) for 5 h. Next, cells were stained with LIVE/DEAD® Fixable Dead Cell Stain Kit (AmCyan, Molecular Probes, Invitrogen), fixed and per- meabilized with the corresponding buffers (eBioscience), and stained for CD3 (PE, DakoCytomation, Denmark), CD8 (PB, DakoCytomation, Denmark), IFNgamma (FITC, BDPharmingen), IL-4 (PE- Cy7, eBioscience) and IL-l7A (Alexa -647, ience) at room temperature. IFN—gamma-, IL and IL-2 levels were also determined by ELISA following the manufacturer’s protocol (Bio- source, Camarillo, California) in e supematants of PHA-expanded cells and in TCC 72 hours after stimulation with PHA or VPl/VLP.

Quantification of mRNA Expression Levels by RT-PCR For mRNA gene expression assays, the primer and probe sets (Tbet, Hs00203436_ml and Gata3, Hs00231122_ml) were sed from Applied tems (Foster City, CA). 18S rRNA was used as endogenous control, and the relative gene expression was calculated by the AACt method using brain-derived PHA-expanded cells as calibrator.

ELISA for VPl/VLP-Specific Antibodies The titer of VPl/VPL-specific immunoglobulin G antibodies in CSF and sera from both IRIS-PML ts was determined as described previously (Weber et al., 1997). Briefly, ELISA plates were coated with 100 ml VPl-VLP (1 mg/ml) and incubated with serial dilutions of CSF or sera. Human IgG was ed by a biotin conjugated anti-human Fc antibody (eBioscience) and detected by an avidin horseradish peroxidase cience). Antibody titers in CSF as well as serum were adjusted to the total amount of IgG in the particular compartment. Results were expressed as arbitrary units, which were standardized using always the same human serum as standard.

HLA-A*0201/JCV VP136 and VP1100 tetramers and tetramer staining HLA-A*02:01 eric complexes were synthesized as previously described. Briefly 02:Ol, B2 microglubluin and epitope peptide were refolded and ed using size on chromatography.

Site-specific biotinylation was achieved through on of the BirA target sequence to the last C al extracellular domain of the HLA-A*0201 molecule. Tetrameric complexes were generated using Extravidin-PE (Sigma). PHA-expanded brain-infiltrating cells were stimulated with anti- CD2/CD3/CD28 MACs beads (Miltenyi Biotec, Auburn, CA) and at day 5 after stimulation cells were washed and resuspended to a concentration of 5 x 106 cells/ml. 100 ul were stained with 3 ul of PE- coupled tetrameric HLA-A*02:Ol/JCV VP136 or HLA-A*02:01/JCV VPl 100. After 30 min incubation at 37 °C the cells were washed and stained with anti-CD3 (PB, eBiolegend, San Diego, CA) and anti- CD8 (FITC, Dako) for additional 30 min on ice. Then cells were washed and fixed with 0.5% para- formaldehyde before analysis by flow cytometry.

Results Two Cases of Natalizumab-Associated PML-IRIS Two male patients of 41 and 43 years with relapsing-remitting MS (RR-MS) presented July 2009 and January 2010 respectively with al signs (visual field defect in patient 1; monoparesis in patient 2) and imaging findings suspicious of PML after 28- and 40 months respectively of natalizumab treat- ment. Natalizumab was stopped immediately, and several rounds of plasmapheresis performed. Both patients subsequently developed PML-IRIS with patchy or band-like areas of st ement on MRI (Fig. la) and worsened clinical s of complete loss of vision in patient 1, and hemiple- gia, hemianopia and neuropsychological deterioration in patient 2. With respect to diagnostic workup, patient 1 was immediatly diagnosed as PML based on CSF JCV viral load, although it was low. Diag- nosis in patient 2 was more complicated with repeatedly negative PCR s for CSF JCV viral load until the third testing was positive just above old levels; 12 copies; threshold 10 copies in the NIH reference laboratory). In contrast to the low or borderline JCV CSF viral loads, antibody testing for JCV major capsid protein (VPl/VLP)-specific antibodies in serum and CSF, which was established during this study, revealed strong intrathecal antibody response with 95 - ld higher VPl/VLP- specific dy titers in the CSF ed to serum after adjusting total IgG concentrations to the same levels. Hence, different from the PCR testing for viral DNA, the intrathecal antibody response left no doubt of CNS infection by JCV at the time of PML-IRIS. The is of the IgG subclasses in patient 2 demonstrated that intrathecal antibodies are mainly IgG1 and IgG3. These data indicate a strong JCV-specific l immune response that is confined to the CNS compartment and directed primarily against the major structural JCV protein VPl/VLP. Whether minor components of the antibody response target other JCV proteins remains to be studied.

Due to the above difficulties to diagnose PML, patient 2 underwent a diagnostic brain biopsy to con- firm or refute PML. Neuropathological examination failed to show the typical signs of PML, i.e. nu- clear inclusions in hyperchromatic oligodendrocytes and bizarre ytes, but rather a paucity of CNS cells and massive perivascular and parenchymal lymphomononuclear infiltrates, reactive s with stellate astrocytes and predominance of diffilse and destructive parenchymal infiltrates of foamy macrophages. The majority of cells stained ve for HLA-DR, which is usually exclusively found on activated microglia and absent in normal brain tissue. T cells and B cells were present in the infil- trate, and a high proportion of the latter d positive for the plasma cell marker CD138. Part of the biopsy tissue was processed, and CNS-derived mononuclear cells were also characterized by flow cytometry. 96.5 % of cells expressed the pan hematopoietic cell marker CD45 (not shown) and among them 42.4 % expressed the pan T cell marker CD3+. Of these 241 % were CD8+ and 70.4 % CD4+ T WO 14134 cells. Almost all of these cells expressed the memory marker CD45RO. 29% of CD45+ CNS- infiltrating cells sed the B cell marker CD19, and among these 86.1% were ve for CD27/CD38, i.e. they were memory B cells/plasma cells. Accordingly, a diagnosis of inflammatory demyelinating e rather than PML was made. Subsequent immunohistochemistry for JCV was negative, but sparse nuclear signals for JCV DNA were found by the second attempt of in situ hybridi- zation (data not shown), which together with the low JCV viral load and strong intrathecal antibody response confirmed the initial suspicion of PML and pointed at IRIS rather than the underlying demye- ng disease as responsible for the neuropathological findings.

Antigen city of Brain-Infiltrating T Cells Next the antigen specificity and frequency of brain-infiltrating T cells were characterized. Brain- derived mononuclear cells were first ed as bulk populations by an unbiased stimulus (PHA).

While our culture conditions favored the expansion of CD4+ over CD8+ T cells the relative i- tion of CD4+ T cells remained stable as demonstrated by staining with monoclonal antibodies t T cell receptor (TCR) le chains VBl-VB22. Due to the almost threefold excess of memory CD4+ over CD8+ T cells at the time of brain biopsy, we focused our attention on CD4+ cells and assessed their specificity for JCV. For this purpose, expanded brain T cells were tested against recombinant JCV capsid protein VPl/VLP and against tetanus toxin protein (TTx) in proliferative assays. We di- rectly compared brain-derived versus CSF- or peripheral blood-derived T cells as well as versus unmanipulated peripheral blood mononuclear cells. As shown in Fig. 1a, derived T cells responded with a stimulation index (SI) > 600 against VPl/VLP protein with no response against TTx. SIs against VPl/VLP and TTx in the CSF were 7 and 14 respectively, and in panded PBMC the responses to VPl/VLP and TTx were ve and moderately positive (SI of 6.5) respectively. Un- manipulated PBMC showed a significantly stronger response to TTx compared to VPl/VLP in a 7 days primary proliferation assay.

Functional Phenotype of Brain-Infiltrating CD4+ T cells The inventors then examined if intracerebral CD4+ T cells belonged to one of the major T helper (Th) subtypes, Thl, Th2 or Thl7 cells, based on their cytokine secretion pattern. Expanded bulk T cell populations from the brain, CSF and PBMC as well as unmanipulated PBMC were examined by intra- cellular cytokine staining against IFN-gamma, IL-4, and IL-17, the signature cytokines of Th1-, Th2- and Thl7 cells. ILproducing cells were hardly able (Fig. lb), while IFN-gamma-secreting cells made up between 46.1 and 53.2% in cells from the brain and CSF (Fig 1b). When combining intracellular staining for mma and IL-4, the situation was remarkably different. In the brain- and CSF-derived population, Th1-, Th2- and bifilnctional Th1-2 cells (secreting both IL-4 and IFN- gamma) were similar in frequency (Fig. 1b), while Th2 cells predominated in the peripheral blood- derived, PHA-expanded cells (Fig. lb). 32.7% of brain-derived CD4+ T cells had a tional Th1-2 phenotype.

Fine Specificity and Frequency of Brain-Infiltrating T Cells To ine which specific JCV peptides are recognized by infiltrating T cells, 204 15-mer peptides spanning all JCV proteins (Agno, VPl, VP2, VP3, Large-T, and small-T) were synthesized and arranged in a set of 82 pools, where each peptide appears twice, but in two ent pools (see s). Brain-derived T cells responded to multiple pools (Fig. 2a; pools 1-41). The inventors iden- tified 15 immunogenic candidate peptides that were then tested individually and lead to the identifica- tion of 11 stimulatory peptides (peptides with SI>10) (Fig 4b). The se was directed against pep- tides 4 (Agnozs, the number s the first amino acid (aa) of the 15-mer peptide), 20 (VP134), 23 (VP154), 27-29 (VP174)(all VP174 peptides; 28 and 29 are variants of e 27 with single aa mutations ), 72 0), 73 9), 76 (VP1335), 191 (LTAg66g), and 195 (STAggz) (peptides with SI > in bold). Thus, brain-derived T cells responded to l JCV proteins (Agno, VPl, LTAg, sTAg), however, by far the strongest against VPl (6 peptides). That VPl is the prime target is supported by an even stronger response against entire VPl/VLP protein (Fig. 1a) and by the higher pre- cursor ncies of VPl-specific T cells (between l/294 and l/7l4 T cells responding to peptides VP134, VP1319 and VP174) when compared to cells responding to Agnozs (1/ 14492) and LTAgGGg (1/ 1449) (Fig. 2c). When the inventors examined PHA-expanded CSF- and peripheral blood-derived T cells, CSF only mounted weak responses against pool 39 and peptide LTAg668 contained in this pool, and PBMC were negative. Remarkably, peptide VP134, the e that elicits the strongest re- sponse with respect to SI (Fig. 2b) and precursor frequency (Fig. 2c), contains the JCV epitope VP136, one of the two epitopes together with VP1100 that are recognized by HLA-class I-restricted CD8+ T cells in the context of HLA-A*02:01 (Du Pasquier et al., 2003b). PML-IRIS patient 2 is HLA-A*02:01+ lass I and —class II types under methods), for this reason the inventors deter- mined the frequency of CD8+ T cells specific of these two HLA-A*02:01 JCV epitopes in the PHA- expanded brain-infiltrating CD8+ T cells by tetramer staining. 0.8% of PHA-expanded brain- infiltrating CD8+ T were specific for VP136 and 0.6% for VP1100 (Fig.4d). With respect to HLA-class II, patient 2 expresses the MS-associated HLA-DR haplotype DRBl*15:01 and DRB5*01:01. The JCV-specific CD4+ T cell response was largely restricted by DRBl *15:01/B5*01 :01, when VPl/VLP protein was presented by APCs from a DRBl*l5:01/B5*01:01 homozygous donor (not shown).

These data demonstrate that, similar to intrathecal antibodies, the CD4+ T cell response is mainly di- rected against the major structural JCV protein VPl and that peptide VP134 contains an e for both virus-specific CD4+ and CD8+ T cells. Such a focus of CD4+ and CD8+ T cells on the same im- munodominant epitope has previously been shown for an influenza nucleoprotein e (Carreno et al., 1992).

Table 1 shows the immunodominant CD4+ T cell epitopes identified by the inventors.

Since PML is characterized by endrocyte lysis and release of myelin and since the patient suffers from MS, it was of interest to examine if brain-derived T cells responded to myelin proteins. PHA- expanded derived T cells were tested against pping peptides spanning the major myelin proteins, myelin basic protein (MBP), proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein (MOG), but none of the myelin peptides was recognized despite a strong response against JCV VPl/VLP protein.

Table 1: List of immunodominant JCV epitopes. The amino acid sequence and length of each JCV peptide is shown. The position of the respective peptide is related to the respective protein of the reference JCV genome 699. Peptides with amino acid mutations are designated as variants (V1, V2 and so forth). Peptides recognized by T cell clones isolated from brain only are marked by bold print. Other peptides were recognized by eral T cell clones.

Len-th SEQ ID No I 2 3 4 5 6 VPI (74-88) VI 15 7 VPI (74-88) V2 15 8 VPI (74-88) V3 15 9 10 II 12 13 14 15 12 16 12 17 12 18 19 20 21 22 23 VPl (151-165) v1 ALELQGVLFNYRTKY 15 24 VPl 65) v2 ALELQGVLFNYRTTY 15 25 VPl 75) V1 YRTKYPDGTIFPKNA 15 26 _——15 27 15 28 16 29 30 16 31 32 33 34 35 36 37 38 39 E_——15 40 41 42 43 14 44 45 46 14 47 E_——15 48 14 49 13 50 51 52 53 m——1254 14 55 56 14 57 58 14 59 60 14 61 62 63 64 14 65 16 66 67 68 14 69 StA (82-96) V1 15 70 2012/064445 StA (82-96) V2 15 71 72 14 73 74 14 75 76 77 78 79 80 81 82 83 84 85 VPl S61L (54-68) HLRGFSKLISISDTF 15 86 VPl K60E (54-68) HLRGFSESISISDTF 15 87 88 89 90 91 92 Fine specificity and functional phenotype of JCV-Specific CD4+ T Cell Clones Prior data had shown that CD4+ differentiate into certain T helper phenotypes such as Thl cells (IFN— gamma producers), Thl7 cells ing IL-l7, Th2 cells expressing the signature cytokine IL-4, or T tory cells based on the expression of certain transcription factors (Zhu et al., 2010). The differ- entiation into Thl 0r Th2 cells is considered mutually exclusive and controlled by the transcription factors T-bet (Thl) and Gata-3 (Th2) (Zhu et al., 2010). Based on these data, our finding of commit- ted memory cells with a bifunctional (Thl-2) phenotype was highly unexpected, and we ore es- tablished VPl/VLP-specific T cell clones to examine this point at the clonal level. VPl/VLP-specific TCC were ted as described in material and methods. Initially 21 VPl/VLP-specific single cell- derived cultures were generated by ng dilution and characterized for TCRV beta expression, fimctional phenotype and fine specificity (Table 2). This characterization allowed the identification of 11 presumed different TCC. The number of single cell g cultures corresponding to each TCC gives an idea about the frequency of each TCC in the brain infiltrate (Fig. 3a). TCC-4 was most abun- dant and represented by 5 colonies emerging from single g wells, followed by TCC-2 with 3 single cell growing cultures, TCC-l, -8, -9 and -10 with 2 single cell growing cultures and finally TCC-3, -5, -6, -7 and -ll with only 1 single cell growing culture. The fine specificity of the ll TCC is summarized in Figure 5b. Each TCC was tested against 64 lS-mer peptides spanning VPl protein and lead to the identification of the following stimulatory peptides: VPl34 (recognized by TCC-l and -2), VP154 (recognized by , VPl74 (all VPl74 peptides, recognized by TCC-4), VP191 nized by TCC-5), VP1143 (recognized by , VPlzzg nized by TCC-7), VP1319 (recognized by TCC-8 and -9) and VPl335 (recognized by TCC-10 and -l 1) Taking into account both the number of different TCC recognizing a specific peptide and the frequency of each TCC in the brain infiltrate the immunodominant peptides recognized by VPl specific brain infiltrating TCC were VPl34, VPl74, Vle and VPl335 confirming the fine specificity obtained using the brain-derived bulk cell population (Fig. 3c). Intracellular cytokine ng of these TCC ed Thl-2 and Thl phenotypes (Fig. 4a). 5 TCC representing the 57% of the brain-derived VPl specific single cell growing cultures showed a Thl and 6 TCC representing the 43% of the brain-derived VPl-specific single cell growing cultures a Thl phenotype (Fig. 4b). Specificity and filnctional phenotype did not correlate. For three of the immunodominant peptides (VPl34, Vle and VPl335) we found TCC with both phenotypes. The T helper phenotype of Thl-2 and Thl TCC was confirmed my measuring IL-4 and IFN—gamma protein secretion by ELISA and by ining the expression of mRNAs of the transcription factors Gata3 and T-bet. Thl-2 TCC (n=5) secreted IL-4 in addition to IFN—gamma, while IL-4 secretion was barely detectable in Thl TCC (n=6) (Figure 6c). Cytokine secretion profiles were not due to stimulation with PHA, since the same stable patterns were observed after specific stimulation with VPl/VLP protein by intracellular cytokine staining or ELISA measurements from culture supematants. Transcription factor expression confirmed the phenotype of the TCC. Thl-2 TCC (n=5) expressed mRNA for Gata3 and T-bet, while Thl TCC (n=6) only sed t-bet (Figure 6d). sion The Viral etiology of PML has been shown almost 40 years ago, but still relatively little is known about the immune mechanisms that control JCV infection. CD8+ JCV-specific xic T cells have been related to recovery from PML (Du Pasquier et al., 2004a; Koralnik et al., 2002), and two viral epitopes have been identified in HLA-A*02:01-positive individuals (Du er et al., 2004a; Du Pasquier et al., 2004b). In contrast, limited information is available on the fine specificity and charac- teristics of JCV-specific CD4+ T cells in PML and even less in PML-IRIS (Jilek et al.). The virus- specific T cell se at the site of infection, i.e. the CNS parenchyma, has not been examined at all.

The inventors’ data provide novel insights into this subject and lead them to propose the following enetic events during PML-IRIS under natalizumab treatment. The anti-VLA-4 antibody inhibits immune surveillance of JCV ion at immunoprivileged sites such as the brain by blocking cell mi- gration (Stuve et al., 2006) and local antigen presentation in the CNS (del Pilar Martin et al., 2008).

As a result, pathologic neurotropic JCV variants may lead to PML in a small number (l/500-l/1000) of treated MS ts for s that are not yet understood (Major, 2010; Ransohoff, 2005). As soon as PML is suspected and natalizumab is stopped or actively removed by plasmapheresis, fully fianctional and activated T cells regain access to the CNS compartment, initiate the strong inflamma- tion that is l for PML-IRIS and ively eliminate Virus-infected cells by a number of mecha- nisms including CD4+ and CD8+ T cells and antibody-forming plasma cells.

Table 2 Characterization of VP1 specific brain infiltrating T cell clones (TCC) TCC # Well # Th Phenotype TCR Vbeta Fine icity TCC—1 17A Th0 V82 VP134 1 8A Th0 V82 VP134 TCC—2 16A Th1 V82 VP134 28A Th1 V82 VP134 18B Th1 vsz VP184 TCC-3 29A Th1 vms VP154 1 TCC-4 10A Th0 V85.1 VP174_1, VP174_2_VP174_3 14A Th0 v35.1 VP1744, VP174,2, VP174_3 27A Th0 v3.5.1 VP1”, VP174_2_ VP174_3 30A Th0 V85.1 VP1744, VP174-2, VP174_3 19B Th0 V85.1 VP1“,1 VP174,2 VP174_3 TCC-5 3A Th1 vrs - VP191 TCC-6 11B Th1 V8 — VP1143 TCC—7 12B Th0 V82 VP1229 TCC-8 21A Th0 V8 - VP1319 25A Th0 V8 - VP1319 TCC-9 36A Th1 V8 - VP1319 1 B Th1 V8 — VP1319 TCC—1 0 19A Th0 V853 VP1335 3B Th0 V85.3 VP1335 TCC-11 24A Th1 V8 - VP1335 Among the CNS-infiltrating T- and B cells, CD4+ T cells with either Thl- or the above bifilnctional Thl-2 phenotype are probably the most critical element based on the ing findings. Their parallel secretion of Th1- (IFN—gamma) and Th2 (IL-4) cytokines probably explains the expression of HLA- class II molecules on resident cells such as infected astrocytes and lia, but also on infil- trating immune cells, since IFN—gamma is the strongest inducer of HLA-class 11. Although colocaliza- tion studies of HLA-DR with an astrocytic marker such as GFAP could not be performed due the paucity of material, the widespread expression of HLA-DR strongly suggests that these are also posi- WO 14134 tive. In y to MS and its animal model experimental autoimmune encephalitis (EAE), where local vation of immigrating T cells has been demonstrated, JCV-specific Thl-2 and also Thl cells are probably locally reactivated by ition of JCV peptides on JCV-infected, HLA-class 11 positive ytes, microglia/macrophages or recruited dendritic cells (DCs). Furthermore, the secretion of large quantities of IL-4 leads to activation and expansion of memory B cells/plasmablasts in the CNS compartment with the consequence of virus-specific antibody secretion. Locally produced JCV capsid protein (VPl)-specific IgG antibodies may recognize virus-infected oligodendrocytes, which could then be lysed by complement- or antibody-mediated cellular xicity. The relative increase in the CSF of IgG1 and IgG3 antibodies, which bind complement with high affinity and have been described in the context of other viral infections, supports this . Since infected oligodendrocytes do not express HLA-class II, but effectively express HLA-class I, it can be ed that JCV-specific, HLA- A2-restricted CD8+ cytolytic T cells nik et al., 2001) (Koralnik et al., 2002) also contribute by killing JCV-infected oligodendrocytes and/or astrocytes. That these previously described cells in the peripheral blood of AIDS patients with PML are ly also participating in the local eradication of JCV in the brain is supported by our observation of CD8+ T cells specific for JCV VP136 and JCV VP100 as defined by e-loaded HLA-A*02:01 tetramers. Infected astrocytes may not only serve as local antigen presenting cells for CD4+ virus-specific T cells, but may also be killed by Thl-2 cyto- lytic cells (Hemmer et al., 1997), but this together with the question of DR expression by astrocytes will require fiarther studies.

The above pathogenetic scenario accounts for the s of IFN—gamma- and IL-4, i.e. the widespread expression of HLA-class II molecules in the brain as well as the strong intrathecal antibody response against JCV, however, it is still puzzling that a large fraction of brain-infiltrating cells show a Thl-2 phenotype. Previously, these cells were referred to as Th0 cells and considered an ediate differ- entiation step before naive cells develop into memory cells committed to either Thl or Th2 lineage (Mosmann and Coffinan, 1989). This notion has, however, already been contended early based on following the cytokine patterns of single clones (Kelso, 1995). Today, Th1- and Th2 cells are under- stood as mutually exclusive fates (Ansel et al., 2006). However, individual TCC with dual ne secretion have been described as Th0 cells in measles virus infection (Howe et al., 2005) and among disease-exacerbating autoreactive T cells during altered peptide ligand-based therapy of MS (Bielekova et al., 2000). The inventors’ t observation of stable Thl-2 clones based on intracel- lular ne staining, cytokine secretion and transcription factor expression point to a defined T helper cell subpopulation in the CNS rather than an intermediate or transient differentiation stage. Due to the abovementioned ill-defined role of Th0 cells and the prior controversy about their nce as terminally entiated cells, we propose here to refer to IFN—gamma/IL-4 T helper cells as bifilnc- tional Thl-2 cell. The context and signals that lead to this Thl-2 differentiation need filrther examina- tion. In a recently published study in a viral infection model, the authors demonstrated that non- protective Th2 cells could be converted to stably IFN—gamma/ILexpressing and protective CD4+ cells by concerted action of antigen-specific TCR , type I and —II interferons and IL-12 (Hegazy et al., 2010) (Zhu and Paul, 2010). The inventors’ findings are the first evidence for the existence of a stable GATA-3+T-bet+ and IL-4+IFN—gamma+ Th2+l ype in vivo in . It is conceivable that these cells were reprogrammed in the brain, and they could well explain the unusually strong im- mune response and fulminant course of PML-IRIS.

Regarding the fine specificity of brain-infiltrating T cells, the inventors’ data are interesting in several aspects. The JCV-specific T cell response is overall broad since peptides from almost all JCV proteins are recognized, which is consistent with the inventors’ efforts to map immunodominant epitopes of JCV for peripheral blood-derived CD4+ T cells in healthy donors and MS patients. However, more than 50% of peptides recognized by brain-derived CD4+ T cells are part of the major ural protein VPl. Furthermore, VPl-specific T cells dominate with respect to strength of proliferation and precur- sor frequency. It is uing that VPl34_4g contains not only a major epitope for cytotoxic, HLA- A*02:01-restricted CD8+ T cells (Du Pasquier et al., 2003b), which the inventors found as well in the brain of the PML-IRIS patients by tetramer staining, but also for HLA-DRBl*lS:01/DRB5*01:01- restricted CD4+ T cells. Furthermore, the recognition of peptide VPl74 and two variants thereof with single amino acid substitutions tes that recognition of this epitope may be relevant to protect the host from immune evasion during persistent JCV infection. This has been shown previously for human immunodeficiency— (Borrow et al., 1997) and lymphocytic choriomeningitis virus infections (Ciurea et al., 2001). The us intrathecal dy response against VPl filrther underscores the role of this structural protein. Therefore, the inventors’ findings show that VPl is ant for protective im- mune responses against JCV-infected brain cells and that these are mediated by antibodies, CD4+ and CD8+ T cells. The strength of this response is probably in part ined by the HLA type of patient 2, who expresses both the major MS risk allele DRBl*lS:01/DRB5*01:01 and 1, which pre- sent an identical VPl epitope to CD4+ and CD8+ T cells. He may therefore have experienced a par- rly pronounced T cell-mediated immune response in the brain with its immunopathologic conse- quences of massive PML-IRIS, brain swelling, and neurological worsening. As already pointed out by others (Cinque et al., 2003) the JCV-specific immune response is a double-edged sword. t a filnctional immune response brain cells are lysed by uncombated viral infection. On the other hand, if unleashed, the vigorous JCV-specific response during PML-IRIS causes brain inflammation and edema, and while it effectively eliminates JCV from the CNS, it may lead to death of the patient if not at least arily attenuated by immunosuppression (Tan et al., 2009b).

The cellular and humoral JCV-specific immune response in the brain during PML-IRIS not only com- plicates the treatment, but may also cloud the diagnosis of PML in the first place. ent from cur- rent routine, which relies on CSF JCV viral load and, if a biopsy is performed, on immunohistochemis- try and in situ hybridization for JCV n and DNA respectively, the intrathecal dy response 2012/064445 against VPl appears more robust and should be examined. In both PML-IRIS patients of this study hecal VPl-specific antibody titers were extremely high despite almost undetectable JCV DNA by PCR and in situ hybridization. The important role of JCV dy testing is supported by prior obser- vations of high antibody titers in AIDS patients with PML (Weber et al., 1997), but also recent data in natalizumab-treated MS patients (Gorelik et al., 2010).

Another important and unexpected observation of this study is that, different fiom the JCV-specific antibody response, pathogenetically relevant T cells are confined to the CNS parenchyma itself, and that the CSF is of little use for investigating T cell specificity and filnction. This finding is probably highly relevant not only to PML-IRIS, but also to MS, where most studies have d on CSF as a surrogate for the responses within the CNS from obvious reasons, i.e. because CNS tissue is rarely available to investigators. Future research should therefore make every possible effort to examine bi- opsy or autopsy tissue if it can be acquired. When studying the brain-infiltrating CD4+ T cells of this MS patient with PML-IRIS, the ors were fiarther surprised to see that none of the es from three major myelin proteins were recognized, suggesting that bystander activation or —recruitment of myelin-specific T cells during massive brain inflammation does not occur, but that cells are exquisitely specific for the causal agent.

Example 2 — sation to JCV An individual healing attempt was performed in a patient with idiopathic CD4+ lymphopenia, a rare constitutive immunodeficiency, who ped PML at the age of 64 years (referred to as "patient Hamburg" in figure 11). The male patient had been y, i.e. not experienced un- usual or frequent infections, throughout his life, and in February 2010 was hospitalized with signs of an encephalitis of unknown origin. He was thoroughly worked up, and a diagnosis of suspected EBV-related encephalitis was made. Following transient improvement during anti- viral therapy, he deteriorated further albeit . During 2010, two brain biopsies were per- formed, and in the second one at the end of 2010, a diagnosis of progressive multifocal leu- koencephalopathy (PML) was made based on positive JCV viral load in the CSF and on dem- onstration of JCV-infected oligodendrocytes and ytes in the brain. In parallel, the inven- tors found a low CD4+ T cell count (around 300/microliter) as well as a D8+ ratio of 0.5 or less, which are both tent with the sis of idiopathic CD4+ lymphopenia. In addition, in vitro experiments in the laboratory documented an absent T cell response to JCV virus VPl in peripheral blood mononuclear cells and an almost complete absence of naive CD4+ T cells. Following these observations, the ors reasoned that the patient ly had developed PML based on a pre-existing and probably genetically determined low CD4+ number, which became further accentuated by entirely physiological immune involution, which sets in and increases above 50 years of age. 2012/064445 The inventors wanted to test if ation with VPl, and, in this case of CD4+ lymphopenia, preferably combined with recombinant IL-7, would increase the number of JCV-specific T cells that the patients must have had, since he is JCV-positive. Further, if this were to occur, the inventors hoped that the vaccine-induced or —boosted JCV VPl-specific T cell se would lead to these cells’ migrating to the CNS and elimination of virus and virus-infected cells from the CNS compartment. The inventors therefore applied for an “individual healing attempt”, discussed this option and its potential risks with the patient and obtained his consent. The use of IL-7 (Cytheris) was fiarther supported by a recent publication in r case of CD4+ lym- phopenia (Patel et al., 2010), in whom recombinant IL-7 together with antiviral drugs had led to ntial improvement of the patient, however, in that patient, no immunological studies were performed, and therefore nothing was known about improvement of antigen-specific im- mune responses.

The vaccination approach in the above patient included the following steps (for timing of vac- cinations and tests see scheme below): Subcutaneous injection with the entire recombinant major capsid protein VPl (provided by the Life Science Inkubator, Bonn) in combination with a dermally applied TLR7 agonist (imiqimod, Aldara; commercially available) and iv. recombi- nant IL-7 (Cytheris). The VPl n was administered in the form of like particles (VLP), as the recombinantly expressed VPl protein associated to such particles under the con- ditions used herein. As shown below, the patient not only showed an in vitro proliferative re- sponse against JCV VPl after only two vaccinations, but also reduced the JCV viral load to 0 and finally began to show slight contrast enhancement around the PML lesions by brain MRI imaging, which all support that the vaccination worked in vivo. He also showed clinical im- provement with slight delay after developing a JCV-specific immune response. rmore, since the inventors’ data from the brain-infiltrating T cells in the PML-IRIS patient described in Example I suggested that JCV-specific CD4+ T cells with a T helper l-2 ype are probably crucial for elimination of JCV virus from the brain, the ors also stained for Thl- 2 CD4+ T cells in the cerebrospinal fluid of the patient, and could demonstrate that these cells are indeed present.

Another dual healing attempt was performed in a t with breast cancer who received chemotherapy and developed acute myeloid leukaemia (AML) as a side effect of the chemotherapy.

The t then received an gous and allogeneic hematopoietic stem cell transplant as treatment of the AML and subsequently developed acute graft-versus-host disease (grade IV). The immunodeficiency acquired as a consequence of these treatments resulted in PML. This case is referred to as "patient " in figure ll. Both ts were treated by recombinant IL-7 (s.c.). 2 days later, the first dose of VPl (s.c. 1 mg, imiquimod cream on the skin) was administered. 2 days after the first dose of VPl, the second dose of rIL-7 was given.

On day 12 after the first IL-7 dose, the patients ed a second vaccination with VPl s.c. plus imiquimod, and in this case simultaneously rIL-7. At week 6, a third dose of VPl/imiquimod and a fourth dose of rIL-7 were administered. Figure 11 shows the results from the below bed proliferation assay performed at the time points indicated in the graph with peripheral blood mononuclear cells (PBMC) obtained from both patients. A stimu- lation index of >2 is considered a positive response. It can be seen that both patients showed a positive immune response against JCV after vaccination.

Material and Methods Blood and CSF samples Biological samples were obtained after informed written t. Peripheral blood mononu- clear cells ) were separated from EDTA-blood by Ficoll (PAA, Pasching, Austria) density centrifugation.

Cerebrospinal fluid (CSF)-derived mononuclear cells were expanded by seeding 2000 cells/well plus 2x105 irradiated (45 Gy) allogeneic feeder cells. 1 ug/ml PHA-L (Sigma-Aldrich, Munich, Germany) and 500 IU/ml IL-2 (kindly provided by Federica Sallusto, Institute for Research in Biomedicine, Bellinzona, CH) was added. The addition of IL-2 was repeated every 3-4 days until day 14.

Proliferation Assays The proliferation se of PBMC to VPl (kindly provided by Viktorya Demina, Life Sci- ence Inkubator, Bonn, Germany) and Tetanus toxoid (TTx, Novartis, Marburg, Germany) was tested by seeding 2x105 cells in a 96-well U-bottom microtiter plates. VPl was used at 2 ug/ml and TTx at 5 ug/ml. After 7 days incubation, oration of 3H-thymidine (Hartmann Ana- lytic, Braunschweig, Germany) was ed. Stimulatory indices (SI) were calculated by dividing the mean CPM (counts per minute) of the wells plus antigen by the mean CPM of the wells without n.

To e proliferative responses to VPl and TTx by flow cytometry the CellTraceTM CFSE Cell Proliferation Kit (Invitrogen, Darmstadt, Germany) was used. Therefore, cells were seeded as described above and restimulated with antigen after six days and d with CFSE following the manufacturer’s instruction. After five days cells were analysed by flow cytome- try.

Flow try Analysis Whole blood ngs were performed by adding the appropriate antibody cocktail in a volume of 50 ul to 100 ul blood. The mixture was incubated for 30 minutes at room temperature, fol- lowed by 10 minutes of red blood cell lysis with FACS Lysing Solution (BD PharMingen).

After washing, the cells were analysed by flow cytometry in a LSR 11 (BD). Following antibod- ies were used: CD4 (APC, RPA-T4, eBioscience), CD8 (PB, DK25, Dako, Glostrup, Den- mark), CD45RO (FITC, UCHL1, eBioscience), CD25 (PE-Cy7, eBioscience), CD3 (PE, Da- koCytomation, Denmark), CD8 (PB, tomation, Denmark), IFN—gamma (FITC, BDPharmingen), IL-4 (PE-Cy7, eBioscience).

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Claims (37)

What we claim is:

1. Use of a protein or peptide for the manufacture of a medicament for treating or preventing progressive multifocal leukoencephalopathy (PML) or ssive multifocal leukoencephalopathy-immune reconstitution inflammatory syndrome (PML-IRIS) in a human subject, wherein said protein or peptide comprises at least one CD4+ epitope derived from polyoma virus JC (JCV), wherein the protein or peptide is present in the form of a virus like particle (VLP), and wherein the protein or e comprises VP1 .

2. The use of claim 1, wherein the VP1 is a fusion protein further comprising at least one epitope selected from the group comprising SEQ ID NO: 1 and SEQ ID NO: 46–76.

3. The use of claim 1 or 2, n said PML treatment comprises the administration of a cytokine which is capable of ing and ining T cells.

4. The use of claim 3, wherein said cytokine is selected from the group consisting of IL-7, IL-2, IL-15 and IL-21.

5. The use of any one of claims 1-4, wherein said PML treatment comprises the administration of an adjuvant.

6. The use of claim 5 , wherein said adjuvant is selected from the group of MF59, aluminium hydroxide, calcium phosphate gel, lipopolysaccharides, imidazo-quinolines, oligonucleotide sequences with CpG motifs, stearyl tyrosine, DTP-GDP, DTP-DPP, threonyl- MDP, 7-allyloxoguanosine, glycolipid bay R1005, antigen peptide system, polymerized haptenic es, bacterial extracts, TLR-7 agonists, TLR-8 agonists and vit-

7. The use of claim 6 wherein the o-quinolines are selected from mod and S- 28463.

8. The use of any one of claims 1 to 7, wherein said human subject is infected with JCV.

9. The use of any one of the claims 1 to 8, wherein said human subject is selected from the group of: a) human ts having a congenital immunodeficiency, b) human subjects having an acquired immunodeficiency resulting from a disease or pathological condition, and c) human subjects having an acquired immunodeficiency resulting from a therapeutic ention.

10. The use according to claim 9, wherein the congenital immunodeficiency is idiopathic CD4+ lymphopenia or Hyper-IgE-Syndrome.

11. The use according to claim 9, wherein the disease or pathological condition is AIDS, ia, lymphoma, multiple myeloma or infection with hepatitis virus B or C.

12. The use according to claim 9, wherein the therapeutic intervention is chemotherapy, radiation or immunosuppressive treatment.

13. The use of any one of claims 8-12, wherein said immunosuppressive treatment comprises treatment with an immunosuppressive antibody.

14. The use of claim 13, wherein said immunosuppressive antibody is selected from the group of natalizumab, efalizumab, rituximab, ocrelizumab and alemtuzumab.

15. The use of any one of the claims 13 to 14, wherein said human t is afflicted with an autoimmune disease.

16. The use of claim 15, wherein said autoimmune e is multiple sclerosis.

17. The use of claim 16, wherein said human subject is to be d with the antibody natalizumab.

18. Use of a protein or peptide for the manufacture of an agent for in vivo diagnosing infection with JCV and/or for in vivo sing PML, wherein said n or peptide comprises at least one CD4+ epitope derived from polyoma virus JC (JCV), wherein the protein or peptide ses VP1 and is present in the form of a virus like particle, and wherein the method of diagnosing comprises detecting a reaction of CD4+ T cells in a sample to a presence of the epitope.

19. Use of a pharmaceutical kit for the manufacture of a medicament for treating or ting PML or PML-IRIS in a subject, wherein said kit comprises a protein or peptide comprising at least one CD4+ epitope derived from JCV, wherein the protein or peptide ses VP1 and is present in the form of a virus like particle, and wherein said kit ses an adjuvant.

20. The use of claim 19, wherein the adjuvant is selected from the group comprising a TLR- 7 agonist and TLR-8 agonist.

21. The use of claim 20, wherein the adjuvant is a TLR-7 agonist.

22. The use of claim 21, wherein the TLR-7 agonist is imiquimod.

23. The use of any one of claims 19 to 21, wherein the VP1 is a fusion protein r comprising at least one epitope selected from the group comprising SEQ ID NO: 1 and SEQ ID NO: 46–76.

24. The use of any one of claims 19 to 23, wherein the kit further comprises IL-7.

25. The use of any one of claims 19 to 24, n the treating or preventing comprises administering the protein or peptide comprising at least one CD4+ epitope derived from JCV subcutaneously.

26. The use of any one of the claims 19 to 25, wherein the treating or preventing comprises stering the adjuvant dermally.

27. The use of any one of claims 19 to 26, wherein the subject is a human subject selected from the group consisting of a human subject diagnosed with PML or a human t at risk of developing PML, and wherein the human subject has optionally been diagnosed to be a carrier of JCV.

28. The use of any one of claims 19 to 27, wherein the subject is a human t selected from the group of: a) compromised or immunodeficient human subjects; and b) human ts eligible for immunosuppressive treatment.

29. The use of claim 28, wherein the immunocompromised or immunodeficient human subjects are carriers of HIV, human subjects having immunosuppressive treatment or congenital immunodeficient patients.

30. The use of claim 29, wherein the congenital immunodeficient patents are patients with idiopathic CD4+ lymphopenia or Hyper-IgE-Syndrome.

31. The use of claim 28, wherein the immunosuppressive ent is selected from the group comprising treatment with zumab, efalizumab, mab, ocrelizumab and alemtuzumab.

32. The use of claim 28 or 31, wherein the immunosuppressive treatment is treatment of a human subject diagnosed with an autoimmune disease or a transplantation patient, wherein the treating or ting PML or PML-IRIS comprises administering the components of the kit to said patient before, after or during immunosuppressive treatment.

33. Use of an adjuvant, in the manufacture of a medicament, for treating or preventing PML or PML-IRIS in a subject, wherein the treating or preventing ses administration of the nt in combination with a protein or peptide comprising at least one CD4+ epitope derived from JCV, wherein the protein or peptide comprises VP1 and is present in the form of a virus like particle.

34. The use according to claim 1, substantially as herein described or exemplified.

35. The use according to claim 18, substantially as herein described or exemplified

36. The use according to claim 19, substantially as herein described or exemplified.

37. The use according to claim 33, substantially as herein bed or exemplified.