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WO2008125366A2 - Nouveaux traitements de maladies - Google Patents

Nouveaux traitements de maladies Download PDF

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Publication number
WO2008125366A2
WO2008125366A2 PCT/EP2008/004992 EP2008004992W WO2008125366A2 WO 2008125366 A2 WO2008125366 A2 WO 2008125366A2 EP 2008004992 W EP2008004992 W EP 2008004992W WO 2008125366 A2 WO2008125366 A2 WO 2008125366A2
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ebv
cells
cell
use according
substance
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PCT/EP2008/004992
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WO2008125366A3 (fr
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Francesca Aloisi
Barbara Serafini
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Istituto Superiore Di Sanita
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/245—Herpetoviridae, e.g. herpes simplex virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16211—Lymphocryptovirus, e.g. human herpesvirus 4, Epstein-Barr Virus
    • C12N2710/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to treatments for chronic inflammatory diseases characterised by autoimmune features.
  • MS multiple sclerosis
  • CNS central nervous system
  • EBV Epstein-Barr virus
  • a variety of abnormalities in humoral immunity are associated with MS, including: intrathecal synthesis of immunoglobulins (predominantly oligoclonal IgG) 12 ; presence of clonally related B cells 13 14 and unusual B-cell subsets (germinal centre centroblasts and centrocytes) 15 in demyelinated lesions and cerebrospinal fluid (CSF); and, in a subset of early-onset MS patients, formation of intrameningeal lymphoid-like structures resembling B-cell follicles 16 ' 17 .
  • EBV Epstein-Barr virus
  • the present invention provides the use of at least one of (a) an anti-EBV substance, and (b) a B cell cidal agent, in the manufacture of a medicament for the treatment of an autoimmune disease.
  • the use includes only an anti-EBV substance.
  • the use includes both an anti-EBV substance and a B cell cidal agent.
  • Autoimmune diseases may include, inter alia, systemic lupus erythematosus, Hashimoto's thyroiditis, Grave's disease, Sjogren's syndrome, multiple sclerosis, rheumatoid arthritis, myasthenia gravis and inflammatory myopathies (dermatomyositis, inclusion body myositis, and polymyositis).
  • Hashimoto's thyroiditis is a disease that commonly causes the condition known as hypothyroidism.
  • the present invention is also useful in treating said condition.
  • the invention also provides at least one of (a) an anti-EBV substance, and (b) a B cell cidal agent for the treatment of an autoimmune disease.
  • a pharmaceutical composition comprising at least one of (a) an anti-EBV substance, and (b) a B cell cidal agent, for the treatment of an autoimmune disease.
  • the invention also provides a method for treating an autoimmune disease, comprising administering, to a patient in need thereof, a therapeutically effective amount of at least one of (a) an anti-EBV substance, and (b) a B cell cidal agent.
  • EBV establishes a low level, persistent, infected state in B cells. In this state, little or no active virus production is seen, and B cells are actively encouraged to replicate, with concomitant suppression of normal apoptotic regulation. This type of infection is referred to herein as latent infection.
  • Infected plasma cells which arise from infected B cells, are the cell type in which viral reactivation occurs and may optionally be further targeted by the medicaments of the present invention.
  • levels of latent infection may be high, or may alternate on an occasional basis with lytic infection, where whole virus is expressed by infected cells.
  • B cells The latent infection of B cells stimulates a cytotoxic T cell response directed at the infected B cells. It is apparent that brain damage is caused predominantly by the antiviral immune response. In multiple sclerosis, the B cells form ectopic follicles in the central nervous system (CNS), especially in the meninges, and these lymphoid-like structures represent the main intracerebral foci of viral reactivation. Without being bound by theory, it appears that the meningeal localisation of B cells leads to extended cortical lesions by causing the clustering of cytotoxic T cells. The effect may be enhanced by the apparent dysregulation, as described above, of EBV infection in susceptible individuals. Around 90% of the population is EBV -seropositive, but the virus is generally efficiently controlled. Any deviation from this delicate host-virus balance is considered to be 'dysregulation', whether it is caused by EBV, by other viruses or by genetic factors.
  • autoimmune disease is associated with regions of the body not restricted to the CNS
  • a B cell cidal agent in a systemic medicament, and to just use one or more substances (a).
  • a B cell cidal agent for the treatment of systemic lupus erythematosus or rheumatoid arthritis, for example, it is preferred to use one or more anti-EBV substances, and no B cell cidal agents.
  • a B cell cidal agent for instance a monoclonal antibody, such as rituximab, for treating both lupus and rheumatoid arthritis. This has been shown to work well.
  • B cell cidal agents may help reduce the pool of infected B cells in the body and lower the EBV-specific immune response which mediates brain damage.
  • Plasma cell cidal agents may also be employed in addition to or separately from B cell cidal agents, although it is generally preferred to target B cells, as these are the progenitors of the plasma cells.
  • Cidal agents as defined herein may be thought of as being capable of reducing or leading to a reduction in the number of B-cells, including those infected with EBV, or rendering said cells inactive.
  • Anti-EBV substances include drugs, antibodies and siRNA, for example.
  • Suitable anti-EBV drugs include nucleotide and nucleoside analogues such as, inter alia, acyclovir, valacyclovir, and ganciclovir, and Type-1 interferons, such as IFN- ⁇ and IFN- ⁇ .
  • nucleotide and nucleoside analogues such as, inter alia, acyclovir, valacyclovir, and ganciclovir
  • Type-1 interferons such as IFN- ⁇ and IFN- ⁇ .
  • Other anti- EBV substances will be apparent to the skilled person.
  • Antibodies against EBV may be against any suitable target. Clinical trials have been performed on vaccines comprising the gp350/220 surface glycoprotein, as well as the HLA B8 restricted epitope from EBNA-3A. When used in treatment, as opposed to prophylaxis, of an autoimmune disease, then antibodies specific for latent markers may be employed, these markers including Epstein Barr Nuclear Antigen 2 and latent membrane protein 1 (LMPl). Where a vaccine against EBV is to be administered to prevent EBV infection and subsequent autoimmune disease, then it is generally preferable to target antigens associated with active infection, such as BFRFl and gp220/350.
  • active infection such as BFRFl and gp220/350.
  • siRNA short interfering RNA
  • EBNAl and EBNA2 genes may be employed that targets any part of the EBV genome necessary for EBV replication, such as the EBNAl and EBNA2 genes.
  • Particularly preferred targets are EBNAl sequences such as 5'-ggaggttccaacccgaaat-3' (SEQ ID NO: 23) and 5'- ggactaccgacgaaggaac-3' (SEQ ID NO: 24), as disclosed in Virology 2006; 346:385, and those derived from LMPl, such as 5'-ggaatttgcacggacaggc-3' (SEQ ID NO: 25), as disclosed in Cancer Letters 2006; 232:189.
  • Other suitable targets will be readily apparent to those skilled in the art.
  • SEQ ID NOs: 26-28 (the RNAi sequences directed at SEQ ID NOs: 23-25 respectively), or variants or fragments capable of hybridising to SEQ ID NOs: 23-25 under highly stringent conditions such as 6 x SSC, are useful for siRNA.
  • the anti-EBV substances may be administered systemically, or directly to the CNS, either by injection into the ventricles or intrathecally.
  • Administration direct to the CNS is referred to herein as intrathecal administration, although intracerebroventricular administration is also contemplated by the present invention.
  • Administration to the CNS is particularly preferred for treatment of MS.
  • administration may be to the area affected, for instance the thyroid or neck, or the affected joint. Delivery may be intravenous or by transdermal, transmucosal by oral means, although suitable encapsulation or formulation may be required in respect of oral delivery.
  • B cell cidal agent in the medicament.
  • Any physiologically acceptable B cell cidal agent may be used, as appropriate.
  • B cell cidal agents are apparent to those skilled in the art, and include monoclonal antibodies targeting B-cell surface proteins, such as the chimaeric monoclonal antibody known as Rituximab developed by IDEC Pharmaceuticals, to activate antibody-dependent cellular cytotoxicity and/or complement-dependent cytotoxicity and or/apoptosis.
  • both the anti-EBV substance or the B cell cidal agent are administered, preferably contemporaneously. However, it is also preferred that only one of these two is administered.
  • Either the anti-EBV substance or the B cell cidal agent alone may be effective in controlling, suppressing, and even eliminating B cell infections, but it is particularly preferable to employ at least one of each where possible, especially intrathecally.
  • a particularly preferred medicament employs both Rituximab and valacyclovir. Another preferred combination is Rituximab and acyclovir. Where the medicament comprises more than one active ingredient, these may be administered together or separately. Where a disease has both CNS and systemic components, then, where a B cell cidal agent is employed, this is advantageously restricted to intrathecal administration, while an anti-EBV substance may be administered both systemically and intrathecally, if desired.
  • Medicaments of the present invention may also comprise, additionally to, or alternatively to, one of the active ingredients, a T cell inhibitory, blocking, suppressing, or cidal substance when administered intrathecally.
  • a T cell inhibitory, blocking, suppressing, or cidal substance when administered intrathecally.
  • Such a substance is preferably targeted at CD8+ T cells.
  • the substance is preferably a specific antibody.
  • Type II topoisomerase inhibitors are effective as CD8+ T cell cidal agents, and include etoposide, which induces apoptosis of EBV-infected lymphocytes.
  • the medicament of the present invention be directed to multiple sclerosis, and it is preferred that any such medicament be suitable for intrathecal administration.
  • the components of the disease in human brain are; the latent EBV infection, the infected B cells, the periodic reactivation of EBV, and the bystander tissue damage caused by cytotoxic T cells in response to the latently and lytically infected B cells. Any one of these aspects is targetable by the medicaments of the present invention.
  • Acyclovir and valacyclovir are particularly preferred, especially when it is desired to only target the lytic infection and not the latent infection.
  • the present invention further extends to a vaccine for the prevention of multiple sclerosis, or any other autoimmune disease, comprising at least one epitope characteristic of EBV.
  • Suitable vaccines are well known to those skilled in the art, and it is preferred to administer such a vaccine in early childhood (or during the first year after birth), before the individual becomes EBV-seropositive.
  • EBERs EBV-encoded small nuclear mRNAs
  • EBN A2 Epstein-Barr nuclear antigen 2 (EBNA2) and latent membrane protein 1 (LMPl) 18 .
  • EBNA2 Epstein-Barr nuclear antigen 2
  • LMPl latent membrane protein 1
  • EBN A2 is the first viral protein to be expressed in EBV-infected B-cells in vitro and acts as a transactivator of viral and cellular genes inducing B-cell proliferation (growth program).
  • LMPl is an integral membrane protein that promotes B-cell proliferation and survival and is expressed during the growth program and the more restricted viral transcription program, named default program 18 .
  • EBV latent proteins provides a mechanism for B-cell expansion and activation in the MS brain and is consistent with the presence of B-cells expressing the proliferation marker Ki67 and the anti-apoptotic molecule be 12 in intrameningeal follicles (Fig.lb,f) and acute lesions (Fig.3c).
  • LMPl may also induce expression of the enzyme activation-induced cytidine deaminase 19 (Fig.ld), which is normally restricted to germinal centre B-cells.
  • BFRFl + cells were present inside and around all intrameningeal follicles and acute lesions analysed in EBV-high MS cases (Fig.2g-m). A few BFRFl + cells were also detected in the small ectopic follicles identified in one EBV-low MS case (not shown).
  • Double immunostainings showed that BFRFl immunoreactivity was present in a substantial proportion of B-cells and plasma cells (30-55%) but was much stronger in plasma cells than in B-cells (Fig.2 h-m), in agreement with findings in healthy carriers where terminal differentiation into plasma cells initiates the viral replicative cycle 21 .
  • Rare gp350/220 + cells were found only in the parenchymal lesions of one EBV- high MS case (Fig.2o), indicating occasional production of infectious virus, probably due to abortive replication or efficient immune surveillance.
  • cytotoxic CD8 + T-cells have a key role in controlling latent EBV infection and in preventing viral replication 22 .
  • the presence of EBV-infected cells in the MS brain would associate with CD8 + T-cell infiltration. Indeed, intracerebral accumulation of CD8 + T-cells was much more prominent in EBV-high than in EBV-low MS cases (Fig. 7). Participation of CD8 + T-cells in the clearance of EBV-infected cells was supported by the findings that these cells did infiltrate all sites where infected B -cells/plasma cells were located and that the frequency of CDS + T-cells in the meninges and white matter strikingly correlated with that of EBER + cells (Fig.3a,b,k).
  • CD8 + T-cells expressing the proliferation marker Ki-67 were detected in some ectopic follicles and all acute lesions, consistent with recent antigen stimulation likely related to viral reactivation (Fig.3c).
  • CD8 + T-cells established close anatomical contacts with B-cells, plasma cells and BFRFl + cells, and about 3% of them expressed perforin, a key component of lytic granules, indicating recognition and attack of EBV-infected B-cells/plasma cells (Fig.3d-g).
  • Fig.3d-g Even in the EBV-low MS cases, some of the scattered CD8 + T-cells were found in direct contact with B-cells (Fig.3h).
  • Prominent macrophage activation was also associated with intracerebral EBER + and CD8 + T-cell accumulation (not shown).
  • the presence of a large number of EBV-infected cells and cytotoxic T-cells in the brain of the MS cases with ectopic follicles is compatible with the severe clinical and neuropathological features of this patient subset 17 .
  • BDCA2 blood dendritic cell antigen 2
  • plasmacytoid dendritic cells which are the main source of type-I interferon and have a key role in antiviral immunity, were detected in many MS immune infiltrates, predominantly in meningeal follicles and acute lesions of EBV-high MS cases (Fig. 3 i,j).
  • EBV DNA was generally undetectable, being found at low copy number in the CSF of only 2 of 16 MS cases (2 primary progressive MS with 1310 and 4900 copies/ml, respectively). None of the other viruses investigated [herpes simplex viruses type 1 and 2 (HSV- 1/2), varicella- zoster virus (VZV), cytomegalovirus (CMV), human herpesvirus 6 (HHV-6) and JC virus (JCV)] were detected in the 16 CSF samples analysed.
  • Oligoclonal bands which are generally CSF-restricted and relatively constant throughout the disease 12 , were found in the CSF of 15 of 16 MS cases. Interestingly, the number of CSF OCBs was significantly higher in EBV-high MS cases (Fig.4a), further indicating a relationship between EBV infection and intracerebral B-cell activation. Consistent with previous findings 8 - 23 , EBV-specific OCBs were detected in 7 of 16 MS cases (44%), but showed no preferential distribution in the EBV-high and EBV-low MS groups (Fig.4b).
  • Perturbed EBV infection also provides an explanation for the dominance and persistence of CD8 + T-cell clones in the MS brain 25 ' 26 , and implies that bystander tissue damage mediated by cytotoxic T-cells is a major determinant of MS lesions, similar to what happens in other EBV-associated diseases 22 .
  • Stimulation of autoreactive B-cell clones by EBV is not excluded and may explain the variable pattern of antibody reactivities toward CNS-restricted and ubiquitous self-antigens observed in MS patients 27 . This also extends to other chronic diseases with autoimmune features, such as systemic lupus erythematosus and rheumatoid arthritis, in which an association with EBV is indicated by epidemiological, serological and DNA studies 28 .
  • MS cases analysed had not received immunosuppressive therapies, except for MSl 14 (treated with ACTH for 12 months), MS80 (treated with ACTH for 11 years), MS5 (treated with mitoxantrone for 3 years), and MS 153 (treated with ACTH for 30 months).
  • Control tissue specimens, lymphoma and normal human lymph node were provided by the Institute of Pathological Anatomy, A. Gemelli Hospital, and the Pathology Department of S. Andrea Hospital, Rome.
  • tissue blocks (2 x 2 cm) from the cerebral hemispheres were analysed and classified by histopathological methods, i.e. hematoxylin/eosin staining, and the combined Luxol fast blue-periodic acid-Schiff reaction, or by immunohistochemistry using anti-myelin oligodendrocyte glycoprotein antibody, to identify areas of inflammation and demyelination 15 ' 16 .
  • Immunohistochemical stainings for T and B lymphocytes, macrophages, and MHC class II molecule expression were performed to evaluate the degree of lesion activity (pre-active, acute, chronic active, and chronic inactive), as previously described 15 ' 16 .
  • EBER Epstein-Barr virus
  • PNA ISH detection kit both from Dako
  • the kit also included negative and positive control fluorescein- conjugated PNA probes.
  • Treatment with proteinase-k was performed on frozen, PFA fixed-frozen and paraffin-embedded sections at the following dilutions in TBS: 1:500 for 10 min, 1 :100 and 1:10 for 20 min, respectively.
  • An EBV-associated B-cell lymphoma paraffin-embedded sections was used as positive control tissue for EBER signals.
  • EBER + , CD20 + and CD8 + cells was counted in 6 adjacent sections per tissue block (two 2 x 2 cm-sections were analysed for each immunohistochemical staining) at 2Ox magnification by one investigator blinded to the case number (B. S.). One-two blocks per MS case were analysed. Values are expressed as the mean number of positive cells per mm 2 in the white matter and per mm in the meninges.
  • the pattern of OCBs in autopsy CSF was investigated with agarose isoelectric focusing (IEF) (pH 3.0-10.0, Cambrex, Rockland, ME, USA) and affinity immunoblotting using a standard protocol 29 .
  • IgG and albumin were determined with turbidimetry (Cobas Integra, Roche, Milan, Italy).
  • EBV-specif ⁇ c OCBs were determined using a previously published IEF/affinity-mediated immunoblotting protocol 29 , using nitrocellulose membranes that had been previously coated with highly pure EBV Antigen, strain P3HR1, and a mouse monoclonal anti-EBV EA-D IgG as a positive control (both from Fitzgerald Industries International, Concorde, MA, USA).
  • CSF samples were tested by real-time PCR for DNA of the following viruses: EBV, HSV-I, HSV-2, VZV, CMV, HHV6-A, HHV6-B, and JCV.
  • PCR protocols are listed in Table 3. In all virus assays, an internal control was used to control for PCR inhibitors in the samples.
  • the Mann Whitney fZ-test was used to compare the median values of EBER + , CD20 + and CD8 + cells counted in adjacent MS brain sections and of CSF OCBs between EBV-low and EBV- high groups of MS cases. Spearman's rank correlation was calculated to analyse the relationship between number Of EBER + cells and number of CD20 + or CD8 + T cells in MS brain sections. A p value lower than 0.05 was considered as significant.
  • RR relapsing remitting
  • PR progressive relapsing
  • SP secondary progressive
  • PP primary progressive.
  • EBER + cells were detected in pre-active, acute and chronic active, but not inactive, lesions.
  • CD3 T lymphocytes Mouse monoclonal/PS 1 Pre-diluted Irnmunotech, Marseille, F CD20 B lymphocytes Mouse monoclonal/L26 Pre-diluted Immunotech
  • CDl 38 Plasma cells 1:100 Serotec, Oxford, UK monoclonal/B-B4
  • Plasmacytoid dendritic cells Mouse monoclonal/ AC 144 1:10 Miltenyi Biotec, CA, USA
  • CNS tissues from 9 patients with SPMS, 2 patients with PPMS, 2 patients with PRMS and 2 control subjects were fixed in 4% paraformaldehyde (PFA) for several days, cryoprotected in sucrose, frozen by immersion in isopentane precooled on a bed of dry ice (-55°C), and stored a - 80°C until use.
  • Brain tissue blocks from 1 patient with PPMS, 2 patients with RRMS, 6 patients with OIND and one control with Alzheimer's disease were fixed in buffered formalin and embedded in paraffin wax. Brain tissue blocks from 4 patients with SPMS were snap frozen. For MS79 and MS 153 cases both PFA-fixed and snap frozen tissue blocks were analysed.
  • Antigen retrieval procedures utilised microwave of PFA-fixed and paraffin sections in citrate buffer (10 mM, pH 6.0).
  • CD8 clone 4Bl 1
  • EDTA solution 1 mM, pH 9.0
  • Sections immunostained for MOG were treated with cold methanol without antigen retrieval procedure.
  • biotinylated secondary Abs rabbit anti-mouse or goat anti-rabbit IgG (Jackson Laboratories) were used.
  • rhodamine-conjugated goat anti-rabbit IgG and fluorescein- conjugated goat anti-mouse IgG were used as secondary Abs.
  • JC virus VPl VPl-F GAG TGT TGG GAT CCT GTGTTTTC (4) 77 100 VPl-R GAG AAG TGG GAT GAA GAC CTG TTT (5) VPl probe 6-FAM-TCA TCA CTG GCA AAC ATT TCTTCA TGG C-TAMRA (6)
  • Herpes simplex US4-US5 HSV-2 Ql F AGA TAT CCT CTTTAT CAT CAG CAC CA (10) 73 1000 virus-2 (HSV-2) HSV-2 Q2 R TTGTGC TGG CAA GGC GA (11) HSV-2 probe 6-FAM-CAG ACAAAC GAA CGC CGC CG-TAMRA (12)
  • HHV-6 probe A 6-FAM-CAG CCA TAT TTC CGGTAT ATGACC TTC GTAAGC T-TAMRA (18)
  • a representative ectopic follicle located in the depth of a cerebral sulcus displays the morphological features of a germinal centre as it is composed of CD20 + B- cells clustered around a network of stromal/follicular dendritic cells expressing the B- cell attracting chemokine CXCLl 3 (a: double immunofluorescence staining for CD20 in red and CXCL 13 in green).
  • the same ectopic follicle also contains numerous proliferating B-cells (b: double labelling for CD20 in green and the proliferation marker Ki67 in red; the inset in b shows 3 double labelled CD20 + /Ki67 + cells at higher magnification) and peripherally located Ig + plasma cells (c).
  • a distinct intrameningeal ectopic follicle contains: (d) numerous cells expressing activation-induced cytidine deaminase, the key enzyme responsible for somatic hypermutation and Ig class-switch recombination in germinal centre B-cells; (e) fewer cells expressing cleaved caspase-3, which is expressed in germinal centre centroblasts undergoing apoptotic cell death; and (f) numerous cells expressing the anti-apoptotic molecule bcl-2, which is expressed in positively selected germinal centre B-cells.
  • In situ hybridisation for EBER shows enrichment of EBER + cells in 2 follicles from 2 different MS cases
  • In situ hybridisation for EBER blue -black nuclei
  • In situ hybridisation for EBER blue -black nuclei
  • immunostaining for the pan B-cell marker CD20 red membrane staining
  • the inset shows a cell double labelled for the plasma cell marker CD 138 (red staining) and EBER (blue-black nucleus).
  • Enrichment of EBER + B-cells and plasma cells in MS follicles suggests that these structures are generated by clonal expansion of EBV-infected B-cells.
  • EBER signals in the MS brain are confined to the cell nuclei, similar to what is observed in a control, EBV-associated B-cell lymphoma (j).
  • EBV latent and lytic proteins in the MS brain, (a-d) Expression of the EBV latent proteins EBNA2 (a, b) and LMPl (c, d) in the brain of EBV-high MS cases.
  • An acute (a) and a chronic (b) white matter lesion containing several EBNA2 + cells in a perivascular position are shown.
  • the inset in b shows that EBNA2 staining is confined to the cell nucleus.
  • a sparse meningeal infiltrate (c) and an intrameningeal ectopic B-cell follicle (d) containing LMPl + cells (membrane staining) are shown; the inset in d shows the same follicle stained for EBER.
  • e, f Perivascular EBNA2 + and LMPl + cells in chronic active lesions from an EBV-low MS case,
  • g-m Expression of the EBV early lytic protein BFRFl in EBV-high MS cases. Numerous BFRFl + cells are present in an intrameningeal ectopic follicle (g).
  • Double immunofluorescence staining for CD20 (green) and BFRFl (red) shows that BFRFl is expressed in a substantial proportion of CD20 + B-cells but its intensity is much stronger in CD20-negative cells (arrows).
  • the insets in g and h highlight the typical perinuclear localisation of BFRFl immunoreactivity.
  • G" m Double immunofluorescence staining for Ig (green) and BFRFl (red).
  • the lytic cycle- associated protein is strongly expressed in a proportion of Ig + plasma cells in the same ectopic follicle shown in i (j; the asterisks in i and j mark the Ig-negative B-cells with lower BFRFl expression in the centre of the follicle).
  • BFRFl is mainly expressed in Ig + plasma cells located at the periphery ofthe large perivascular inflammatory cell infiltrate (k).
  • Two double labelled Ig + /BFRF1 + plasma cells in the same infiltrate are shown at higher magnification (1, m).
  • the same perivascular cuff contains numerous proliferating cells some of which were identified as B-cells (double positive CD20 + /Ki67 + cells in c, arrows) and some as CD8 + T-cells (double positive CD8 + /Ki67 + cells in the inset in c).
  • Double immunostainings for CD8 (red) and CD20, CD8 (red) and Ig (green), and CD8 (green) and BFRFl (red) show that CD8 + T cells with an activated, lymphoblastoid morphology cluster around and extend cytoplasmic processes that contact CD20 + B- cells (d), Ig + plasma cells (e) and BFRFl + cells (f and insets), (g)
  • the presence of perform granules (green) in a CD8 + T-cell (red) identified in the same BFRFl + perivascular cuff shown in panels b and c and in Fig.2k confirms the cytotoxic activity of CD8 + T-cells.
  • OCBs oligoclonal IgG bands
  • An additional control for binding specificity included CSF from the MS 102 case that was blotted onto casein-coated nitrocellulose paper (absence of reactivity) (lane 5). Faint (lane 1) and and both faint and strong (lane 2) EBV-specific OCBs are present in MS 154 and MS 102 cases, respectively (arrows indicate OCBs).
  • MS 102 was also positive for EBV DNA and had the highest frequency of EBER + and CD8 + cells in both white matter lesions and meninges among the EBV-low MS cases.
  • MS cases with and without ectopic follicles were a posteriori classified as EBV-high and EBV-low, respectively.
  • the graphs show significant differnces in the number of CD20+, EBER+ and CD 8+ cells in the white matter (A) and meninges (B) between the two groups.
  • Dot points represent values for each MS case. Each value represents the mean of cell counts performed in adjacent brain sections (2 sections for each marker) for each brain tissue block; 1-2 blocks for each MS case were analysed.
  • EBV Epstein-Barr virus
  • Ectopic B cell follicles stained with the pan B-cell marker CD20 in the thyroid of patients affected by Hashimoto's thyroiditis A, B. Numerous cells expressing the EBV latency protein LMPl were observed inside ectopic B follicles (C and inset; the same follicle shown in C is circled in red in panel B), whereas immunopositivity for EBNA2 was restricted to a small number of cells distributed at the edge of the follicle and in areas of hyperplasia (D, E). Numerous cells expressing the replication factor BALF2 (F) and the early lytic cycle-associated proteins BMRFl and BFRFl (G, H) were found within and around the B cell follicles. Conversely, cells expressing the most abundant protein of the viral envelope, gp350/220, and the viral capsid antigen pl60 were observed only occasionally (I, J, respectively).
  • Ectopic B cell follicles stained with the pan B-cell marker CD20 in the thymus of patients affected by myasthenia gravis (A-C).
  • In situ hybridization for EBER reveals the presence of numerous EBV-infected cells inside ectopic follicles (D) and as well as in sparse B-cell inflitrates (E).
  • Rarely, isolated EBNA2+ cells were observed at the edge of ectopic follicles (F); the latter comprised numerous cells positive for the EBV latency protein LMPl (G and inset. Numerous cells expressing the early lytic cycle-associated proteins BMRFl and BFRFl were found within and outside the B cell follicles (H-J).
  • EBV-encoded latent membrane protein 1 cooperates with BAFF/BLyS and APRIL to induce T cell-independent Ig heavy chain class switching. J. Immunol. 171, 5215-5224 (2003).

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Abstract

Une infection aberrante de cellules B par le virus Epstein-Barr (EBV) conduit à une maladie auto-immune, notamment la sclérose en plaques, la myasthénie grave et la thyroïdite d'Hashimoto. Cette maladie pouvant être traitée ou l'immunité contre cette maladie pouvant être acquise.
PCT/EP2008/004992 2007-04-13 2008-04-11 Nouveaux traitements de maladies WO2008125366A2 (fr)

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US20230193398A1 (en) * 2020-04-22 2023-06-22 Nicca Chemical Co., Ltd. Nucleic acid detection method and oligonucleotide probe
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CN114990240A (zh) * 2022-06-01 2022-09-02 昆明理工大学 用于检测妇科疾病外源病原体的多重qPCR检测试剂
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