WO1995012313A1 - Virus associated multiple sclerosis: treatments, prevention and diagnosis thereof - Google Patents
Virus associated multiple sclerosis: treatments, prevention and diagnosis thereof Download PDFInfo
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- WO1995012313A1 WO1995012313A1 PCT/US1994/012655 US9412655W WO9512313A1 WO 1995012313 A1 WO1995012313 A1 WO 1995012313A1 US 9412655 W US9412655 W US 9412655W WO 9512313 A1 WO9512313 A1 WO 9512313A1
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- A61K31/52—Purines, e.g. adenine
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- A61K31/7072—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid having two oxo groups directly attached to the pyrimidine ring, e.g. uridine, uridylic acid, thymidine, zidovudine
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- A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
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- C12N2710/16011—Herpesviridae
- C12N2710/16511—Roseolovirus, e.g. human herpesvirus 6, 7
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- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16511—Roseolovirus, e.g. human herpesvirus 6, 7
- C12N2710/16534—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral
Definitions
- This invention relates to the discovery of the primary viral association for multiple sclerosis [MS].
- the virus is a member of the Herpesvirus family and correlates statistically with patients suffering from MS.
- the invention also provides for methods of treating MS by conventional antiviral therapy and vaccines, for the predominant viral types associated with MS and for methods of diagnosing virally induced MS.
- This invention is the discovery of a virus associated type of multiple sclerosis (MS) and of methods for treating virally induced MS.
- MS multiple sclerosis
- MS is an inflammatory disease of the central nervous system. MS, with onset typically in the 3rd or 4th decade of life, is characterized clinically by a variable relapsing and remitting course and pathologically by the progressive accumulation of plaques of demyelination within the white matter of the brain, optic nerves, and spinal cord. Evidence suggests that the demyelination is mediated by macrophages and subsets of
- T-lymphocytes that are sensitized to components of myelin, which surrounds the axons of the neurons.
- the disease is pleomorphic in its presentation, but classic features include impaired vision, nystagmus, dysarthria, decreased perception of vibration and position sense, ataxia, intention tremor, weakness or paralysis of limbs, spasticity, and bladder problems.
- Conventional diagnosis typically requires proof of at least two episodes of neurologic deficit and lesions at more than one site within the central nervous system.
- adjuncts 1) immunoglobulins are elevated in the CSF
- oligoclonal banding pattern in 70-90% of patients which can vary both between patients or between plaques; 2) magnetic resonance imaging (MRI) or nuclear magnetic resonance (NMR) of the brain and spinal cord reveal lesions in nearly every case of clinically defined MS, especially in periventricular regions; and 3) the visual evoked response is abnormal in about 75% of cases, with a decrease in the initial amplitude and prolongation of the latency of the major positive peak.
- Other ancillary tests which may be useful include a decreased suppressor or
- MBP myelin basic protein
- Herpes simplex virus Type I HSV1
- HSV-2 herpes simplex virus 2
- HHV-6 Human herpesvirus-6 was reportedly found by PCR in only 1/31 patients' peripheral blood leukocytes by PCR, and in 1/24 normal
- herpesvirus with MS as disclosed herein permits clinicians to provide for more definitive diagnoses for persons suspected of having virus associated MS.
- herpesvirus is not thought to be the sole cause of MS.
- the virus is thought to trigger a complex immunological response in the host that produces the symptoms associated with MS.
- the described herpesvirus may not be the sole cause of MS symptoms; but, the results provided herein clearly provide for the first time, statistical evidence that a substantial number of MS patients harbor the virus and that the titers can be higher in the MS patients than in the persons infected with the virus yet not having the disease.
- HHV-6 or their particular subtypes are the primary causal agent of MS in humans.
- the family Herpesviridae includes over 100 double stranded DNA viruses, of which six have been well characterized in humans; HSV-1, HSV-2, human cytomegalovirus (CMV), Varicella Zoster virus (VZV), Epstein-Barr virus (EBV), and HHV-6.
- the members of this family of viruses have been classified into three subfamilies: the Alphaherpesvirinae (including HSV-1, HSV-2, and VZV), the Betaherpesvirinae (CMV, HHV-6), and the
- HHV-6 a member of the Betaherpesvirinae
- Betaherpesvirinae has been demonstrated to share 66% hybridization homology with CMV within a conserved region, but shares little homology with other herpesviruses.
- HHV-6 a recently described member of the Herpesviridae, is the causative agent of exanthema subitem, a viral infection which is highly prevalent in children and can be associated with hepatitis, encephalitis, and
- HHV-6 isolates that are related to the Z29 strain isolated from a patient from Zaire (and associated with most cases of
- HHV-6 has fairly broad tropism to cells including T and B lymphocytes, glial cells, and megakaryocytes and has been detected in the CSF of infected children with neurological symptoms (Ablashi et al . , 1989 Dev. Virol . Stand. 70:139). Buchwald et al . have also reported the detection of actively replicating HHV-6 in the lymphocytes of 70% of 113 patients with a chronic fatigue syndrome characterized by an immunologically mediated
- HHV-6 in a number of diseases other than exanthema subitem and chronic fatigue syndrome, including Hodgkin's disease, inflammatory bowel disease, Sjogren's syndrome, sarcoidosis, retinitis, lymphoma, in conjunction with HTLV-1 leukemia, and coinfecting a high percentage of patients with AIDS (Buchbinder et al . , 1988, J. Virol . Meth. 21 (1-4):191). Except for exanthema subitem, however, the exact role of HHV-6 in the pathogenesis of these diseases has not been established. A report that investigated the role of HHV-6 in MS described screening peripheral blood leukocytes which failed to show an
- Immunosuppressants such as glucocorticoids, adrenocorticotropic hormone (ACTH) to stimulate endogenous glucocorticoid production, azathioprine, cyclophosphamide or cyclosporine have been standard therapy. More recent attempts to modulate the immune response have included therapeutic regimens such as total lymphoid
- interferon- ⁇ increases the exacerbation rate of the disease, whereas both IFN- ⁇ and IFN- ⁇ have been found to inhibit disease activity (Panitch, H.S., 1992, Drugs 44(6):946; Knobler, R., 1988, Neurology 38 (7 Suppl. 2):58; Noronha, A., 1992, Ann. Neurol . 31(1):103).
- HHV-6 is a member of the Herpesviridae that comprises two types: A and B, and within the B strains, types B1 and B2.
- MS is caused by HHV-6 and particular substrains that include characteristics such as nucleic acid changes that are identifiable as genetic markers, quantitative differences in viral titer and localization to particular cell types within the brains of patients with MS.
- a substrain is determined to be neurotropic we designate it HHV-6 n .
- This invention provides for methods for treatment of multiple sclerosis (MS) comprising administering to a patient having virus associated MS a pharmaceutically effective amount of an antiviral agent in a pharmaceutically acceptable carrier said agent wherein the agent is effective against HHV-6 n , with the proviso that the agent not be an interferon unless said interferon is in combination with another antiviral agent or applied directly to cerebrospinal fluids.
- MS multiple sclerosis
- antiviral agent is a nucleoside analogue such as those
- acyclovir selected from the group consisting of acyclovir, cygalovir, and ganciclovir.
- the invention also provides for the use of the anti-herpesvirus agents as identified herein for the manufacture of medicaments or therapuetics for the purpose of treating, preventing or alleviating the symptoms of multiple sclerosis.
- the medicaments themselves are also described herein. Especially preferred are those medicaments designed for intrathecal administration to the cerebrospinal fluid.
- this invention provides for an isolated subtype of the
- Herpesviridae family wherein the virus has the following DNA subsequence in its genome:
- This invention further provides for a method of screening for pharmaceutical agents useful in reducing the symptoms of multiple sclerosis (MS) comprising: contacting a cell infected with HHV-6 with an anti-viral agent; and, assaying the anti-viral agent activity by determining the effect of the agent upon viral titer in the cell.
- the method may be specifically designed to detect a member of the HHV-6 n subfamily of Herpesviridae especially those members having Seq. ID. No. 17.
- the methods can be designed to screen for agents in in vitro assays against cell lines infected with the virus, against cells producing an enzyme from a virus or against a purified viral enzyme. Alternatively, the agents may be screened in in vivo assays where the virus is hosted by a mammal.
- This invention also provides for methods of diagnosising multiple sclerosis (MS) in a patient comprising the step of testing for the presence or absence of HHV-6 in a sample from a patient to be tested for MS.
- the virus to be detected can be any of the viruses described above.
- the method of detecting the virus can optionally involve nucleic acid hybridization methods which are selective and specific for the viruses described herein.
- the detection methods can also involve conventional immunoassays such as Western Blots, enzyme linked immunoassays (ELISA) and radioimmuno assays
- RIA RI
- Preferred tissues samples include nerve tissue such as brain samples.
- the diagnosis for MS can include conventional criteria or diagnostic factors in combination with those disclosed herein. The impact of treatment upon the disease progression can be assessed using these methods at different times.
- a method of prophylaxis for multiple sclerosis by administering to a patient at risk for MS, a vaccine against MS comprising inactivated viral particles or peptides of a virus from HHV-6 in a
- the vaccine is effective against HHV-6 wherein the virus is as described herein and is typified by MSV-1206.
- the invention further provides for DNA probes and PCR primers that are specific to the Herpesviridae family of virus in that they can, under appropriately stringent
- Table 1 provides a list of preferred primers, regions for probe to bind and probes that can distinguish between MS associated HHV-6 and non-MS associated HHV-6.
- This invention also provides for diagnostic kits for the diagnosis of multiple sclerosis (MS) comprising a
- HHV-6 n identified herein as associated with MS such as MSV-1206.
- Figure 1 is a map of the HHV-viral genome.
- PZVH14 contains a ⁇ indIII site which distinguishes HHV-6 Types A and B.
- the polymerase region is designated "pol”.
- glycoprotein B region is designated "gB”.
- the DNA binding protein region is designated "DBP” and contains a ⁇ indIII site that is diagnostic for virus MSV-1206.
- DBP DNA binding protein region
- glycoprotein region is designated "vg”.
- the glycoprotein H region is designated “gH” and allows the discrimination of HHV-6 Type B group 1 and group 2 viruses.
- the immediate early gene region is designated “IE” and also allows the
- Figure 2 is a map of marker sites within the HHV-6 genome that distinguish between Group B2 viruses. These markers are used to distinguish and characterize the MS virus from those found in controls. DETAILED DESCRIPTION
- antiviral agents Due to the viral nature of the disease, antiviral agents have application here for treatment, such as
- Antiviral agents include agents or
- compositions that directly bind to viral products compositions that directly bind to viral products
- Antiviral agents do not include immunoregulatory agents that do not directly affect viral titer or bind to viral products. Antiviral agents are effective if they inactivate the virus, otherwise inhibit its infectivity or multiplication, or alleviate the symptoms of MS.
- antiherpesvirus agents that will be useful for treating virus-induced MS can be grouped into broad classes based on their presumed modes of action. These classes include agents that act (i) by inhibition of viral DNA
- Antiviral agents may also be used in combination (i.e., together or sequentially) to achieve synergistic or additive effects or other benefits.
- Several compositions have been reported to modulate the immune system and affect the progression of MS. These compounds include ⁇ -interferon and its analog Betaseron.
- the immunosupressant drug cladribine a lympholytic agent, was also reported to be of use for treating MS. All of these drugs are applied intravenously and are not applied directly to cerebrospinal tissue or fluids. In contrast, this
- interferons ⁇ or ⁇
- other compositions would be enhanced if applied directly to such tissue or fluids for patients suffering from HHV-6 infections leading to MS.
- drugs such as ⁇ -interferon, Betaseron and cladribine have antiviral effects
- the direct administration or application of these agents to the cerbrospinal fluids or tissues for treating HHV infections is a part of this
- an agent may act on more than one target in a virus or virus-infected cell or through more than one mechanism
- nucleoside analogs believed to act through inhibition of viral DNA replication, especially through inhibition of viral DNA polymerase. These nucleoside analogs act as alternative substrates for the viral DNA polymerase or as competitive inhibitors of DNA polymerase substrates. Usually these agents are preferentially
- TK viral thymidine kinase
- the analogue may act as a chain terminator, cause increased lability (e. g. , susceptibility to breakage) of analogue-containing DNA, and/or impair the ability of the substituted DNA to act as template for transcription or replication (see, e. g. , Balzarini et al . (1990) Mol . Pharm. 37, 402-7).
- acyclovir is triphosphorylated to its active form, with the first phosphorylation being carried out by the herpes virus thymidine kinase, when present.
- Other examples are the reported conversion of the compound HOE 602 to ganciclovir in a three-step metabolic pathway (Winkler et al . (1990)
- ganciclovir is preferred over acyclovir for treatment of CMV infection because CMV does not express a viral thymidine kinase that phosphorylates acyclovir.
- HHV-6 In the case of HHV-6, early reports suggest that HHV- 6 is more sensitive to ganciclovir than to acyclovir, although acyclovir does inhibit HHV-6 viral DNA replication (Di Luca et al. (1990) Virology 175 (1) : 199-210; Burns and Sandford, J. Infect. Dis. (1990) 162 (3) : 634-7).
- acyclovir does inhibit HHV-6 viral DNA replication
- Anti-herpesvirus medications suitable for treating viral induced MS include, but are not limited to, nucleoside analogs including acyclic nucleoside phosphonate analogs
- phosphonylmethoxyalkylpurines and -pyrimidines e.g. phosphonylmethoxyalkylpurines and -pyrimidines
- cyclic nucleoside analogs e.g. , phosphonylmethoxyalkylpurines and -pyrimidines
- drugs such as:
- vidarabine (9- ⁇ -D-arabinofuranosyladenine; adenine
- HPMPA [(S)-9-(3- hydroxy-2-phosphonylmethoxypropyl)adenine] and its cyclic form (cHPMPA);
- S)-HPMPDAP [ (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)-2,6-diaminopurine];
- PMEDAP [9-(2-phosphonyl-methoxyethyl)-2,6-diaminopurine]; HOE 602 [2-amino- 9-(1,3-bis(isopropoxy)-2-propoxymethyl)purine];
- PMEA [9-(2-phosphonylmethoxyethyl)adenine]; bromovinyl-deoxyuridine
- acyclovir 9-([2-hydroxyethoxy]methyl)guanine; e.g. , Zovirax (Burroughs Wellcome)]; penciclovir (9-[4-hydroxy-2-(hydroxymethyl)butyl]-guanine); ganciclovir [(9- [1,3-dihydroxy-2 propoxymethyl]-guanine) e.g. , Cymevene,
- Cytovene (Syntex), DHPG (Stals et al . (1993) Antimicrobial Agents Chemother. 37(2):218-23]; isopropylether derivatives of ganciclovir (see, e.g. , Winkelmann et al . , 1988, Drug Res. 38, 1545-48); cygalovir; famciclovir [2-amino-9-(4-acetoxy-3- (acetoxymethyl)but-1-yl)purine (Smithkline Beecham)];
- valacyclovir (Burroughs Wellcome); desciclovir [(2-amino-9-(2-ethoxymethyl)purine)] and 2-amino-9-(2-hydroxyethoxymethyl)-9H-purine, prodrugs of acyclovir]; CDG (carbocyclic 2'-deoxyguanosine); and purine nucleosides with the
- cyclobut-A [(+-)-9-[1 ⁇ , 2 ⁇ , 3 ⁇ )-2,3-bis(hydroxymethyl)-1-cyclobutyl]adenine]
- cyclobut-G [(+-)-9-[1 ⁇ ,2 ⁇ ,3 ⁇ )-2,3-bis(hydroxymethyl)-1-cyclobutyl]guanine]
- BHCG (R)- (1 ⁇ , 2 ⁇ ,1 ⁇ )-9-(2,3-bis(hydroxymethyl)cyclobutyl]guanine]
- an active isomer of racemic BHCG, SQ 34,514 [1R-1 ⁇ ,2 ⁇ ,3 ⁇ )-2-amino-9-[2,3-bis(hydroxymethyl)cyclobutyl]-6H-purin-6-one (see, Braitman et al. (1991) Antimicrob. Agents and
- Triciribine and triciribine monophosphate are potent inhibitors against HHV-6 (Ickes et al . , Seventh International Conf. on Antiviral Research 1994, Abstract No. 122 in
- An exemplary protocol for these agents is an intravenous injection of about 0.35 mg/meter 2 (0.7 mg/kg) once weekly or every other week for at least two doses, preferably up to about four to eight weeks.
- Acyclovir and ganciclovir are of particular interest and are preferred because of their accepted use in clinical settings.
- Acyclovir an acyclic analogue of guanine, is phosphorylated by a herpesvirus thymidine kinase and undergoes further phosphorylation to be incorporated as a chain
- herpesvirus replication It has therapeutic activity against a broad range of herpesviruses, including HHV-6, Herpes simplex Types 1 and 2, Varicella- Zoster, Cytomegalovirus, and Epstein-Barr Virus, and is used to treat disease such as herpes
- Exemplary intravenous dosages or oral dosages for MS are 250 mg/kg/m 2 body surface area, every 8 hours for 7 days, or maintenance doses of 200-400 mg IV or orally twice a day to suppress recurrence.
- Ganciclovir has been shown to be more active than acyclovir against herpesviruses, including HHV-6 and CMV. See, e.g. , Oren and Soble, 1991, Clinical Infectious Diseases 14:741-6.
- Treatment protocols for ganciclovir are 5 mg/kg twice a day IV or 2.5 mg/kg three times a day for 10-14 days. Maintenance doses are 5-6 mg/kg for 5-7 days.
- HPMPC HPMPC is reported to be more active than either acyclovir or
- HSV-1, HSV-2, TK- HSV, VZV or CMV infections in animal models (De Clercq, supra).
- Compounds with anti-CMV activity are likely to be particularly useful in treatment of viral induced MS because, apart from HHV- 7, CMV appears to be the
- herpesvirus most closely related to HHV- 6.
- Nucleoside analogs such as BVaraU are potent inhibitors of HSV-1, EBV, and VZV that have greater activity than acyclovir in animal models of encephalitis.
- fluroidoarbinosyl cytosine and its related fluroethyl and iodo compounds (e.g. , FEAU, FIAU) have potent selective activity against herpesviruses, and HPMPA ((S)-1-([3-hydroxy-2-phosphorylmethoxy]propyl)adenine) has been demonstrated to be more potent against HSV and CMV than acyclovir or
- ganciclovir and are of choice in advanced cases of MS.
- Cladribine (2-chlorodeoxyadenosine) is another nucleoside analogue known as a highly specific antilymphocyte agent
- Cladribine has been found useful in treatment of chronic progressive MS (Sipe et al . , (1994) Lancet 344:9-13).
- 5-thien-2-yl- 2'-deoxyuridine derivatives e.g. , BTDU [5-5(5-bromothien-2 ⁇ yl)-2'-deoxyuridine] and CTDU [b-(5-chlorothien-2-yl)-2'-deoxyuridine]; and OXT-A [9-(2-deoxy-2-hydroxymethyl- ⁇ -D-erythro-oxetanosyl)adenine] and OXT-G [9-(2-deoxy-2-hydroxymethyl- ⁇ -D-erythro-oxetanosyl) guanine].
- 5-thien-2-yl- 2'-deoxyuridine derivatives e.g. , BTDU [5-5(5-bromothien-2 ⁇ yl)-2'-deoxyuridine] and CTDU [b-(5-chlorothien-2-yl)-2'-deoxyuridine]
- OXT-A 9-(2-deoxy-2-hydroxymethyl-
- OXT-G is believed to act by inhibiting viral DNA synthesis its mechanism of action has not yet been elucidated.
- thymidine analogs e.g. , idoxuridine (5-ido-2'-deoxyuridine)
- triflurothymidine have antiherpes viral activity, but due to their systemic toxicity, are largely used for topical herpesviral infections, including HSV stromal keratitis and uveitis, and are not preferred here unless other options are ruled out.
- Bodor (U.S. Patent No. 5,177,064) has described compounds wherein one moiety is the residue of a drug, such as an antiherpesvirus agent, and the second moiety is a drug, such as an antiherpesvirus agent, and the second moiety is a drug, such as an antiherpesvirus agent, and the second moiety is a drug, such as an antiherpesvirus agent, and the second moiety is a drug, such as an antiherpesvirus agent, and the second moiety is a
- phosphonate ester These phosphonate derivatives are adapted for targeted delivery, especially to the brain and will be useful in treatment of virus-induced MS.
- Foscarnet sodium trisodium phosphonoformate, PFA, Foscavir (Astra)
- PAA phosphonoacetic acid
- Foscarnet is an inorganic pyrophosphate analogue that acts by competitively blocking the pyrophosphate-binding site of DNA polymerase.
- Foscarnet is reported to be less toxic than PAA.
- the antiherpes-virus agents described above are believed to act through inhibition of viral DNA polymerase.
- viral replication requires not only the replication of the viral nucleic acid but also the production of viral proteins and other essential elements
- the present invention contemplates treatment of MS by the inhibition of viral proliferation by targeting viral proteins other than DNA polymerase (e . g. , by inhibition of their synthesis or activity, or destruction of viral proteins after their synthesis).
- viral proteins other than DNA polymerase e . g. , by inhibition of their synthesis or activity, or destruction of viral proteins after their synthesis.
- agents that inhibit a viral serine protease e. g. , such as one important in development of the viral capsid will be useful in treatment of viral induced MS.
- a viral serine protease e. g.
- An example of such a serine protease is the HHV-6 homolog of the HSV U L 26 gene product described in EP 0 514 830 A2, which is herein incorporated by reference.
- decarboxylase inhibitors a target of, e.g. , parazofurin
- CTP synthetase inhibitors targets of, e.g., CTP synthetase inhibitors
- cyclopentenylcytosine IMP dehydrogenase
- ribonucleotide reductase a target of, e.g. , carboxyl-containing Nalkyldipeptides as described in U.S. Patent No. 5,110,799 (Tolman et al . , Merck)
- thymidine kinase a target of, e.g. , 1-[2-(hydroxymethyl)cylcoalkylmethyl]-5-substituted -uracils and -guanines as described in, e.g. , U.S. Patent Nos.
- Kutapressin has been recently reported as having efficacy against HHV-6 virus infections.
- Kutapressin is a liver derivative available from Schwarz Parma of Milwaukee,
- HHV-6 is from 200 to 25 mg/ml per day for an average adult of 150 pounds.
- Poly (I) ⁇ Poly(C 12 U) an accepted antiviral drug known as Ampligen from HEM Pharmaceuticals of Rockville, MD has been shown to inhibit HHV-6 and is another antiviral agent suitable for treating MS.
- Intravenous injection is the preferred route of administration. Dosages from about 100 to 600 mg/m 2 are administered two to three times weekly to adults averaging 150 pounds. It is best to administer at least 200 mg/m 2 per week.
- antiviral agents reported to show activity against herpes viruses (e.g. , varicella zoster and herpes simplex) and will be useful for the treatment of viral induced MS include mappicine ketone (SmithKline Beecham); Compounds A, 79296 and A, 73209 (Abbott) for varicella zoster, and
- Interferon has known toxicity problems and it is expected that second generation derivatives will soon be available that will retain interferon's antiviral properties but have reduced side affects.
- viral induced MS may be treated by administering a HHV-6 reactivating agent to induce reactivation of the latent virus.
- a HHV-6 reactivating agent to induce reactivation of the latent virus.
- the reactivation is combined with simultaneous or sequential administration of an anti-HHV-6 agent. Controlled reactivation over a short period of time or reactivation in the presence of an antiviral agent is believed to minimize the adverse effects of certain herpesvirus infections (e.g., as discussed in PCT Application WO 93/04683).
- Reactivating agents include agents such as estrogen, phorbol esters, forskolin and ⁇ -adrenergic blocking agents.
- ganciclovir is an example of a antiviral guanine acyclic nucleoside of the type described in US Patent Nos. 4,355,032 and 4,603,219.
- Acyclovir is an example of a class of antiviral purine derivatives, including 9-(2-hydroxyethylmethyl)adenine, of the type described in U.S. Pat. Nos. 4,287,188, 4,294,831 and 4,199,574.
- Brivudin is an example of an antiviral deoxyuridine derivative of the type described in US Patent No. 4,424,211.
- Vidarabine is an example of an antiviral purine nucleoside of the type described in British Pat. 1,159,290.
- Brovavir is an example of an antiviral deoxyuridine derivative of the type described in US Patent Nos. 4,542,210 and 4,386,076.
- BHCG is an example of an antiviral carbocyclic nucleoside analogue of the type described in US Patent Nos.
- HPMPC is an example of an antiviral phosphonyl methoxyalkyl derivative with of the type described in US
- CDG Carbocyclic 2'-deoxyguanosine
- CDG Carbocyclic 2'-deoxyguanosine
- Amira describes the use of creatine analogs as antiherpes viral agents.
- U.S. Patent No. 5,306,722 (Kim et al . ; Bristol-Meyers Squibb) describes thymidine kinase inhibitors useful for treating HSV infections and for inhibiting herpes
- inhibitory nucleic acid therapeutics which can inhibit the activity of HHV-6 viruses in patients with MS.
- Inhibitory nucleic acids may be
- RNA-RNA single-stranded nucleic acids, which can specifically bind to a complementary nucleic acid sequence.
- an RNA-RNA, a D ⁇ A-D ⁇ A, or R ⁇ A-D ⁇ A duplex or triplex is formed.
- These nucleic acids are often termed “antisense” because they are usually complementary to the sense or coding strand of the gene, although recently approaches for use of "sense” nucleic acids have also been developed.
- inhibitory nucleic acids refers to both “sense” and “antisense” nucleic acids.
- the inhibitory nucleic acid By binding to the target nucleic acid, the inhibitory nucleic acid can inhibit the function of the target nucleic acid. This could, for example, be a result of blocking D ⁇ A transcription, processing or poly(A) addition to mR ⁇ A, D ⁇ A replication, translation, or promoting inhibitory mechanisms of the cells, such as promoting R ⁇ A degradation.
- Inhibitory nucleic acid methods therefore encompass a number of different approaches to altering expression of, for example, HHV-6 n genes that operate by different mechanisms. These different types of inhibitory nucleic acid technology are described in Helene, C. and Toulme, J., 1990, Biochim. Biophys . Acta . 1049:99-125, which is hereby incorporated by reference and is referred to hereinafter as "Helene and
- inhibitory nucleic acid therapy approaches can be classified into those that target DNA sequences, those that target RNA sequences (including pre -mRNA and mRNA), those that target proteins (sense strand approaches), and those that cause cleavage or chemical modification of the target nucleic acids.
- Nucleic acids can be designed to bind to the major groove of the duplex DNA to form a triple helical or "triplex" structure.
- inhibitory nucleic acids are designed to bind to regions of single stranded DNA
- inhibitory nucleic acids are designed to bind to mRNA or mRNA precursors. Inhibitory nucleic acids are used to prevent maturation of pre-mRNA. Inhibitory nucleic acids may be designed to interfere with RNA
- the inhibitory nucleic acids can be targeted to mRNA.
- the inhibitory nucleic acids are designed to specifically block translation of the encoded protein.
- the inhibitory nucleic acid can be used to selectively suppress certain cellular functions by inhibition of translation of mR ⁇ A encoding critical proteins.
- an inhibitory nucleic acid complementary to regions of c-myc mR ⁇ A inhibits c-myc protein expression in a human promyelocytic leukemia cell line, HL60, which
- inhibitory nucleic acids introduced into the cell can also encompass the "sense" strand of the gene or mRNA to trap or compete for the enzymes or binding proteins
- inhibitory nucleic acids can be used to induce chemical inactivation or cleavage of the target genes or mRNA. Chemical inactivation can occur by the induction of crosslinks between the inhibitory nucleic acid and the target nucleic acid within the cell. Other chemical modifications of the target nucleic acids induced by appropriately derivatized inhibitory nucleic acids may also be used.
- Cleavage, and therefore inactivation, of the target nucleic acids may be effected by attaching a substituent to the inhibitory nucleic acid which can be activated to induce cleavage reactions.
- the substituent can be one that affects either chemical, or enzymatic cleavage.
- cleavage can be induced by the use of ribozymes or catalytic RNA.
- the inhibitory nucleic acids would comprise either naturally occurring RNA (ribozymes) or synthetic nucleic acids with catalytic activity.
- inhibitory nucleic acids to specific cells of the immune system by conjugation with targeting moieties binding receptors on the surface of these cells can be used for all of the above forms of inhibitory nucleic acid therapy.
- This invention encompasses all of the forms of inhibitory nucleic acid therapy as described above and as described in Helene and Toulme.
- This invention relates to the targeting of inhibitory nucleic acids to sequences of HHV-6 for use in treating MS.
- An example of an antiherpes virus inhibitory nucleic acid is ISIS 2922 (ISIS Pharmaceuticals) which has activity against CMV [see, Biotechnology News 14(14) p. 5].
- antiherpes drugs e.g. , combinations of zidovudine [3'-azido-3'- oxythymidine, AZT] with HPMPC, ganciclovir, foscarnet or acyclovir or of HPMPC with other antivirals.
- antiherpes drugs e.g. , combinations of zidovudine [3'-azido-3'- oxythymidine, AZT] with HPMPC, ganciclovir, foscarnet or acyclovir or of HPMPC with other antivirals.
- antiherpes drugs e.g. , combinations of zidovudine [3'-azido-3'- oxythymidine, AZT] with HPMPC, ganciclovir, foscarnet or acyclovir or of HPMPC with other antivirals.
- induction with ganciclovir followed by maintenance with foscarnet has been suggested as a way to maximize efficacy while
- the antiviral compositions for treating MS are preferably administered to human patients via oral,
- Intrathecal administration is also contemplated or are administered as discussed above in connection with the
- compositions to be employed by the physician are administered to the physician.
- a preferred route is via a permanent intrathecal catheter for administration (both intermittent and continuous) of antiviral agents.
- compositions of this invention may be in the dosage form of solid, semi-solid, or liquid such as, e.g. , suspensions, aerosols or the like.
- the compositions are administered in unit dosage forms suitable for single administration of precise dosage amounts.
- the compositions may also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration.
- the diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological saline,
- compositions or formulation may also include other carriers, adjuvants; or nontoxic,
- nontherapeutic, nonimmunogenic stabilizers and the like are nontherapeutic, nonimmunogenic stabilizers and the like.
- Effective amounts of such diluent or carrier are those amounts which are effective to obtain a pharmaceutically acceptable formulation in terms of solubility of components, or
- immunosuppressive therapies that can modulate the immunologic dysfunction that arises from the presence of viral infected nerve tissue.
- agents that block the immunological attack of the viral infected brain cells will ameliorate the neurological symptoms of MS and/or reduce the disease progress.
- Such therapies include antibodies that specifically block the targeting of viral infected cells.
- Such agents include antibodies which bind to cytokines that upregulate the immune system to target viral infected cells.
- the antibody may be administered to a patient either singly or in a cocktail containing two or more antibodies, other therapeutic agents, compositions, or the like,
- Immunosuppressive agents useful in suppressing allergic reactions of a host.
- Immunosuppressive agents of interest include prednisone, prednisolone, DECADRON (Merck, Sharp & Dohme, West Point, PA), cyclophosphamide,
- cyclosporine 6-mercaptopurine, methotrexate, azathioprine and i.v. gamma globulin or their combination.
- Potentiators of interest include monensin, ammonium chloride and chloroquine. All of these agents are administered in generally accepted efficacious dose ranges such as those disclosed in the Physi cian Desk Reference, 41st Ed. (1987), Publisher Edward R.
- Immune globulin from persons previously infected with HHV-6 or related viruses can be obtained using standard techniques. Appropriate titers of antibodies are known for this therapy and are readily applied to the treatment of MS. Immune globulin can be administered via parenteral injection or by intrathecal shunt. In brief, immune globulin
- preparations may be obtained from individual donors who are screened for antibodies to HHV-6, and plasmas from
- high-titered donors are pooled.
- plasmas from donors are pooled and then tested for antibodies to HHV-6; high-titered pools are then selected for use in MS patients.
- Antibodies may be formulated into an injectable preparation.
- Parenteral formulations are known and are suitable for use in the invention, preferably for i.m. or i.v. administration.
- the formulations containing therapeutically effective amounts of antibodies or immunotoxins are either sterile liquid solutions, liquid suspensions or lyophilized versions and optionally contain stabilizers or excipients.
- Lyophilized compositions are reconstituted with suitable diluents, e.g. , water for injection, saline, 0.3% glycine and the like, at a level of about from .01 mg/kg of host body weight to 10 mg/kg where appropriate.
- suitable diluents e.g. , water for injection, saline, 0.3% glycine and the like, at a level of about from .01 mg/kg of host body weight to 10 mg/kg where appropriate.
- the diluents e.g. , water for injection, saline, 0.3% gly
- compositions containing the antibodies or immunotoxins will be administered in a therapeutically
- a preferred therapeutically effective dose of the pharmaceutical composition containing antibody or immunotoxin will be in a range of from about 0.01 mg/kg to about 0.5 mg/kg body weight of the treated mammal administered over several days to two weeks by daily
- Antibody may be administered systemically by injection i.m., subcutaneously, intrathecally or
- a permanent intrathecal catheter would be a convenient means to administer therapeutic antibodies. The dose will be dependent upon the properties of the antibody or immunotoxin employed, e . g., its activity and biological half-life, the concentration of antibody in the formulation, the site and rate of dosage, the clinical tolerance of the patient involved, the disease afflicting the patient and the like as is well within the skill of the physician.
- the antibody of the present invention may be administered in solution.
- the pH of the solution should be in the range of pH 5 to 9.5, preferably pH 6.5 to 7.5.
- the antibody or derivatives thereof should be in a solution having a suitable pharmaceutically acceptable buffer such as
- the solution of antibody may also contain a salt, such as sodium chloride or potassium chloride in a concentration of 50 to 150 mM.
- a stabilizing agent such as an albumin, a globulin, a gelatin, a protamine or a salt of protamine may also be included and may be added to a solution containing antibody or immunotoxin or to the composition from which the solution is prepared.
- Antibody or immunotoxin may also be administered via microspheres, liposomes or other microparticulate delivery systems placed in certain tissues including blood.
- the dosages of compounds used in accordance with the invention vary depending on the class of compound and the condition being treated.
- the dosage of an immunoglobulin can range from about 0.1 milligram per kilogram of body weight per day to about 10 mg/kg per day for
- the immunoglobulin can be administered once daily as an intravenous infusion.
- the dosage is repeated daily until either a therapeutic result is achieved or until side effects warrant discontinuation of therapy.
- the dose should be sufficient to treat or ameliorate symptoms or signs of MS without producing unacceptable toxicity to the patient.
- An effective amount of the compound is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer.
- the dosing range varies with the compound used, the route of administration and the potency of the particular compound.
- RDA representational difference analysis
- PCR polymerase chain reaction
- adaptors are present on the 5' ends of the nucleic acid fragments from the tester population and not present on fragments from the driver population.
- the driver (in excess) and tester nucleic acid fragments are combined under hybridization conditions.
- tester molecules that self-anneal contain adaptors on both ends, so that on subsequent PCR, only these molecules amplify from the complex reaction mix.
- Molecules in common between driver and tester form heteroduplexes lacking one priming site, so they are not exponentially amplified.
- the driver sequences contain no priming sites, so they are not amplified during PCR.
- high molecular weight DNA of the same genotype in the tester and driver was used, with spiked model pathogens in the tester (adenovirus-2, bacteriophage lambda).
- RDA Retriet al . , supra at page 949.
- the RDA proponents stated that, "[t]hese DNA's [tester and driver DNA] cannot derive from unrelated individuals, as the abundant polymorphic differences in their DNA's would obscure the detection of the pathogen.” Id.
- RDA was developed using the same genotype as driver and tester, in many cases with human samples, constitutional non-diseased tissue cannot be obtained simultaneously with diseased tissue from the same patient.
- This invention is based upon an improvement of the above technology where a pooled nucleic acid driver is
- PARFs polymorphic amplifiable restriction fragments
- “Healthy” driver and tester (with suspected pathogens) nucleic acid sources are each separately fragmented (e.g., endonucleases) and linked to adaptors which can be hybridized to PCR primers.
- the driver and tester are each separately fragmented (e.g., endonucleases) and linked to adaptors which can be hybridized to PCR primers.
- the driver and tester are each separately fragmented (e.g., endonucleases) and linked to adaptors which can be hybridized to PCR primers.
- the adaptors are removed from the amplicons of the both tester and driver fragments and new adaptors are ligated to the 5' ends of the testers amplicon fragments which are then combined with the driver fragments present in excess.
- the tester and driver amplicons are denatured and permitted to reanneal.
- the pathogenic DNA can only anneal to itself and is the only amplifiable material in the sample.
- HHV- 6 can be propagated in vitro.
- MSV-1206 and other like strains inducing MS can be propagated by the standard techniques for growing HHV-6 strains (Ablashi, D.V., 1991, etal. Virology 184 :545-552). Briefly, PHA stimulated cord blood mononuclear cells,
- macrophage, neuronal, or glial cell lines are cocultivated with cerebrospinal fluid, plasma, peripheral blood leukocytes, or tissue extracts containings viral infected cells or
- the recipient cells are treated with 5 ⁇ g/ml polybrene for 2 hours at 37° C prior to infection. Infected cells are observed by demonstrating morphological changes, as well as being positive for HHV-6 antigens using HHV-6
- the virus is either harvested directly from the culture fluid by direct centrifugation, or the infected cells are harvested, homogenized or lysed and the virus is separated from cellular debris and purified by standard methods of isopycnic sucrose density gradient
- the detection of the herpesvirus and the diagnosis of virus associated MS are essentially identical processes.
- the basic principle is to detect the virus using specific ligands that bind to the virus but not to other proteins or nucleic acids in a normal human cell or its environs.
- the ligands can either be nucleic acid or antibodies.
- the ligands can be naturally occurring or genetically or physically modified such as nucleic acids with non-natural or antibody derivatives, i.e., Fab or chimeric antibodies.
- Samples from patients with MS can be taken wherein the virus or evidence of the virus is suspected (e.g., antibodies to the virus) or where the nucleic acid to be detected is likely to be resident.
- the samples are taken from blood (cells, serum and/or plasma), cerebrospinal fluid (CSF), or, most preferably, from tissue in the brain.
- An intrathecal catheter would be a convenient means to sample and monitor virus.
- the most accurate diagnosis for MS will occur if elevated titers of the virus are detected in the brain or CSF, which is the focal point of the disease.
- the detection of high titers of the virus in the brain is by itself indicative of the disease, though confirmation of other MS diagnostic factors are also desirable such as those already known.
- MS is also indicated if the indicative nucleic acid sequence (as opposed to antibodies) is detected in samples from other tissue locations.
- MS is further indicated if antibodies to the virus are detected and at least one of the other diagnostic factors for MS is
- the invention can be used in conjunction with other tests to corroborate the diagnosis of MS.
- application of the invention in addition to certain laboratory parameters which are generally associated with MS can be used to correlate with MS.
- the finding of elevated immunoglobulin levels in the CSF is diagnostically corroborative and can be used in conjunction with the
- demyelination and the visual evoked response, which may show a decreased initial amplitude and a prolonged latency of the major positive peak.
- tests which can corroborate the diagnosis of MS include those that detect decreased suppressor or cytotoxic T cell activity and
- the detection and assay of viral titer is also useful for measuring the progression of the disease or the effect of treatment. By measuring viral titers at different points, one can assess the progress or treatment of the disease.
- MS is known to vary in frequency by geographic location. We can therefore expect that different substrains of HHV-6 that cause MS will differ in nucleotide sequences detectable as markers, as well as in pathogenicity. Included are markers that identify the substrains that cause MS in the Los Angeles, California population, which are typified by MSV-1206.
- HHV-6n Because there are different substrains of HHV-6n, it is useful to identify, study and classify these substrains. Any strain of HHV- 6, including variants described here and elsewhere, may be detected for in general assays, including those useful to discriminate between type A and type B, group 1 and group 2. Several HHV substrains have been described. See, for example, Aubin et al . , 1991, J. Clin. Microbiol .
- the assay is performed on samples taken during periods of active disease as opposed to periods of remission, including from such sources as CSF, brain biopsies or peripheral blood.
- the diagnostic assays utilize nucleic acid assays such as nucleic acid hybridization assays and assays which detect amplification of specific nucleic acid to detect for a nucleic acid sequence of HHV-6 n , and particularly HHV-6B2 strains by detecting for one or more of the markers described herein and/or sequences set out in Table 1.
- a preferred marker is named MDBP providing a HindIII marker in the DNA binding protein gene. Sequences homologous to those, as described below, will be understood to be effective as well. Marker MDPB was found in 10/27 MS samples and 2/26 control samples.
- MSV-1206 is a herpes-like agent identified herein which contains nucleic acid sequences representing Sequence ID NOS. 17 (MDPB), 25 (38A), 31 (38E), 33 (38EP), 39 (38D,BST711) and 45 (UGL, UrGly Nla1), It is a member of a subset of the HHV-6B group 2 subfamily of HHV-6 viruses.
- MDPB Sequence ID NOS. 17
- 25 38A
- 31 (38E) 31
- 38EP 33
- 39 38D,BST711
- UNL UrGly Nla1
- sequence listing corresponds to its complementary sequence and those described including allowances for minor sequencing errors, single base changes, deletions, substitutions and the like, such that any such sequence variation corresponds to the nucleic acid sequence of the pathogenic organism or disease marker to which the relevant sequence listing relates.
- nucleic acid sequence “homologous to” or
- a DNA sequence which is homologous to a target sequence can include sequences which are shorter or longer than the target sequence so long as they meet the functional test set forth. Hybridization conditions are specified along with the source of the cDNA library.
- the hybridization is done in a Southern blot protocol using a 0.2XSSC, 0.1% SDS, 65°C wash.
- SSC refers to a citrate-saline solution of 0.15 M sodium chloride and 20 mM sodium citrate. Solutions are often expressed as multiples or fractions of this concentration. For example, 6XSSC refers to a solution having a sodium chloride and sodium citrate concentration of 6 times this amount or 0.9 M sodium chloride and 120 mM sodium citrate.
- 0.2XSSC refers to a solution 0.2 times the SSC concentration or 0.03 M sodium chloride and 4 mM sodium citrate.
- primers are designed to target a specific portion of the nucleic acid of the targeted agent. For example, primers for amplifying a subsequence for
- MSV-1206 including any of the subsequences having one of the genetic markers identified herein or as set out in Sequence ID Nos. 1, 14, 24 and 25, would be effective.
- Preferred primers are those set out in Table 1.
- the primers are about 14 to about 24 nucleotides in length. From the
- Target specific probes may be used in the nucleic acid hybridization diagnostic assays for MS.
- the probes are specific for or complementary to the target of interest.
- probes to one of the nucleic acid sequences for MSV-1206 would be effective.
- nucleic acid probes are about 50 to about 1000 nucleotides, most preferably about 200 to about 400 nucleotides.
- a sequence is "specific" for a target organism of interest if it includes a nucleic acid sequence which when detected is determinative of the presence of the organism in the presence of a heterogeneous population of proteins and other biologies.
- a specific nucleic acid probe is targeted to that portion of the sequence which is determinative of the organism and will not hybridize to other sequences especially those of the host where a pathogen is being detected.
- the specific nucleic acid probe can be RNA or D ⁇ A polynucleotide or oligonucleotide, or their analogs.
- the probes may be single or double stranded nucleotides.
- the probes of the invention may be synthesized enzymatically, using methods well known in the art (e.g., nick translation, primer extension, reverse transcription, the polymerase chain reaction, and others) or chemically (e.g. , by methods such as the ph ⁇ sphoramidite method described by Beaucage and
- the probe must be of sufficient length to be able to form a stable duplex with its target nucleic acid in the sample, i . e. , at least about 14 nucleotides, and may be longer (e. g. , at least about 50 or 100 bases in length). Often the probe will be more than about 100 bases in length. For example, when probe is prepared by nick-translation of D ⁇ A in the presence of labeled nucleotides the average probe length may be about 100-600 bases.
- the probe will be capable of specific hybridization to a specific MS viral nucleic acid.
- Such "specific hybridization” occurs when a probe hybridizes to a target nucleic acid, as evidenced by a detectable signal, under conditions in which the probe does not hybridize to other nucleic acids (e.g. , animal cell or other bacterial nucleic acids) present in the sample.
- nucleic acids e.g. , animal cell or other bacterial nucleic acids
- a variety of factors including the length and base composition of the probe, the extent of base mismatching between the probe and the target nucleic acid, the presence of salt and organic solvents, probe concentration, and the temperature affect hybridization, and optimal hybridization conditions must often be determined empirically. For discussions of nucleic acid probe design and annealing conditions, see, for example, Sambrook, supra,
- the probe will have considerable sequence identity with the target nucleic acid.
- sequence identity required for specific hybridization will depend on the length of the probe and the hybridization conditions, the probe will usually have at least 70% identity to the target nucleic acid, more usually at least 80% identity, still more usually at least 90%
- a probe can be identified as capable of hybridizing specifically to its target nucleic acid by hybridizing the probe to a sample treated according the protocol of this invention where the sample contains both target virus and animal cells (e.g., nerve cells).
- a probe is specific if the probe's characteristic signal is associated with HHV-6 n DNA in the sample and not generally with the DNA of the host cells and non-biological materials (e.g. , substrate) in a sample.
- a specific probe e.g. , a fluorescently labeled DNA probe
- nucleic acid hybridizations i.e., in si tu, Southern, or other
- detergents e. g. , sodium dodecyl sulfate
- chelating agents e. g. , EDTA
- other reagents e. g. , buffers, Denhardt's solution, dextran sulfate
- the probes can be tested on host cells containing the MS-associated virus and compared with the results from cells containing non MS-assocated virus.
- a convenient method for determining whether a probe is specific for a MS-associated viral nucleic acid utilizes a Southern blot (or Dot blot) using DNA prepared from one or more HHV-6 viruses. Briefly, to identify a target specific probe DNA is isolated from the virus such as
- Test DNA either viral or cellular is transferred to a solid (e.g. , charged nylon) matrix.
- the probes are labelled following conventional methods. Following denaturation and/or
- the probe is
- Stringent hybridization conditions will depend on the probe used and can be estimated from the calculated T m (melting temperature) of the hybridized probe (see, e.g. , Sambrook for a description of calculation of the T m ).
- T m melting temperature
- hybridization conditions is hybridization in a solution containing denatured probe and 5x SSC at 65°C for 8-24 hours followed by washes in 0.1x SSC, 0.1% SDS (sodium dodecyl sulfate) at 50-65°C.
- the temperature and salt concentration are chosen so that the post hybridization wash occurs at a temperature that is about 5°C below the T M of the hybrid.
- the temperature may be selected that is 5°C below the T M or conversely, for a particular temperature, the salt
- a preferred method for detecting MS associated HHV-6 is the use of PCR and/or dot blot hybridization.
- the presence or absence of an MS causal agent as a diagnosis for MS includes Southern transfers, solution hybridization or non-radioactive detection systems, all of which are well known to those of skill in the art. Hybridization is carried out using probes. Visualization of the hybridized portions allows the qualitative determination of the presence or absence of the causal agent.
- RNA transfer may be used for the detection of message in samples of RNA or reverse
- transcriptase PCR and cDNA can be detected by methods
- a preferred method for diagnosing MS is to first determine if the person suspected of having MS is hosting a herpesvirus and then determining if the virus has any of the above described genetic markers associated with previous MS patients.
- a preferred method of detecting for the presence or absence of the HindIII site found within the major DNA binding protein of MSV-1206 See Figure 2 and the Example section below). This detection can be readily accomplished, for example, by using PCR primers that flank the MSV-1206 HindIII site, such as two of the primers provided in Sequence ID ⁇ os. 13-16 to amplify a portion of the MSV-1206 sequence bearing that site in a patient sample.
- the amplified product can then be subjected to HindIII digestion.
- the products of the digestion can then be separated by, for example, gel
- In situ PCR is described in ⁇ euvo et al . , 1993, Intracellular localization of polymerase chain reaction (PCR)-amplified Hepatitis C cD ⁇ A, American Journal of Surgical Pathology, 17 (7) , 683-690; Bagasra et al . , 1992, Detection of Human Immunodeficiency virus type 1 provirus in mononuclear cells by in situ polymerase chain reaction J. New England Journal of Medicine, 326(21) , 1385-1391; and Heniford et al . , 1993, Variation in cellular EGF receptor mRNA
- in situ hybridization assays are well known and are generally described in Angerer, et al., 1987, Methods Enzymol . 152:649-660 incorporated by reference herein.
- in situ hybridization cells are fixed to a solid support, typically a glass slide. The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of target-specific probes that are labelled.
- the probes are preferably labelled with radioisotopes or
- lymphocyte/100,000 lymphocyte/100,000. Cone et al., 1993, J. Clin. Micro. 31: (5) 1262.
- antibodies to HHV-6 can be used to detect for the agent in the sample.
- the sequence being targeted is expressed in transfected cells, preferably
- immunoassays include competitive immunoassays, radioimmunoassays, Western blots, ELISA, indirect
- Monoclonal antibodies or recombinant antibodies may be obtained by various techniques familiar to those skilled in the art. Briefly, spleen cells or other lymphocytes from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (see, Kohler and
- Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal
- antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host. New techniques using recombinant phage antibody expression systems can also be used to generate monoclonal antibodies. See for example: McCafferty, J et al. 1990, Nature, 348: 552 ; Hoogenboom, H.R. et al ., 1991, Nuc. Acids Res. 19:4133, 1991; and Marks, J.D. et al . 1991. J. Mol Biol . 222: 581 - 597.
- Monoclonal antibodies prepared against HHV-6 have been described, see for example, Balachandran et al . , 1989, J. Virol . 63:2835-2840. It is preferred, however, that
- monoclonal antibodies be raised to peptides derived from
- Such peptides may be produced by expressing the specific sequence in a recombinantly engineered cell such as bacteria, yeast, filamentous fungal, insect (especially employing baculoviral vectors), and mammalian cells.
- a recombinantly engineered cell such as bacteria, yeast, filamentous fungal, insect (especially employing baculoviral vectors), and mammalian cells.
- promoter which is either constitutive or
- the vectors are suitable for replication or integration in either prokaryotes or eukaryotes.
- Typical cloning vectors contain antibiotic resistance markers, genes for selction of
- transformants inducible or regulatable promoter regions, and translation terminators that are useful for the expression of viral genes.
- Suitable eukaryote hosts may include plant cells, insect cells, mammalian cells, yeast, and filamentous fungi.
- peptides described herein produced by recombinant technology may be purified by standard techniques well known to those of skill in the art. Recombinantly produced viral sequences can be directly expressed or
- the protein is then purified by a combination of cell lysis (e.g. , sonication) and affinity chromatography. For fusion products, subsequent digestion of the fusion protein with an appropriate proteolytic enzyme releases the desired peptide.
- the purified viral protein or other peptides when described as “isolated” or “substantially pure” describes a protein that has been separated from components which
- a monomeric protein is substantially pure when at least about 85% or more of a sample exhibits a single polypeptide backbone. Minor variants or chemical modifications may typically share the same
- Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by
- the proteins may be purified to substantial purity by standard techniques well known in the art, including selective precipitation with such substances as ammonium sulfate, column chromatography, immunopurification methods, and others. See, for instance, R. Scopes, Protein
- the antibodies raised against the viral strain or peptides may be detectably labelled, utilizing conventional labelling techniques well-known to the art.
- antibodies may be radiolabelled using, for example,
- radioactive isotopes such as 3 H, 125 I, 131 I, and 35 S.
- the antibodies may also be labelled using fluorescent labels, enzyme labels, free radical labels, or bacteriophage labels, using techniques known in the art.
- Typical fluorescent labels include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, alophycocyanin, and Texas Red.
- enzymes may be coupled to other molecules by covalent links, the possibility also exists that they might be used as labels for the production of tracer materials.
- Suitable enzymes include alkaline phosphatase, beta-galactosidase, glucose- 6-phosphate dehydrogenase, maleate dehydrogenase, and peroxidase.
- ELISA enzyme-linked immunosorbent assay
- homogeneous enzyme immunoassay also known as
- EMIT enzyme-multiplied immunoassay
- separation may be achieved, for example, by the use of antibodies coupled to a solid phase.
- the EMIT system depends on deactivation of the enzyme in the tracer-antibody complex; the activity can thus be measured without the need for a separation step.
- chemiluminescent compounds may be used as labels.
- Typical chemiluminescent compounds include
- bioluminescent compounds may be utilized for labelling, the bioluminescent compounds including luciferin, luciferase, and aequorin.
- the antibody may be employed to identify and quantify immunologic counterparts (antibody or antigenic polypeptide) utilizing techniques well-known to the art.
- RIA radioimmunoassay
- HI hemagglutination Inhibition
- CF Complement Fixation
- the serological methods are useful when one wishes to detect antibody to a specific variant. For example, one may wish to see how well a vaccine recipient has responded to the new variant. Alternatively, one may take serum from a patient to see which variant the patient responds to the best. The CF and ELISA tests, in the present standard
- the invention also provides substances suitable for use as vaccines for the prevention of MS and methods for administering them.
- the vaccines are directed against HHV-6, and most preferably, neurotropic HHV-6 such as MSV-1206.
- the vaccines comprise HHV- 6 antigen, most
- HHV-6 preferably peptides encoded by a sequence of a neurotropic HHV-6.
- Vaccines can be made recombinantly. Typically, a vaccine will include from about 1 to about 50 micrograms of antigen or antigenic protein or peptide. More preferably, the amount of protein is from about 15 to about 45 micrograms.
- the vaccine is formulated so that a dose includes about 0.5 milliliters.
- the vaccine may be administered by any route known in the art.
- the route is parenteral. More preferably, it is subcutaneous or intramuscular.
- an antigen can be conjugated to a suitable carrier, usually a protein molecule.
- a suitable carrier usually a protein molecule.
- This procedure has several facets. It can allow multiple copies of an antigen, such as a peptide, to be conjugated to a single larger carrier molecule.
- the carrier may possess properties which facilitate transport, binding, absorption or transfer of the antigen.
- Suitable carriers are the tetanus toxoid, the diphtheria toxoid, serum albumin and lamprey, or keyhole limpet, hemocyanin because they provide the resultant conjugate with minimum genetic restriction.
- Conjugates including these universal carriers can function as T cell clone activators in individuals having very different gene sets.
- conjugation between a peptide and a carrier can be accomplished using one of the methods known in the art. Specifically, the conjugation can use bifunctional cross-linkers as binding agents as detailed, for example, by Means and Feeney, "A recent review of protein modification
- Vaccines against a number of the Herpesviruses have been successfully developed.
- Vaccines against Varicella-Zoster Virus using a live attenuated Oka strain is effective in preventing herpes zoster in the elderly, and in preventing chickenpox in both immunocomprised and normal children (Hardy, I., et al., Inf. Dis. Clin. N. Amer. 4(1):159 (1990); Hardy, I. et al., New Engl . J. Med. 325 (22):1545 (1991); Levin, M.J. et al . , J. Inf. Dis. 166(2):253 (1992); Gershon, A.A., J. Inf. Dis.
- Vaccines against Herpes simplex Types 1 and 2 are also commercially available with some success in protection against primary disease, but have been less successful in preventing the establishment of latent infection in sensory ganglia (Roizman, B. Rev. Inf . Disease, 13 Suppl. 11:S892 (1991); Skinner, G.R. et al . , Med.
- Vaccines against HHV-6 can be made by isolating extracellular viral particles from infected cell cultures, inactivating the virus with formaldehyde followed by
- envelope glycoproteins can be expressed in E. coli or transfected into stable mammalian cell lines, the proteins can be purified and used for vaccination (Lasky, L.A., J. Med. Virol . 31(1):59 (1990)).
- MHC - binding peptides from HHV-6 infected cells can be identified for vaccine candidates per the methodology of Marloes et al . , supra .
- the antigen may be combined or mixed with various solutions and other compounds as is known in the art.
- it may be administered in water, saline or buffered vehicles with or without various adjuvants or immunodiluting agents.
- adjuvants or agents include aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (alum), beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions, oil-in-water emulsions, muramyl dipeptide, bacterial endotoxin, lipid X, Corynebacterium parvum
- polyribonucleotides sodium alginate, lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole, DEAE-dextran, blocked copolymers or other synthetic adjuvants.
- adjuvants are available commercially from various sources, for example, Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.) or Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Michigan), Other suitable adjuvants are Amphigen (oil-in-water), Alhydrogel (aluminum hydroxide), or a mixture of Amphigen and Alhydrogel. Only aluminum is approved for human use.
- the proportion of antigen and adjuvant can be varied over a broad range so long as both are present in effective amounts.
- aluminum hydroxide can be present in an amount of about 0.5% of the vaccine mixture (Al 2 O 3 basis).
- the amount of the antigen can range from about 0.1 ⁇ g to about 100 ⁇ g protein per patient.
- a dose for intramuscular injection for example, would comprise 0.5 ml containing 45 ⁇ g of antigen in admixture with 0.5% aluminum hydroxide.
- the vaccine may be incorporated into a sterile container which is then sealed and stored at a low temperature, for example 4°C, or it may be freeze-dried. Lyophilization permits long-term storage in a stabilized form.
- the vaccines may be administered by any conventional method for the administration of vaccines including oral and parenteral (e. g. , subcutaneous or intramuscular) injection.
- Intramuscular administration is preferred.
- the treatment may consist of a single dose of vaccine or a plurality of doses over a period of time. It is preferred that the dose be given to a human patient within the first 8 months of life.
- the antigen of the invention can be combined with appropriate doses of compounds including influenza antigens, such as influenza type A antigens. Also, the antigen could be a component of a recombinant vaccine which could be adaptable for oral administration.
- Vaccines of the invention may be combined with other vaccines for other diseases to produce multivalent vaccines.
- a pharmaceutically effective amount of the antigen can be employed with a pharmaceutically acceptable carrier such as a protein or diluent useful for the vaccination of mammals, particularly humans.
- Other vaccines may be prepared according to methods well-known to those skilled in the art.
- the epitopes are typically segments of amino acids which are a small portion of the whole protein.
- Such derivatives may include peptide fragments, amino acid substitutions, amino acid deletions and amino acid additions within the amino acid sequence for a virus from HHV-6.
- HHV-6 proteins have significant tertiary structure and the epitopes are usually conformational.
- Therapeutic, intravenous, polyclonal or monoclonal antibodies can been used as a mode of passive immunotherapy of herpesviral diseases including perinatal varicella and CMV.
- Immune globulin from persons previously infected with HHV-6 and bearing a suitably high titer of antibodies against HHV- 6 can be given in combination with antiviral agents (e.g.
- Antibodies to HHV-6 can be administered. Antibodies specific for an epitope expressed on HHV- 6 infected cells are preferred and can be obtained as described above. See also I.B above.
- MS drug screening assays which determine whether or not a drug has activity against HHV-6, and more particularly MSV-1206 are contemplated in this invention.
- Such assays comprise incubating a compound to be evaluated for use in MS treatment with cells which express MS associated HHV-6 viral proteins or peptides and determining therefrom the effect of the compound on the activity of such agent.
- In vitro assays in which the virus is maintained in suitable cell culture are preferred, though in vivo animal models would also be effective.
- Example 4 is typical of these in vitro assays.
- In vitro assays include infecting peripheral blood leukocytes or susceptible T cell lines such as MT-4 with the agent of interest in the presence of varying concentrations of compounds targeted against viral replication, including nucleoside analogs, chain terminators, antisense oligonucleotides and random
- Infected cultures and their supernatants can be assayed for the total amount of virus including the presence of the viral genome by
- a culture of susceptible cells could be infected with HHV-6 in the presence of various concentrations of drug, fixed on slides after a period of days, and examined for viral antigen by indirect immunofluorescence with
- MT-4 cell monolayers can be used for an infectious agent assay using indirect
- purified enzymes isolated from HHV-6 can be used as targets for rational drug design to determine the effect of the potential drug on enzyme activity, such as thymidine
- HHV-6 isolates previously reported, can be assigned to one of two groups on the basis of the following criteria (Arch. Virol 129:363, 1993): 1) all HHV-6 isolates are tropic for CD4+ T lymphocytes, but the group characterized by strains GS and U1102 (variant A) or by strain Z29 (variant B) may preferentially infect different cell lines; 2) each group has a distinct pattern of reactivity to monoclonal antibodies; 3) the two groups have characteristic restriction endonuclease sites due to restriction site polymorphisms that extend across the entire genome; and 4) genomic sequence analysis of eight different regions of the genome revealed sequence identity ranging from 97-100% within groups and from 94-96% between groups, and predicted amino acid sequences from 98-100% within groups and 92-96% between groups.
- HHV-6 sequences isolated from infants with exanthema subitem and related febrile illnesses have been Z29-like, with the exception of two patients from whom both types were isolated, and HHV-6 isolated from immunocompromised adults may belong to either or both groups.
- HHV-6 Type B2 strains we have found that the vast majority of individuals are in fact infected with HHV-6 Type B2 strains, and that these viruses can be further subtyped by molecular markers into strains that are associated with MS.
- the MSV-1206 virus is a prototype for the MS associated virus that is found in the Los Angeles population.
- the partial nucleic acid sequences for MSV-1206 and other MS associated HHV- 6 as set out in Table 1 are unique and are particularly useful for, inter alia, diagnosis of MS.
- the MSV-1206 sequences and the unique peptides they encode in substantially pure form are embodiments of this invention.
- This invention further embraces diagnostic kits for detecting the presence of an MS agent in tissue samples, such as CSF or serum, comprising a container containing anti-HHV-6 antibodies, and instructional material for performing the test.
- tissue samples such as CSF or serum
- inactivated viral particles or peptides derived from MSV-1206 may be used in a diagnostic kit to detect for antibodies specific to MS associated virus.
- Diagnostic kits for detecting the presence of an MS agent in tissue samples comprising a container containing a nucleic acid sequence specific for neurotropic HHV-6 and instructional material for detecting for MS are also included.
- a container containing nucleic acid primers to any one of such sequences is optionally included as is HHV specific antibodies described herein.
- Antigen refers to an entity or fragment thereof which can induce an immune response in a mammal.
- the term includes immunogens and regions responsible for antigenicity or antigenic determinants.
- Antigenic determinant refers to a region of immunodominance.
- Binding agent refers to either DNA, antibody or antibody-like agents that can specifically attach to HHV products in the presence of biological material from normal, healthy human cells and fluids. Such agents are
- “Complementary” means that one nucleic acid hybridizes selectively to, or specifically to another nucleic acid. Selectivity of hybridization exists when hybridization occurs that is more selective than total lack of specificity. Typically, selective hybridization will occur when there is at least about 55% identity over a stretch of at least 14-25 nucleotides, preferably at least about 65%, more preferably at least about 75%, and most preferably at least about 90%. See, M. Kanehisa, Nucleic Acids Res. 12:203 (1984), incorporated herein by reference.
- Effective refers to agents that ameliorate the symptoms of the disease by impacting the virus directly
- isolated or substantially pure when referring to nucleic acids, refers to those that have been purified away from other chromosomal or extrachromosomal DNA or RNA by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, and other techniques well known in the art or as otherwise described herein. See, F. Ausubel, et al . , ed., Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York (1987), incorporated herein by reference.
- Nucleic acids may be DNA or RNA and may be single- or double-stranded. Additionally,
- nucleic acid sequence identity exists when a nucleic acid segment will hybridize, under stringent
- Nucleic acid probes may be RNA or DNA fragments prepared, for example, by digesting plasmid nucleic acid, or by PCR, or synthesized by methods such as the phosphoramidite method described by Beaucage and Carruthers, Tetrahedron Lett. 22:1859-1862 (1981), or by the triester method according to Matteucci, et al . , J. Am. Chem. Soc. 103:3185 (1981), both incorporated herein by reference. Where a specific nucleic acid sequence is given, it is understood that the
- complementary strand is also identified and included, for the complementary strand will work equally well in situations where the target is a double-stranded nucleic acid.
- a nucleic acid probe is complementary to a target nucleic acid if it will anneal only to a single desired position on that target nucleic acid under stringent
- hybridization conditions e.g. 1 x SSC at 65°.
- annealing conditions depend, for example, upon a probe's length, base composition, and the number of mismatches and their position on the probe, and must often be determined empirically.
- nucleic acid sequence refers to a single- or double-stranded polymer of deoxyribonucleotide or
- ribonucleotide bases read from the 5' to the 3' end. It includes both self-replicating plasmids, infectious polymers of DNA or RNA and nonfunctional DNA or RNA. Unless otherwise stated, where a single nucleotide sequence is described, its complement is implicitly described.
- Specifically immunoreactive refers to a binding reaction between an antibody and antigen which is selectively determinative of the presence of the antigen in the presence of a heterogeneous population of proteins and other biologies.
- Genomic analysis was performed using a DNA pool of 24 PBLs from normal blood donors as driver against brain tissue samples obtained from five patients with MS. The samples were obtained from patients who had died with a clinical diagnosis of MS, underwent autopsy and brain
- plaques were used for tester DNA extraction, and the plaques were dissected from these tissues with a sterile scalpel.
- DNA was extracted from these tissues as described above, digested with the restriction endonuclease HindIII, ligated onto adaptors and amplified by PCR. After 20 cycles, aliquots of the amplicons from each patient were stored at -70°C and the remainder was used as tester in an RDA
- fragments were 98% homologous to the major DNA binding protein (KA2L) gene of Herpesvirus-6B.
- the virus bearing sequence ID. No. 48 were as previously undescribed and has been named
- the oligonucleotides of Table 3 were designed from the KA2L gene sequence of HHV-6B in GeneBank/EMBL and used to screen for the presence of HHV-6B related sequences in the RDA derived amplicons from samples from five MS patients.
- One microliter of each amplicon 50 ng was placed into a 50 microliter PCR reaction with 5 pg each of primer 1 and 4, 1 and 2, or 2 and 3, 50 mMol/L KCl, 2 mMol/L magnesium chloride, 2.5 U of Taq polymerase, 100 mMol/L deoxynucleoside
- Example 2 Using PCR to diagnose HHV-6b related Multiple Sclerosis.
- a brain tissue sample from a MS patient is isolated and the DNA isolated using the alkaline SDS method described above.
- the MSV-1206 type virus is detected by amplification of the markers with the primers listed, followed by detection of the MSV-1206 dot blot format, in which the PCR product is transferred to a Nylon membrane and hybridized with a labeled internal oligonucleotide probe that is complementary to the sequence containing the nucleotides specific for the virus under high stringency hybridization and wash conditions
- MS1206 specific sequence includes the use of allele-specific MS1206 sequence.
- oligonucleotides during PCR the oligonucleotide ligation assay, or ligase chain reaction.
- the latter three methods can be designed to target the individual nucleotides that
- HHV-6 Type B Group 2 viruses which differ qualitatively by signature nucleotide alterations from the viruses that are most prevalent in the literature, Z29 (Type B Group 1 virus) and U1102 (Type A virus). More importantly, among the Type B Group 2 viruses, these viruses can be further subtyped into a class that is highly associated with multiple sclerosis, and a separate class that is found in individuals without MS.
- MSV-1206 is therefore a member of a subset of the HHV-6B Group 2 subfamily of HHV-6 viruses, and has linked markers that can be used to characterize this virus and its closest relatives.
- the markers and their location on the MSV-1206 genome is shown in Figure 2 and Table 1.
- the MS viruses appear to be in higher titers in patients with MS.
- the HHV-6 family comprises a large genome and it is impractical and unnecessary to sequence the entire genome of each viral isolate or variant.
- the map of the HHV-6 genome including regions analyzed is shown in Figures 1 and 2.
- Phylogenetic analysis of the pZVH14 locus and variable glycoprotein region (vg), which allows differentiation of Type A versus Type B viruses demonstrated that the MSV-1206 virus belonged to the Type B Group of HHV-6.
- Analysis of the immediate early region (IE) and glycoprotein H region (gH) demonstrated that MSV-1206 is most homologous to the group 2 branch (isolates H622, H623 from Chou, S. and Marousek, G., 1994, Virology 198:370-376; Gompels, et al., 1993, J. Gen. Virol .
- MSV-1206 In order to classify MSV-1206 as a Type A versus Type B strain, five independent regions of the MSV-1206 virus were .amplified and characterized with respect to their sequence similarity to corresponding regions of HHV-6A and HHV-6B strains, including the immediate early region, the DNA polymerase region, a HindIII restriction fragment length polymorphism within a BamHI fragment, the major DNA binding protein region (described above as the segment identified by RDA, and containing the characteristic HindIII restriction site), and a segment of a variable glycoprotein gene.
- MSV-1206 is within the HHV-6 family, qualitative differences were found between the virus isolated from patient MSV-1206 and the prototype strains for HHV-6A (U1102) and HHV-6B (Z29). MSV-1206 differed from the most prevalent HHV-6B virus reported in the literature
- strain Z29 in three of these regions, including nucleotide differences in the immediate early region, the major DNA binding protein, and the HindIII site within a BamHI fragment that is used to type differences between HHV-6A and HHV6-B strains. In all three of these regions, the MSV-1206 virus does not type as Z29, which is the prototype strain for the class of HHV-6B viruses that cause roseola.
- the most diagnostic region that differed between the published Z29 sequence and MSV-1206 is the HindIII site within the major DNA binding protein (MDBP) identified in MSV-1206 by RDA. We constructed PCR primers that flanked this site.
- MDBP major DNA binding protein
- bases 1141 - 2880 from GenBank record HH6KAHC This is the portion of the major DNA binding protein gene from strain Z29 that includes the homologue of the
- HindIII fragment that we isolated by RDA from the MSV-1206 virus.
- the HindIII sites that define the fragment are AAGCTT and AAGCTC.
- the second site at position 1810 is AAGCTT in MSV-1206, and therefore a cleavage site for HindIII, while the corresponding sequence in Z29 is AAGCTC, and
- MSV-1206 represents a subset of HHV-6 Type B viruses that are associated with MS, and this site
- HST HH6IMMEA
- Z29 HH6IEXB
- H322 HH6IEXC
- H387 HH6IEXD
- H622 HH6IEXE
- H623 HH6IEXE
- H6D21 5'-TTGAAACTCAACCCGACTC-3' (Sequence ID No. 55)
- H6D24 5'-CAGTTTCATAACCAAATG-3' (Sequence ID No. 56)
- Flanking distal primers were necessary in a nested two-stage PCR to obtain products in some cases.
- a sequence was obtained from eight individuals with MS: MS473, MS536, MS573, MS579, MS1206, MS1721, MS1862, MS1864. All eight individuals contained the following sequence (in the immediate early region listed 5' to 3') designated "8/8MS" in Table 4 below.
- Sequences 8/8MS, U1102, pblA, DA, Z29, H322, H387, and HST correspond to Sequence ID Nos. 57 to 64, respectively.
- Sequences pb1F * , Stim * , A1245 * , H622 and H623 are all
- MBE have the site. Although the genes located in this region have not been defined, the RFLP has been mapped to an 850 bp
- primers are: primer A 5'-GATCCGACGCCTACAAACAC-3' (Sequence ID No. 67) primer B 5'-TACCGACATCCTTGACATATTAC-3' (Sequence ID No. 68)
- glycoprotein gene The protein coding sequence of glycoprotein gene "BHLF2" from strain U1102 has been reported in Gompels, et al . , DNA seq. 3:25-39 (1992) (1943- 3898 in GenBank record HV6IDDNA).
- the homologous glycoprotein gene "KA8LA” from strain Z29 can be found in GenBank record
- HH6KAHC bases 12678-14894.
- the alignment of bases 13328- 13941 from Z29 with bases 2413-2858 from U1102 shown below reveals two regions with insertions in the Z29 sequence relative to the U1102 sequence that can be used in a PCR-based assay to distinguish these two strains:
- primer 1 primer 2>
- primer 11 primer 12>
- sequence ID Nos. corresponding to primers 1, 2, 4-6, 11-14 above are 70-78, respectively.
- the expected sizes of the PCR products produced with specific pairs of the primers will be:
- sequence ID No. 83 we have sequenced the entire glycoprotein gene of the MSV-1206 virus and present it as sequence ID No. 83.
- MS drugs Human umbilical cord blood cells (5 ⁇ 10 ⁇ 5 cells) are mixed with a 50% TCID (tissue culture infection dose as measured by cytopathic effects after 5-6 days) and spun at 1800 rpm for 10 min. The inoculated cells are incubated at 37°C for 30 min. before transferring them to 12 well plates in 2 ml of cord blood media (+/- drug). After incubation in a CO 2 incubator for 5-12 days, aliquots of the cells are examined for cytopathic effects. To quantitate the effect on the drug on replication, aliquots of cells are fixed onto slides and stained with anti-HHV-6 human positive control serum and anti-human Ig-FITC with Evan's Blue as a counter stain. The total cell count and stained cell count is determined by examination under a fluorescence microscope. The percentage of bright cells relative to the zero drug control are plotted vs. drug concentration on a semi-log plot. The IC50 is reported as the concentration of drug in
- Example 5 Detection of HHV-6 in brain tissue.
- HHV-6 was localized in brain tissues by
- ICC immunocytochemistry
- murine monoclonal antibodies to antigens p101 and p41/38 were used as the primary antibody (Chemicon Corp.), followed by a biotinylated goat anti-mouse secondary antibody.
- the detection system consists of a streptavidin-horseradish peroxidase conjugate with diaminobenzidine as the colorimetric marker (BioGenex
- the in situ hybridization probe is 8 kilobases of PCR amplified MS-1 viral DNA, in which dig ⁇ xigenin is
- the detection system consisted of a sheep anti-digoxigenin Fab fragment conjugated to alkaline phosphatase.
- the colorimetric marker is nitroblue tetrazolium/bromochloroindole phosphate (NBT/BCIP, Boehringer Mannheim Corp.)
- HHV-6 was primarily localized in microglia and astrocytes of the subpial and subependymal regions, with patchy low level staining of the overlying ependymal cells and meningoepithelial cells.
- the cortex and white matter from normal brains showed sporadic low level staining of neurons (10-20%) and glial cells ( ⁇ 1/1000).
- MS cases particularly those with active inflammation or plaques, both the intensity of staining and percentage of positive neurons and glial cells were greater than in controls (Table 5).
- the regions of gray matter surrounding both inactive and active plaques contained a high percentage (>90%) of stained neurons.
- HHV-6 is neurotropic and is associated or causative of the lesions that are characteristic of MS.
- MOLECULE TYPE DNA (primer)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
Abstract
Description
Claims
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Cited By (5)
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US5874531A (en) * | 1995-03-07 | 1999-02-23 | President And Fellows Of Harvard College | Identification of self and non-self antigens implicated autoimmune disease |
US5883254A (en) * | 1996-11-08 | 1999-03-16 | Hoffmann-La Roche Inc. | Process for making pyrimidine derivatives |
US7084247B2 (en) | 2004-03-11 | 2006-08-01 | Peptimmune, Inc. | Identification of self and non-self antigens implicated in autoimmune diseases |
EP2274042A1 (en) * | 2008-04-11 | 2011-01-19 | Epiphany Biosciences, Inc. | Treatment and/or prevention of multiple sclerosis |
EP2569441A1 (en) * | 2010-05-14 | 2013-03-20 | Stålhandske, Per | Kit and method |
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1994
- 1994-11-04 AU AU10878/95A patent/AU1087895A/en not_active Abandoned
- 1994-11-04 CA CA 2175806 patent/CA2175806A1/en not_active Abandoned
- 1994-11-04 EP EP95901761A patent/EP0726708A1/en not_active Withdrawn
- 1994-11-04 WO PCT/US1994/012655 patent/WO1995012313A1/en not_active Application Discontinuation
Non-Patent Citations (12)
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5874531A (en) * | 1995-03-07 | 1999-02-23 | President And Fellows Of Harvard College | Identification of self and non-self antigens implicated autoimmune disease |
US7255861B1 (en) | 1995-03-07 | 2007-08-14 | President And Fellows Of Harvard College | Preparations for inducing immunotolerance and uses therefor |
US5883254A (en) * | 1996-11-08 | 1999-03-16 | Hoffmann-La Roche Inc. | Process for making pyrimidine derivatives |
US6121447A (en) * | 1996-11-08 | 2000-09-19 | Hoffmann-La Roche Inc. | Process for making pyrimidine derivatives |
US7084247B2 (en) | 2004-03-11 | 2006-08-01 | Peptimmune, Inc. | Identification of self and non-self antigens implicated in autoimmune diseases |
EP2274042A1 (en) * | 2008-04-11 | 2011-01-19 | Epiphany Biosciences, Inc. | Treatment and/or prevention of multiple sclerosis |
EP2274042A4 (en) * | 2008-04-11 | 2012-03-14 | Epiphany Biosciences Inc | Treatment and/or prevention of multiple sclerosis |
EP2569441A1 (en) * | 2010-05-14 | 2013-03-20 | Stålhandske, Per | Kit and method |
EP2569441A4 (en) * | 2010-05-14 | 2013-11-27 | Per Staalhandske | Kit and method |
US9429518B2 (en) | 2010-05-14 | 2016-08-30 | Csens Ab | Kit and method |
US10190149B2 (en) | 2010-05-14 | 2019-01-29 | Biovica International Ab | Kit and method for measuring the activity of deoxynucleoside kinase |
Also Published As
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AU1087895A (en) | 1995-05-23 |
EP0726708A1 (en) | 1996-08-21 |
CA2175806A1 (en) | 1995-05-11 |
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