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  • C12N2740/10022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

• the invention relates to the field of diagnosing and treating diseases associated with endogenous retroviruses.
• a retrovirus that is found in a majority of human patients suffering from demyelinating diseases such as multiple sclerosis is transmissible to human cells including mononuclear blood cells, and to non-human cells.
• a specific splice variant comprising the env region and a part of the pol region is present in retroviral mRNA isolated from blood cells of MS patients and patients suffering from autoimmune diseases and malignancies, but not in mRNA isolated blood cells from healthy subjects.
• MS Multiple sclerosis
• retrovirus particles possess reverse transcnptase (RT) activity (Christensen et al., 1997; Christensen et al., 1999) and share a few antigenic determinants with HTLV-1 , but are distinct from the known exogenous retroviruses at the antigenic level (Christensen et al., 1997).
• Endogenous retroviruses that are primarily transmitted vertically are found ubiquitously in the mammalian genome and constitute several percent of the genome. It has been suggested that endogenous retroviruses may play a role in autoimmune diseases (Hohenadl et al., 1979; Krieg et al., 1992; Dalgliesh, 1997).
• HTDV/HERV-K Only once before has a human endogenous retrovirus, HTDV/HERV-K, been clearly demonstrated as particles with possible pathological connotation - in teratocarcinoma cell lines (T ⁇ njes et al., 1996; Boiler et al., 1993). It has not yet been determined whether HTDV/HERV-K occurs as particles in patient plasma.
• HERV-H sequences with high homology to the HERV-H variant RGH-2 were also found specifically at particle level in cell-free plasma from 24 out of 33 MS patients but could neither be demonstrated in 29 plasma samples from patients with autoimmune or other diseases nor in 20 plasma samples from healthy controls. It was found that nucleic acid binding proteins analogous to the nucleocapsid protein, Gag NC, of other retroviruses are found in the above retrovirus particles.
• WO 99/53103 discloses methods of diagnosing demyelinating disease including MS, subgrouping patients suffering from such disease and/or monitoring the stage of activity of demyelinating disease including MS based on the detection, in a retroviral particle or an encapsidated virion-like particle, of the RGH virus-derived SEQ ID
• Diagnostic agents comprising probes capable of hybridising to such sequences, subsequences or variants, antibodies reacting with epitopes or proteins or peptides encoded by the sequences, subsequences or variants and/or such epitopes for such methods are also disclosed in WO 99/53103.
• RGH viruses belong to the RTVL-H/HERV-H (priming of transcription with tRNA His ) family of endogenous retroviruses.
• This family of type-C like retroviruses is related to the onco- retroviruses human T-cell leukemia virus (HTLV-1/-2), to bovine leucosis virus (BLV), and to ERV-9.
• HTLV-1/-2 human T-cell leukemia virus
• BLV bovine leucosis virus
• ERV-9 ERV-9.
• a genomic RTVL-H clone which potentially encodes functional proteins has been described (Wilkinson et al.) and most interestingly, a HERV-H element with an intact open env reading frame (and highly homologous to RGH) has been described recently (Lindeskog et al., 1999; Blond et al., 1999).
• HERV-H/RT ⁇ /L-H RNA is found in normal lymphocytes, placental tissue and some neoplasias (Medstrand et al., 1992; Mager et al., 1987; Kelleher et al., 1996; Lower et al., 1993; Johansen et al., 1989), but RGH particles as such have not been reported previously, nor has their potential transmissibility.
• RGH is also separate from the RTVL-H viruses according to the classification based on LTR- types (Goodchild et al., 1993) as the RGH-2 LTR region contains three class 1 repeats rather than two (LTR type 1) or one (LTR type 1a and 2).
• RGH-1 clone is 4869 bp long and encompasses a partial pol and an env-3'LTR region and thus lacks gag
• published RGH-2 clone is 8715 bp long and includes the complete coding potential: 5'LTR gag-pol-env 3'LTR.
• the majority of cDNA clones isolated from MS patient material are closely related to the full-length clone RGH-2 as they contain gag sequences. RGH-like sequences were reported present in about 100 copies/haploid genome (Hirose et al., 1993).
• HERV-H clone RGH-2 Hirose et al., 1993
• HERV-H clone with intact open Env reading frame described in Lindeskog et al., 1999.
• the retroviral particles are also described in: Christensen et al., 1997; Christensen et al., 1999; M ⁇ ller-Larsen et al., 1998; Christensen et al., 1998; Christensen at al., 2000.
• the HERV-H RGH2 sequences present in virus particles produced in MS cell lines or isolated from the blood of MS patients show >80% homology to the RGH-2 sequence and the HERV-H Env reading frame.
• blood cells from MS patients, but not from healthy controls contain an aberrantly spliced HERV-H env mRNA which may represent a genetic marker of cells producing or able to produce the infectious HERV-H RGH2 retrovirus particles.
• a comparison of a number of such aberrantly spliced mRNA molecules from different individuals provides indications that the genetic information encoding the mRNA may originate at a single endogenous HERV-H locus or at a small number of such loci.
• the invention pertains in a first aspect to a human endogenous retrovirus belonging to the RTVL-H/HERV-H family in isolated form or in retroviral particle form that is capable of infecting a mononuclear cell and of replicating therein.
• the invention provides a method of detecting intra-species infectivity (transmissibility) of such a retrovirus, the method comprising the steps of isolating the retrovirus from a patient suffering from a demyelinating disease such as multiple sclerosis, a patient suffering from an autoimmune disease, a patient suffering from diabetes or a patient suffering from a malignancy, contacting said isolated virus with a culture of mononuclear cells derived from a human subject not suffering from any of the above diseases, and detecting infection of at least a proportion of the mononuclear cells with the retrovirus.
• a demyelinating disease such as multiple sclerosis
• a patient suffering from an autoimmune disease a patient suffering from diabetes or a patient suffering from a malignancy
• contacting said isolated virus with a culture of mononuclear cells derived from a human subject not suffering from any of the above diseases, and detecting infection of at least a proportion of the mononuclear cells with the retrovirus.
• a method of detecting inter-species infectivity (transmissibility) of the retrovirus comprising the steps of isolating the retrovirus from a patient as also defined above, contacting said isolated viruses with a culture of mononuclear cells derived from a non-human individual, and detecting infection of said cells with the retrovirus.
• MS multiple sclerosis
• a method of diagnosing a disease selected from the group consisting of multiple sclerosis, an autoimmune disease, diabetes and a malignancy comprising isolating mononuclear blood cells from a patient suspected of suffering from any of said diseases and detecting in said cells a spliced HERV-H RGH retrovirus env mRNA sequence comprising a region from the protease encoding (pol) region of said retrovirus, including a mRNA sequence comprising a region from the protease encoding region that is selected from the group consisting of SEQ ID NO:44; SEQ ID NO:45; SEQ ID NO:46; SEQ ID NO:47; SEQ ID NO:48; SEQ ID NO:49; and SEQ ID NO:50 as defined herein, and variants and subsequences hereof;
• a method of diagnosing a disease associated with the retrovirus comprising the steps of isolating the retrovirus from a human subject suffering from any of the above diseases and testing its infectivity according to the above methods;
• a method of treating a patient suffering from a disease, the clinical manifestations of which are associated with the presence of the retrovirus according to the invention comprising administering to the patient an effective amount of a pharmaceutical agent that is capable of at least partially preventing the infection of cells in the patient with the retrovirus, adverse immunological responses to the infection and/or the migration of the retrovirus to cells in the patient that can be infected by the retrovirus;
• a method of treating a patient suffering from a disease, the clinical manifestations of which are associated with cells transformed (immortalised) by infection with the retrovirus according to the invention comprising administering to the patient an effective amount of a pharmaceutical agent that is capable of at least partially eliminating such transformed (immortalised) cells;
• an antiviral agent for use as a medicament in the control of a disease, the clinical ' manifestations of which are associated with the presence of the retrovirus according to the invention
• an antiviral agent in the manufacturing of a medicament for controlling a disease associated with the retrovirus according to the invention
• a method of controlling a disease comprising administering to the patient an effective amount of a pharmaceutically active compound that is capable of at least partially inhibiting said immunological responses; and a pharmaceutical composition comprising a pharmaceutically active compound that is capable of at least partially inhibiting in a human subject suffering from any of the above diseases an immunological response to the retrovirus according to the invention.
• the invention relates to :
• composition comprising a pharmaceutically active compound that is capable of at least partially preventing the infection of cells in a patient with the retrovirus according to the invention and/or the migration of the retrovirus to cells in the patient that can be infected by the retrovirus;
• composition comprising a pharmaceutically active compound that is capable of at least partially eliminating in a patient cells transformed (immortalised) by infection with the retrovirus according to the invention
• an allelically occurring spliced HERV-H RGH retrovirus env mRNA sequence comprising a region from the protease (pot) region of said RGH retrovirus as a marker for the occurrence or the state of a disease selected from the group consisting of a demyelinating disease including multiple sclerosis, an autoimmune disease, diabetes and a malignancy.
• the present invention is i.a. based on the unexpected finding that certain human endogenous retroviruses belonging to the RTVL-H/HERV-H family are capable of being transmitted to (infect) mononulear cells and of replicating therein.
• this unexpected and surprising finding renders it likely that such infective retroviruses play a role in the development of demylelinating diseases, in particular multiple sclerosis, and possibly also in the development of autoimmune diseases such as e.g. diabetes, and malignancies. Accordingly, these achievements of the inventors imply that novel therapeutic approaches to the treatment of such diseases can be designed and additionally, the present findings can be the basis for improved diagnostic and prognostic methods.
• retrovirus belonging to the RTVL-H/HERV-H family in isolated form or in retroviral particle form that is capable of infecting a mononuclear cell and of replicating therein.
• a retrovirus is a retrovirus that encodes a spliced HERV-H RGH retrovirus env mRNA sequence comprising a region from the protease (pol) region of said RGH retrovirus.
• Such a retrovirus includes a retrovirus wherein the spliced mRNA sequence comprises a region from the protease encoding region that is selected from the group consisting of SEQ ID NO:44; SEQ ID NO:45; SEQ ID NO:46; SEQ ID NO:47; SEQ ID NO:48; SEQ ID NO:49; and SEQ ID NO:50 as defined herein, and variants and subsequences hereof.
• sequence is meant a sequence which comprises at least 10, 15, 20, 30, 40 or 50 nucleotides of any of the above specific sequences.
• variant is meant a sequence which is not identical to any of these specific sequences, but which has a sequence identity of at least 80%, 85%, 90%, 95%, 98%, 99% or 99.5% with any of these sequences.
• the invention comprises detection of a variant of any of SEQ ID NO:44; SEQ ID NO:45; SEQ ID NO:46; SEQ ID NO:47; SEQ ID NO:48; SEQ ID NO:49; and SEQ ID NO:50 which has a sequence identity of at least 80% with a subsequence of any of the sequences of at least 10, 15, 20, 30, 40 or 50 nucleotides. It will be appreciated that the person of skill in the art will know how to make subsequences and variants of the specific sequences, e.g. by deletion, substitution and insertion of one or more nucleotides.
• sequence identity indicates that a given sequence is compared with a similar part of any of the above specific sequences. The best possible alignment of the sequences is made and then the degree of identity is calculated.
• a computer program such as GCG, Wisconson Package, Version 9.1 , Genetics Computer Group (GCG), 575 Science Drive, Madison, Wisconsin, USA 53711 using the defaults proposed in the program Bestfit with respect to gap weight (50), length weight (3), average match 10,000, average mismatch (-9,000) may be useful in this respect.
• All of such spliced mRNA sequences that were isolated had a size of about 950 bp and were spliced env mRNA sequences containing the same splice donor site in the leader region downstream from the PBS sequence and a splice acceptor site in the part of the pol that encodes the integrase.
• a region of 104 bp from the protease encoding region was inserted between the splice donor site and the splice acceptor site.
• the spliced mRNA sequences were detected in retroviruses isolated from MS patients and from a patient suffering from diabetes, whereas they could not be detected in healthy subjects.
• a retrovirus according to the invention can e.g. be isolated from a patient suffering from a demyelinating disease such as multiple sclerosis (MS), an autoimmune disease, diabetes mellitus or a malignancy including leukaemia, e.g. from blood cells, blood plasma or a cell culture derived from peripheral blood of said human subject.
• a demyelinating disease such as multiple sclerosis (MS)
• an autoimmune disease such as diabetes mellitus or a malignancy including leukaemia
• blood cells e.g. from blood cells, blood plasma or a cell culture derived from peripheral blood of said human subject.
• blood cell cultures derived from such patients constantly comprised a significant proportion of a specific type of T-cells which were characterised according to CD antigens on their surface as being CD3+, CD4- and CD8- T-cells.
• Retroviruses according to the invention include retroviruses obtainable by ultracentrifugation of a cell culture supernatant or a cell-free plasma sample in OptiprepTM density gradient medium followed by recovery of the fraction containing intact retroviral particles. Protocols for purification of retroviral particles from cell culture supernatants or cell-free plasma samples using ultracentrifugation on an Optiprep gradient are provided in the accompanying Examples.
• Retroviruses according to the invention include human endogenous retroviruses of the RTVL-H/HERV-H family in isolated form.
• isolated form includes, but is not necessarily limited to, retrovirus purified from patient serum or a cell culture supernatant, for example using the protocols supplied in the accompanying examples.
• the retrovirus according to invention is, as it is demonstrated in the following examples, capable of infecting a blood cell derived from a human subject not suffering from MS, an autoimmune disease, diabetes or a malignancy, and of replicating therein. Additionally, it has been found that the retrovirus is also capable of infecting non-blood human cells such as e.g. liver cells.
• the retrovirus is also capable of infecting a non-human cell, i.e. the retrovirus shows inter-generic infectivity.
• the retrovirus can be transmitted to cells derived from bats, rabbits and rodents.
• the infective endogenous retrovirus according to the invention is an RGH virus including a retrovirus comprising a nucleic acid sequence that is at least 80% identical to the RGH-2 virus sequence published by Hirose et al., Virology, 1993, 192:52-61.
• the invention provides a method of detecting intra-species infectivity (transmissibility) of the endogenous retrovirus according to the invention.
• the method comprises that the isolated retrovirus is contacted with a culture of mononuclear cells such as blood cells derived from a human subject not suffering from MS, an autoimmune disease, diabetes or a malignancy, and detecting infection of said cells with the retrovirus.
• the detection of the infection can be carried out in several ways.
• the infection is detected by the appearance or increase of RT activity in the infected culture using methods for detection of RT activity which are known in the art including the very sensitive assays for detecting RT activity generally referred to as RT-PCR and Product Enhanced Reverse Transcriptase (PERT) assay, or by an assay based on co-cultivating of cells suspected of harbouring the infective retrovirus e.g. cell cultures from peripheral blood from an MS patient with a cell line harbouring a non- transmissible retroviral construct followed by an assay for rescue of the infecting retrovirus into indicator cells such as it is described in details in the following examples.
• RT-PCR very sensitive assays for detecting RT activity
• PROT Product Enhanced Reverse Transcriptase
• the infection of the culture of mononuclear cells is detected by the appearance in cells of said culture of a spliced HERV-H RGH retrovirus env mRNA sequence comprising a region from the protease (pol) region of said RGH retrovirus, including a region from the protease encoding region that is selected from the group consisting of SEQ ID NO:44; SEQ ID NO:45; SEQ ID NO:46; SEQ ID NO:47; SEQ ID NO:48; SEQ ID NO:49; and SEQ ID NO:50 as defined herein, and variants and subsequences hereof.
• infection of cell cultures by the retroviruses of the invention can be detected by the occurrence of clonal growth of the infected cells.
• This method comprises the steps of isolating the retrovirus from a human subject suffering from a demyelinating disease, an autoimmune disease, diabetes or a malignancy, followed by contacting the isolated virus with a culture of mononuclear cells including blood cells derived from a non-human individual, and detecting infection of said cells with the retrovirus using any of the above methods for detection of infection of a cell with the retrovirus.
• the mononuclear recipient cells may be derived from any non- human animal such as a rodent, a rabbit or a bat.
• the recipient mononuclear cells being contacted with the retrovirus are in the form of a culture of mononuclear cells that are pre-stimulated with a mononuclear cell stimulating compound including PHA.
• a further objective of the invention is to provide a culture of mononuclear cells derived from a human subject selected from the group consisting of a multiple sclerosis (MS) patient, a patient suffering from an autoimmune disease, a patient suffering from diabetes and a patient suffering from a malignancy, said cells are capable of expressing a spliced HERV-H RGH retrovirus env mRNA sequence comprising a region from the protease encoding (pol) region of said retrovirus.
• MS multiple sclerosis
• the culture comprises cells wherein the spliced mRNA sequence being expressed comprises a region from the protease encoding region that is selected from the group consisting of SEQ ID NO:44; SEQ ID NO:45; SEQ ID NO:46; SEQ ID NO:47; SEQ ID NO:48; SEQ ID NO:49; and SEQ ID NO:50 as defined herein, and variants and subsequences hereof.
• Such cultures include cultures comprising a proportion of CD3+, CD4- and CD8- T-cells.
• the proportion of such T-cells in the mononuclear cell cultures producing the retrovirus according to the invention is typically in the range of 1-20% of mononuclear cells, e.g. at least 2% including the range of 2-15%.
• One important objective of the invention is to provide a method of diagnosing a disease selected from the group consisting of multiple sclerosis, an autoimmune disease, diabetes and a malignancy.
• This method comprises as a first step the isolation of mononuclear blood cells from a patient suspected of suffering from any of such diseases followed by detecting in these cells the expression of a spliced HERV-H RGH retrovirus env mRNA sequence comprising a region from the protease encoding (pol) region of said retrovirus including such a sequence that comprises a region from the protease encoding region that is selected from the group consisting of SEQ ID NO:44; SEQ ID NO:45; SEQ ID NO:46; SEQ ID NO:47; SEQ ID NO:48; SEQ ID NO:49; and SEQ ID NO:50 as defined herein, and variants and subsequences hereof.
• the invention relates to a method of diagnosing a disease associated with the retrovirus of the invention, the method comprising the steps of isolating the retrovirus from a human subject suspected of suffering of a demyelinating disease including multiple sclerosis, an autoimmune disease, diabetes or a malignancy, and testing its infectivity according to any of the methods defined above and explained in details in the following examples.
• the invention provides a method of treating a patient suffering from a disease, the clinical manifestations of which are associated with the presence of the retrovirus according to the invention which method comprises administering to the patient an effective amount of a pharmaceutical agent that is capable of at least partially preventing the infection of cells in the patient with the retrovirus and/or the migration of the retrovirus to cells in the patient that can be infected by the retrovirus.
• the term "migration” includes the targeting of the retrovirus to a susceptible cell in the body by the transfer of a cell harbouring the infective retrovirus to the susceptible target cell whereby the retrovirus may infect the susceptible target cell by cell-to-cell contact.
• any compound that is capable of interfering with at least one step in the infection of new cells in the body including compounds that, at least partially, eliminates the retrovirus- carrying immortalised cells, preferably in a selective manner, compounds that prevent or inhibit migration of such cells, compounds that interfere with the cell-to-cell contact or which interfere with the transmission of the retrovirus into the target cells (infection), can be used in the treatment methods of the invention.
• the compounds which can be used in such methods include antiviral agents, antibodies, anti-sense nucleic acids, nucleic acid binding proteins and ribozymes.
• an immune response may be "direct”, i.e. directed against an antigen on the infected cell or "indirect”, i.e. the immune response is directed against other cell antigens.
• the invention pertains in another aspect to a method of controlling the above diseases which comprises administering to a patient suspected of suffering from such a disease an effective amount of a pharmaceutically active compound that is capable of at least partially inhibiting such immunological responses.
• useful compounds include compounds that inhibit cells of the immune system that are activated to generate the immunological responses.
• the pharmaceutically active compound that is capable of at least partially inhibiting immunological responses to the retroviral infection is a compound that is directed against an immunological response elicited specifically by a retrovirus expressing a spliced HERV-H RGH retrovirus env mRNA sequence comprising a region from the protease encoding (pol) region of said retrovirus including such a sequence that comprises a region from the protease encoding region that is selected from the group consisting of SEQ ID NO:44; SEQ ID NO:45; SEQ ID NO:46; SEQ ID NO:47; SEQ ID NO:48; SEQ ID NO:49; and SEQ ID NO:50 as defined herein, and variants and subsequences hereof.
• the pharmaceutically active compound is a compound directed against inhibiting the biological activity of activated T-cells including CD3+, CD4- and CD8- T-cells or against eliminating such T-cells.
• an antiviral agent for use as a medicament in the control of a disease, the clinical manifestations of which are associated with the presence of the retrovirus according to the invention and use of an antiviral agent in the manufacturing of a medicament for controlling a disease associated with such a retrovirus such as a demyelinating disease, a malignancy, diabetes and an autoimmune disease.
• a disease associated with such a retrovirus such as a demyelinating disease, a malignancy, diabetes and an autoimmune disease.
• the expression "antiviral agent” should be understood in its broadest sense as indicating any compound that prevents or inhibits the infectivity and replication of the retroviruses and/or the pathological manifestations of the infection including adverse immunological manifestations, optionally resulting in an autoimmune state of the patient.
• antiviral agents that selectively inhibits or eliminates the retrovirus according to the invention can be developed or selected from known antiviral agents. It is within the scope of the invention to use antibodies, anti-sense nucleic acids and ribozymes as antiviral agents.
• It is another objective of the invention to provide a pharmaceutical composition comprising a pharmaceutically active compound that is capable of at least partially inhibiting in a human subject as defined in claim 4 an immunological response to the retrovirus according to the invention, including a composition wherein the pharmaceutically active compound is directed against an immunological response elicited specifically by a retrovirus according to the invention including a retrovirus expressing a spliced HERV-H RGH retrovirus env mRNA sequence as defined hereinabove and a composition wherein the pharmaceutically active compound is a compound directed against inhibiting the biological activity of activated T-cells including CD3+, CD4- and CD8- T-cells or against eliminating such T-cells.
• compositions comprising a pharmaceutically active compound that is capable of at least partially preventing the infection of cells in a patient with the retrovirus according to the invention and/or the migration of the retrovirus to cells in the patient that can be infected by the retrovirus and a pharmaceutical composition comprising a pharmaceutically active compound that is capable of at least partially eliminating in a patient cells transformed (immortalised) by infection with the retrovirus.
• Compounds having potential antiviral activity may be identified with the aid of a screening assay based on the production of retroviral particles from a culture of mononuclear cells. Compounds that inhibit production of retrovirus from cells which normally (i.e. in the absence of the compound) produce retroviral particles would be scored as having potential anti-retroviral activity.
• a typical assay may be carried out using a B- lymphoblastoid MS derived cell line established in culture. The cells are dispensed into the wells of a standard microtiter plate and produce virus particles in the cell culture medium. Virus particles may be detected by identifying their structure in Optiprep gradients prepared by ultracentrifugation.
• Candidate compounds are then added and the production of viral particles in the presence of the test compound analysed by suitable means. For example, production of intact viral particles may be analysed by ultracentrifugation on an Optiprep gradient. Typically, the effect of the candidate compound will be tested at several different concentrations. Inhibition of virus production is scored as an indication that the candidate compound has potential antiviral activity. Compounds identified as having potential antiviral activity using such a screening assay may be useful as antiviral agents or as lead compounds in the development of antiviral agents with pharmaceutical potential.
• screening assays for compounds having potential antiviral activity may be based on an infectivity assay.
• a typical assay may comprise exposing target cells susceptible to infection with a virus according to the invention, for example cultured PHA stimulating lymphocytes from healthy control subjects, to sample containing a retrovirus according to the invention in the presence or absence of a candidate compound.
• the sample containing the retrovirus may be, for example, a cell supernatant from a cell line which produces the retroviral particles (e.g. B-lymphoblastoid MS derived cell lines, as discussed above) or a cell-free plasma sample containing retrovirus.
• Candidate compounds which prevent or substantially reduce the infection of the lymphocytes by retroviral particles present in the cell supernatants are scored as having potential antiviral activity. Such compounds may again be useful as antiviral agents or as lead compounds in the development of antiviral agents with pharmaceutical potential.
• An infectivity assay may be based on the biochemical tests for indicating retroviral activation in PHA stimulated lymphocytes already established and described in the accompanying Examples.
• Cultures of MS derived cell lines producing infectious retroviral particles may also be used to identify compounds that prevent the synthesis of viral polypeptides, including those polypeptides that may act as a superantigen in the body, thereby triggering an autoimmune response.
• an immunoassay may be set up as a screening test using the cell lines themselves, or cell supernatants, indicating the production of the viral specific epitopes in the MS derived cell lines.
• the invention relates to the use of an allelically occurring spliced HERV-H RGH retrovirus env mRNA sequence comprising a region from the protease (pol) region of said RGH retrovirus as a marker for the occurrence or the state of a disease selected from the group consisting of a demyelinating disease including multiple sclerosis, an autoimmune disease, diabetes and a malignancy.
• a demyelinating disease including multiple sclerosis, an autoimmune disease, diabetes and a malignancy.
• such use is based on mRNA sequences comprising a region from the protease encoding region that is selected from the group consisting of SEQ ID NO:44; SEQ ID NO:45; SEQ ID NO:46; SEQ ID NO:47; SEQ ID NO:48; SEQ ID NO:49; and SEQ ID NO:50 as defined herein, and variants and subsequences hereof.
• Fig.1 shows examples of RGH sequences in particulate form from MS cell lines.
• RNA templates were from Optiprep-gradient purified retroviral particles from the MS cell lines MS1533, MS1845 and MS 1851.
• the corresponding sequences from RGH-2 (Hirose et al., 1993) are included in the alignment (above: RGH-2 1589 - 1 87 2 (SEQ ID NO:1); below: RGH-2 2141 _ 231 3 (SEQ ID NO:2) .
• the MS1533/MS1845 sequences (above) SEQ ID NO:3/SEQ ID NO:4 were obtained with primer-set I; whereas the MS1533/MS1851 sequences (below) (SEQ ID NO:5/SEQ ID NO:6) were obtained with primer-set II.
• Clones appear to be true variants of RGH-2 (the full-length RGH clone), as the variation is too great to be attributed to the Taq error-rate. ... indicates gaps introduced to optimise alignment;
• Fig. 2 shows, as examples of RGH sequences in particulate form from MS patient plasma, env primed RT-PCR derived sequences from cell-free, filtered, ultracentrifuged plasma samples from the MS patients 2094 (SEQ ID NO:9) and 2095 (SEQ ID NO: 10).
• the corresponding sequences from RGH-1 (SEQ ID NO:7) and RGH-2 (SEQ ID NO:8) (Medsrand et al., 1992) are included in the alignment. Clones appear to be true variants of RGH-2 (the full-length RGH clone), as the variation is too great to be attributed to the Taq error-rate indicates gaps introduced to optimise alignment;
• Fig. 3 illustrates South-Westem analyses of ssDNA binding proteins in retroviral particles isolated from MS cell lines. Retroviral pellets obtained by ultracentrifugation of growth media from the indicated MS cell lines were analysed for the presence of ssDNA binding proteins by comparative South-Westem analysis. The M r marker is shown to the left. Left arrow: 35 kD protein also present in unconditioned medium with human serum (HS). j: triple ssDNA binding bands.
• Lane 1 human serum, 2: MS1533, 3: MS1851 , 4: B95- 8(EBV + ), 5: C91-PL(HTLV-1 + ), 6: FLK(BLV +) , 7:MS1845, 8: C8166-(HTLV-1 ⁇ gag).
• the gels were run under conditions ensuring the best resolution in the low M r range, thus cross-reactivities for the higher M r proteins (> 40 kD) are poorly resolved.
• the three low molecular weight bands (M r 10-20 kD) in the MS cell lines are also found with the positive control retroviruses HTLV-1 and BLV, but the MS band pattern exhibits slight differences in size.
• the cell line C8166 contains three mutated HTLV-1 genomes and it is known that no expression occurs from the gag and env regions (Bhat et al., 1993). In the lane with the EBV-producing line B95-8 only two bands are present in the low M r region. The middle band of the three is absent, clearly demonstrating that this cannot be an EBV-related band. The primary reason for using B95-8 was to confirm that the middle of the low M r ssDNA bands of the MS cell lines is not of EBV origin, but it should be kept in mind that B95-8 contains some transmissible SMRV-H type D retrovirus-like sequences (Sun et al., 1995).
• the HTLV-1 tax gene product p40 Tax is a trans-regulatory protein containing other zinc-finger motifs. Tax-related bands would not be expected in this assay as p40 Tax is rarely packaged in virions;
• Fig. 4 are translations of the CX 2 CX 4 HX 4 C corresponding gag sequence regions. Sequences (upstream of the protease domain) from HTLV-1 (SEQ ID N0:11) and RTVL- H (SEQ ID NO:12) (Mager et al., 1987) and RGH-2 (SEQ ID NO:13) (Hirose et al., 1993) (translated) and examples of translated gag-primed RT-PCR derived sequences from Optiprep-purified retrovirus particles from the MS1533 donor (D) (SEQ ID NO: 14) and an infected recipient (R) (SEQ ID NO:15/SEQ ID NO: 16).* indicates termination codons indicates gaps introduced to optimise alignment. Note the high proline content. The motif CX 2 CX HX 4 C....CX 3 HX 4 C is conserved in both donor and recipient clones;
• Fig. 5 illustrates a Product Enhanced Reverse Transcriptase (PERT) assay indicating cell- free transmission of purified retroviral particles from long-term cell cultures from peripheral blood from MS patients using PHA-stimulated lymphocytes from healthy human individuals as recipient. The results are indicated as OD (PERT/10 6 cells)
• PERT Product Enhanced Reverse Transcriptase
• Fig. 6 illustrates another PERT assay indicating cell-free transmission of purified retroviral particles from long-term cell cultures from peripheral blood from MS patients using PHA- stimulated lymphocytes from healthy human individuals as recipient. The results are indicated as OD (PERT/10 6 cells); and
• Fig. 7 illustrates a PERT assay indicating cell-free transmission of purified retroviral particles from long-term cell cultures from peripheral blood from MS patients using PHA- stimulated lymphocytes from rabbit as recipient. The results are indicated as OD (PERT/10 6 cells).
• FLK-BLV Fetal Lamb Kidney-Bovine Leucosis Virus
• BLV + Bactetal Lamb Kidney-Bovine Leucosis Virus
• HTLV-1 + HTLV-1 +
• C8166 HTLV-1 ⁇ gag
• the EBV-producing cell line B95-8 was obtained from ATCC.
• the cell lines were supplemented with foetal calf serum instead of human serum and were grown in a separate laboratory.
• Retroviral RNA was purified from cell culture supernatants and from plasma samples: 1.3 1 suspension cultures were centrifuged at 4°C, 30 min. at 2500 x g. The cell-free supernatant was aspirated, underlayered with a cushion of 4 ml 50% Optiprep in NaCI/Hepes (Nycomed) in 60 ml tubes and ultracentrifuged at 4°C, 2 hrs. at 45.000 x g. Optiprep is an iodinated, non-ionic density gradient medium (Nycomed Pharma, Norway) which, in contrast to sucrose, maintains intact retroviral particles (M ⁇ ller-Larsen et al., 5 1998).
• the cushion and proximal overlaying retrovirus-containing medium is mixed (5-6 ml), filtered through a 0.45 ⁇ m filter (Acrodisc 32, Gelman Sciences) to remove cellular debris, adjusted to 20% Optiprep and centrifuged in 11.2 ml Optiseal (Beckmann) tubes in a 0 Beckmann NVTi rotor at 364.000 x g for 3.5 hrs. 400 - 500 ⁇ l fractions (12 drops) were collected after centrifugation.
• RT activity was measured for each fraction and poly-A RNA was purified by lysing the 5-6 fractions with major RT activity directly in 5 volumes of lysis- binding buffer (Dynal, Norway), adding 100 ⁇ l beads/sample (mRNA Direct kit, Dynal, Norway) and following the manufacturer's instruction. 5
• RNA-coupled beads were stored in 80% EtOH at -80°C. Before use, each RNA sample was treated with amplification grade DNAse (Life Technologies) according to the manufacturer's instructions. After cooling on ice the DNAse was removed by exchanging the buffer containing the RNA-complexed paramagnetic beads with DEPC- 5 treated ddH 2 0.
• the ultrasensitive RT assays (PERT (Product Enhanced Reverse Transcriptase)). were 0 performed directly on 5 ⁇ l gradient fraction in 5 ⁇ l buffer A, essentially as described previously (Christensen et al., 1999; Lugert et al., 1996; Silver et al., 1993; Pyra et al., 1994).
• RT-PCR was performed using the GeneAmp RNA PCR kit (Perkin Elmer) according to the manufacturer's suggestions, except that 1 ⁇ of Taq polymerase was used , per reaction.
• First strand synthesis was primed with random hexamers, in conjunction with the bead-oligo-dT complexed RNA-template.
• control cDNA synthesis primed with the corresponding upstream primer was negative.
• Primer sets for PCR initially, we used various retroviral consensus primers at low stringency conditions. The results lead us to assay for endogenous retrovirus sequences, following which we obtained positive results with the HERV-H/RTVL-H gag consensus primers of the endogenous retrovirus panel (Medstrand et al., 1992); the primers were kindly donated by P. Medstrand and J. Blomberg:
• PCR products were analysed by agarose gel electrophoresis, and each product was cloned in pUC using the SureClone Ligation kit (Pharmacia) and sequenced using the ABI Prism kit (Applied Biosystems) using an automatic sequenator (ABI 377).
• the nitrocellulose filters were equilibrated in a buffer containing 0 mM Tris-HCl pH 7.0, 1 mM EDTA, 1 X Denhardts buffer, 50 mM NaCl, and incubated with radioactively labelled probe in the same buffer for 1 hr at room temperature.
• the filters were washed extensively in the same buffer, air dried and exposed to Fuji X-ray film (Santax) at -80°C.
• Lymphocyte samples isolated by Ficoll-lsopaque density gradient centrifugation of citrate blood from healthy volunteers were seeded and cultured as described previously (Munch et al., 1995) (10 7 cells/aliquot). Two ⁇ l PHA (Difco)/10 ml was added. After 2 days purified retroviral particles from an MS cell line, pelleted by ultracentrifugation, suspended in TNE and filtered was added (particles from 60 ml culture/aliquot), this representing a cell-free infection. The suspensions were cultured and examined in the microscope twice weekly and supernatant samples for PERT (Christensen et al., 1999) were collected regularly.
• B-Lymphoblastoid cell lines were established from venous blood from MS patients (Table 1.1). The cell lines arose spontaneously after long-term cultivation. All cell lines were tested for mycoplasma contamination and were found negative by repeated analyses.
• Particles were purified from cell culture supernatants by ultracentrifugation in Optiprep gradients. Their retroviral origin was confirmed by negative staining electron microscopy (EM) on each gradient fraction and by the sensitive PERT (Product Enhanced Reverse Transcriptase) assays (Christensen et al., 1999; Lugert et al., 1996; Silver et al., 1993; Pyra et al., 1994) also performed on each gradient fraction. The presence of RNA in the particles was demonstrated by chasing the culture with [5- 3 H]-uridine (Amersham) for 24 hrs before harvest, followed by TCA precipitation of each gradient fraction, and counting in a ⁇ -.counter. PERT activity, presence of retroviral particles in EM and RNA content co- located in the gradients (M ⁇ ller-Larsen et al., 1998).
• EM negative staining electron microscopy
• PERT Product Enhanced Reverse Transcriptase
• Wistar rats immunised with purified, retroviral particles exhibit a specific serological response towards potentially immunogenic synthetic peptides translated from the HERV-H sequences but it remains, however, to be assessed whether all the retroviral proteins in the particles are encoded by HERV-H.
• the virion proteins may also be encoded by other retroviral sequences in the genome as heterologous RNA co-packaging in virions (Linial, 1990) is a well-known phenomenon.
• MS cell line particle RNAs purified by our standard procedure, using the recently reported ERV-9-MS related nested primer set ST1-1/-2 and RT-PCR conditions (Perron et al., 1997). The results were negative.
• the cell lines were established as part of an extensive culturing series and arose after more than 2 months of cultivation. They are continuously growing. No external EBV source was involved. Their origin was confirmed by RFLP mapping and by HLA-DO ⁇ typing (Christensen et al., 1997).
• HERV-H sequences specifically present in plasma from MS patients
• RGH-homolbgous sequences at particle level could also be associated with MS in vivo
• RNA extracted from particles isolated from plasma was assayed by RT-PCR with subsequent sequencing of the cloned amplicons.
• the results of the plasma RT-PCR and sequencing analyses are presented in Table 1.1. Examples of MS patient sequences are shown in Fig. 2 (SEQ ID NOS:9-10). Expression of RGH sequences at particle level was specific for MS (24 out of 33 cell-free plasma samples from MS patients). These sequences were absent in all of 29 cell-free plasma samples from patients with autoimmune diseases and in all of 20 cell-free plasma samples from healthy controls.
• NC Gag nucleocapsid
• nucleic acid binding protein equivalent to the Gag NC proteins of HTLV and BLV, is also present in the retrovirus from 3 MS cell lines assayed (Fig. 3).
• the middle of the three ssDNA binding bands present in the 10- 20 kD range corresponds to the retroviral nucleocapsid protein: the band-patterns for the MS cell lines (lanes 2, 3 and 7), HTLV-1 (lane 5) and BLV (lane 6) are similar; and the lack of a corresponding band in the lane with the EBV-producer B95-8 (lane 4) excludes that the middle band is of EBV-origin. Furthermore, when the same filter is used in a Western blot with HTLV-1 + serum, the p15 band corresponds exactly to the middle-band of the three ssDNA-binding bands of HTLV-1 (not shown).
• this endogenous retrovirus may be transmissible is demonstrated by the cell-free transmission of purified virions to lymphocytes from healthy individuals.
• the activation of endogenous sequences already present in the recipient can not be ruled out, but we consider it doubtful that such an activation could be due to EBV.
• EBV is unlikely to co-purify with the retrovirus, and spontaneous EBV transformation of the healthy lymphocytes seems implausible as i) by no means all healthy lymphocyte samples are activated/infected, ii) the cells are cultured for a short time (4-8 weeks), iii) we observe clonal growth and RT activity concomitantly, and iv) the controls all die out.
• MSRV ERV-9/type-C-related sequences
• Retroviral association has been reported recently for several autoimmune diseases: in addition to the previously mentioned implication of a HERV-K variant in insulin dependent diabetes mellitus (IDDM) (Conrad et al., 1997), in Sj ⁇ gren's syndrome, pol sequences from a putative exogenous type-D-like retrovirus were found in salivary glands (Griffith et al., 1997); in another study, the pol sequences from a putative exogenous type-D-like retrovirus were found in lymph nodes (Yamano et al., 1997); in a systemic lupus erythematosus study seroreactivity towards short, type-C-like endogenous retroviral Env peptides and a HTLV-1 -de rived Gag peptide was described (Bengtsson et al., 1996). Finally, in rheumatoid arthritis, differential expression of multiple endogenous retroviral pol sequences in synovial fluid
• MS is associated with production of otherwise replicatively quiescent endogenous retroviruses. Whether this represent a causal factor by eliciting an autoimmune response is not fully substantiated yet.
• the presence of HERV-H/RGH sequences at particle level is clearly MS specific. This could indicate a direct and specific implication in the disease process, also because all plasma samples from MS patients with active disease were HERV-H/RGH positive.
• the epidemiology of the disease implies that if replication of endogenous retroviruses is a causal factor, the activation mechanism(s) is/are likely to be of external origin.
• retroviral particles associated with sequence variants of the human endogenous retrovirus HERV-H/RGH and produced by spontaneously formed long-term cell cultures from peripheral blood mononuclear cells from several MS patients can be transmitted to other, i.e. non-blood human cells and non-human cells. It is also shown that these long-term cell cultures consist of a mixed population of mainly B cells, and a smaller fraction of CD3+, CD4-, CD8- T cells.
• Intra-species transmission was demonstrated by co-cultivation of long-term cell cultures from peripheral blood from MS patients with a retroviral vector construct-harbouring cell line followed by assays for reverse transcriptase (RT) activity and assays for rescue of the retroviral vector-construct in indicator cells.
• RT reverse transcriptase
• the principles behind this co- cultivation assay are as follows: Transmission of retroviruses is to some extent dependent on cell-to-cell contact; i.e. cell-free transmission is possible, but occurs at a low frequency (de Rossi et al., 1985) (see Example 6).
• LacZ-donor cells harbour a retroviral vector- construct with the LacZ marker-gene. This construct is not transmissible in itself, but can be mobilised in trans.
• the LacZ-donor cells are co-cultivated with cells that produce retroviral particles. If the LacZ-donor cells are productively infected with retrovirus, a proportion of the subsequently produced infectious retroviral particles will contain the (mobilised) construct. These retroviral particles are produced into the co-cultivation supernatant.
• Blue cells and decrease in RT activity possible transmission as construct is mobilised; retroviral production may be low;
• the retrovirus producing long-term MS cell cultures are subcultured three times a week at a density of 0.5 x 10 6 cells/ml and cultivated in RPM1 1640 (Whittaker) supplemented with 200 i.u./ml penicillin (Leo), 0.2 mg/ml streptomycin (Rosco), 290 mg/ml glutamine (Sigma), 10 mM HEPES (Bioproduct) and 10% heat inactivated human serum (serum-pool obtained from the blood bank at Skejby Hospital, Denmark) in Costar bottles.
• the indicator cells are the human rhabdomyosarcoma cell line TE671 , and the LacZ-donor cells are TELacZ-cells.
• TELacZ is a clone of TE671 harbouring the MFGnlsLacZ retroviral vector (Takeuchi et al., 1994).
• TE671 and TELacZ are subcultured once weekly at a density of 0.5 x 10 6 cells/ml and cultivated in D-MEM (Dulbecco's modified Eagle's medium, Gibco, Life Technologies) supplemented with 5% FCS (foetal calf serum, Gibco, Life Technologies) and supplemented with 200 i.u./ml penicillin (Leo), 0.2 mg/ml streptomycin (Rosco) and 145 mg/ml glutamine (Sigma) in Costar bottles.
• D-MEM Dulbecco's modified Eagle's medium, Gibco, Life Technologies
• 6-well trays (Falcon 1146, non-tissue culture coated) are coated with human fibronectin (Sigma F-0895) at 10 ⁇ g/ml in PBS (standard phosphate buffered saline: 150 mM NaCl, 150 mM Na-phosphate, pH 7.4) for 2 hrs at room temperature.
• PBS standard phosphate buffered saline: 150 mM NaCl, 150 mM Na-phosphate, pH 7.4
• the fibronectin is aspirated and the wells are blocked with 2% BSA (FAF, Boehringer Mannheim) in PBS for 30 min. Then the wells are washed in culture medium, and TE671 or TELacZ are seeded at 3 x 10 4 cells/2ml medium/well and incubated at 37°C in a C0 cabinet.
• Fibronectin facilitate the possibility of transmission through co-localization of virus particles and target cells (Williams, 1999) After 3 days, the medium is aspirated and the retrovirus producing MS cells are added (in their own growth medium but with 5% HS only) at 3 x 10 5 cells/2ml medium/ well. After addition of the MS cells, the trays are centrifuged at 185 x g at room temperature for 10 min. and the trays are incubated again at 37°C in a C0 2 cabinet. A typical experiment include the following duplicate cell combinations:
• the controls without fibronectin are trays blocked with BSA, or Costar bottles. After 5 days of co-cultivation, half of the wells are washed gently 3 times with PBS and all wells are supplemented with fresh medium (with 5% HS) up to 5-6 ml. This washing step removes most MS cells, but not the MS cells associated with the well walls or with the indicator cells. After further 3 days of co-cultivation, a 1 ml sample is taken from each well and prepared for RT-assay.
• the remaining culture from each well is aspirated and filtered through a 0.45 ⁇ m filter (Acrodisc, Gelman Science) (saturated with PBS).
• This cell-free supernatant is plated onto TE671 indicator cells in Nunclon gridded culture dishes (Nunc 169558A), 1-2 dish(es)/well.
• the indicator cells are seeded at 2.5 x 10 5 cells/5ml medium/dish the day before, and incubated at 37°C in a C0 2 cabinet.
• 3.5 ml medium is aspirated from the dish before the addition ofthe filtered, cell free supernatant.
• the dishes are then incubated at 37°C in a C0 2 cabinet for 4-5 days.
• the supernatants are aspirated from the dishes and a 1 ml sample is taken from each dish and prepared for RT- assay.
• the cells are fixed by addition of 5 ml/dish of 0.05% glutaraldehyde in PBS.
• the dishes are incubated for 15 min at room temperature and then washed twice in PBS.
• the cells are then histochemically stained for ⁇ -galactosidase expression by the following procedure: 2 ml stain solution is added to each dish and they are wrapped air- and light-tight and incubated at 37°C for 24 hrs.
• the stain solution is aspirated, and the dishes are washed twice with 5 ml PBS.
• the stain-solution is prepared immediately before use and consists of a 1 :40 dilution of 40mg/ml X-Gal (5-bromo-4-chloro-3-indolyl- ⁇ -D- galacto-pyranoside; Sigma) in DMSO in 5 mM K-ferro cyanide, 5 mM K-ferri cyanide, 2 mM MgCI 2 , pH 7.
• a typical time-chart for the assay can be outlined as follows (if all of LacZ-donor cells, retrovirus producing cells and indicator cells are in culture on day 1):
• Day 1 Fibronectin coating of 6-well trays and seeding of TE621 and/or TELac; Day 3: Addition of retrovirus producing MS cells for co-cultivation;
• Scoring can be performed both as positives/negatives and using multiplicity of infection (MOI).
• the probability of any cell for infection by one of the viruses is 1/n c , therefore the probability of any cell being uninfected (P)is [1- (1/n c )] ⁇ v .
• log P n v - log [1 - (1/nc )].
• n c > 3 x 10 3
• n c • log [1 - (1/n c )] -0.4343.
• MOI corrects for the non- linearity of a transmission assay with a simple positive-negative scoring.
• the specific activity of ⁇ -galactosidase can also be determined using the following quantitative photometric assay (Feigner et al., 1994):
• Cells are washed in PBS and lysed in 350 ml of 250 mM Tris-HCl (pH 8.0), 0.1% Triton X- 100, and the lysates are stored at -80°C for at least 1 h.
• 50 ml of lysates and dilutions are combined with 50 ml of PBS, 0.5% BSA and 150 ml 60 mM Na 2 HP0 4 (pH 8.0),1 mM MgS0 4 , 10 mM KCI, 50 mM ⁇ - mercaptoethanol, 0.1% chlorophenol red galactopyranoside (Boehringer).
• RT reverse transcriptase activity
• RT is a retrovirus specific enzyme, which is present in retroviral particles. Monitoring of the RT activity thus constitutes a method for monitoring de novo production of retroviral particles.
• PCR-based RT assays are necessary for measuring directly on small volumes, of supernatant.
• the general assays and the RT profiles of the MS long- term cell cultures have been described in Christensen et al., 1999.
• the ultrasensitive RT assays are based on the conversion of an exogenous RNA template to amplifiable DNA by the sample-contained RT.
• the assays are performed essentially as described by Pyra et al., 1994:
• 1 ml aliquots of cell culture supernatant is obtained by aspiration.
• the samples are passed through a 0.2 ⁇ m filter (Sartorius Minisart) and centrifuged at 4°C 63.000 x g for 100 min (Sigma 3K-30) or 100.000 x g 1 hr in a SW 55 (Beckmann).
• Supernatants are aspirated and pellets suspended in 5 ⁇ l buffer A/ml aliquot of supernatant (buffer A: 50 mM KCI, 25 mM Tris-HCl pH 7.5, 5 mM DTT, 0.25 mM EDTA, 0.025% Triton X-100; 50% Glycerol). Samples must be taken and processed on the same day to prevent loss of RT activity. Samples in buffer A are stored at -70°C.
• CATAGGTCAAACCTCCTAGGAATG-3' (SEQ ID NO:25) are annealed before each assay by adding 0.28 pmol MS2 RNA to 9 pmol in a volume of 1.4 ⁇ l in an RNAse-free 0.5 ml Eppendorf-tube. RNA/primer is heated 95°C 5 min, incubated 37°C 30 min and cooled at 4°C (5 min), spun briefly and kept on ice during the next steps.
• the mixture is incubated 30 min at 37°C, followed by 25 cycles of 94°C 30 sec, 55°C 100 sec, 72°C 110 sec; cooled at 4°C 5 min all in a Perkin Elmer Cetus 480 Thermocycler.
• PERT assays can be performed with two variants of detection: either by hybridisation of amplified products with hapten conjugated oligonucleotides followed by ELISA, essentially as described or by adjustment to the TaqMan technology (Perkin Elmer Cetus).
• the annealed probe is hydrolysed by the 5' nuclease activity of Taq polymerase, thereby enabling the differential measurement of emitted fluorescence in a spectrophotometer.
• ELISA detection is performed as follows: NUNC 96-well titer plates are coated with 50 ⁇ l Avidin-solution 50 ⁇ g/ ⁇ l in coating-buffer (200 mM NaCl, 100 mM Tris-HCl pH 7.4), wrapped light-tight and incubated at room temperature overnight.
• the plates are washed 3 times with 200 mM NaCl, 100 mM Tris-HCl pH 7.4, 0.3% Tween 20, blocked with 200 ⁇ l blocking-buffer in each well for 30 min at room temperature (200 mM NaCl, 100 mM Tris- HCI pH 7.4, 0.05% Tween 20, 20% FCS; washed 3 times and are ready for hybridisation: 25 ⁇ l of PCR-reaction are added to a 0.5 ml Eppendorf-tube with 10 ⁇ l hybridising-mix (0.625 ⁇ l RT-3Bio (10 pmol, 5'-TTAATGTCTTTA-GCGAGACGC-3' (SEQ ID NO:27), 0.4 ⁇ l 500 mM EDTA pH 8, 0.375 ⁇ l 1 M KCI, 0.15 ⁇ l RT-5Dig (10 pmol, 5'- ATGGCTATCGCTGTAGGTAGC-3' (SEQ ID NO:28), 1.0 ⁇ l 100 M Tris-HCl pH 8.3, 7.45
• Fab anti-digoxigenin (Boehringer Mannheim, dil. 625 x in TBS/0.5mM EDTA pH 8) is added to each well, incubated 1 hr at room temperature, washed 5 times and 50 ⁇ l substrate (p-nitrophenyl phosphate) is added in substrate-buffer (Boehringer Mannheim), incubated 20 min, and OD 405 is measured in an ELlSA-reader.
• MS1533, MS1845, MS1851, MS1874, MS1946 are long-term cell cultures from MS patients; C91-PL is a human cell line producing HTLV-1 ; Relative RT values around 1 and above indicates de novo particle production; MOI values for the MS cell cultures are typically about 10 "6 to10 "5 , for C91-PL they are around 10 "3 .
• intra-species transmission was demonstrated by cell-free transmission of purified retroviral particles from long-term cell cultures from peripheral blood from MS patients to stimulated or non-stimulated peripheral blood lymphocytes from healthy individuals followed by assays for reverse transcriptase (RT) activity and characterisation of the transformed recipient cultures.
• RT reverse transcriptase
• the principles behind this cell-free transmission assay are as follows: Transmission of retroviruses is to some extent dependent on cell-to-cell contact; i.e. cell-free transmission is possible, but it occurs at a low frequency (de Rossi et al., 1985; Fan et al.,1992).
• Purified retroviral particles from long-term cell cultures from peripheral blood from MS patients are attempted transmitted to cultured, PHA-stimulated or non-stimulated lymphocytes from healthy individuals.
• Concomitant occurrence of RT-activity and continuous clonal growth in about one third of the experiments in which the retrovirus was attempted transmitted to such lymphocytes from healthy individuals was interpreted as indication of transmission, substantiated by the lack of activity and growth in the respective control cultures.
• Continuous clonal growth refers to a highly increased growth rate as illustrated by the need for feeding and subculturing 3 times a week contrasting feeding once a week without subculturing, accompanied by the occurrence of many free-floating clusters (clones) of cells together with free single blast-like cells. Transmission of the retrovirus is not always successful. Around one third of the recipient cultures will start to grow continuously as described above. Absence of contaminating cells from the donor culture is confirmed by chromosomal analysis of sex chromosomes (the transfer is from male donor to female or vice versa) The corresponding controls eventually die out.
• the cells are prepared for standard analysis: cells in growth phase are treated with colcemide (100 ⁇ l to 10 ml culture, incubated for 30 min at 37°C) for arrest of mitosis in the metaphase, then treated with hypotonic KCI (10 ml 0.56%) to 10 ml culture for 5 min) to spread the chromosomes, followed by fixation (in methanol.glacial acetic acid 3:1) and quinacrine banding (Q-banding): highly AT-rich DNA, such as poly(dA)-po!y(dT), markedly enhance quinacrine fluorescence while GC containing DNA quench fluorescence. (Dr. U. Friedrich, personal comm, see also Comings et al., 1975) before visualisation in the microscope.
• HERV-H/RGH sequence variants were demonstrated in gradient-purified retroviral particles produced from the putatively transformed cell cultures more than one month after infection.
• lymphocytes were isolated from 50 ml citrate blood samples from healthy volunteers by Ficoll-lsopaque (Amersham) density gradient centrifugation (900 x gfor 30 min at room temperature), the cells were seeded and cultured as described in Example 5 (10 7 cells/aliquot).
• Six bottles were prepared of each, to three of which 2 ⁇ l PHA (Difco)/10 ml was added. After 2 days, purified retroviral particles from the supernatant from a long-term cell culture from an MS patient (100 ml culture supernatant/aliquot) was added to 4 of the bottles, this representing a cell-free infection.
• the MS cells were cultured as described in Example 5. They have a fluctuating and rather low yet continuous production of retrovirus particles. Retroviral particles from the cell cultures are released into the supernatants which were collected for retrovirus preparation and purification. Twenty-four hours before harvest of the supernatants, the cells were diluted with one third fresh medium to obtain optimal growth conditions and thereby optimal virus production. 400-500 ml of cell-suspension: 1.2 - 1.4 x 10 6 per ml was used for one round of virus purification: cells and cell debris were removed from the supernatants by centrifugation at 1000 x g for 30 min at 4°C.
• OptiPrep is the trade name for lodixanol, an iodinated, nonionic density gradient medium from Nycomed Pharma, Oslo, Norway. OptiPrep leaves the retroviral particles intact, as opposed to sucrose.
• the tubes are centrifuged in a Sorvall rotor A641 at 45.000 x g for 2 h at 4°C and the supernatant removed from the tubes by aspiration, leaving a volume of 4-5 ml of the medium in immediate proximity of the cushion.
• the cushion and the overlying medium are mixed and the volume of the mixture is measured for final regulation of the concentration of the gradient which is optimal at 20% of OptiPrep.
• a buffered saline solution is used (0.8 % NaCl, 10 mM HEPES-NaOH, pH 7.4).
• the mixture is filtered through 0.45 ⁇ m (Acrodisc, Gelman Science) filters and transferred to 11.2 ml Ultraclear Tubes (Beckmann) and run in a near vertical rotor (Beckmann NVT 65) at 364.000 x g at 4°Cfor 3.5 h. Following centrifugation, the fractions were harvested in 0.5 ml aliquots by puncture of the bottom of the tubes.
• Fractions 2 - 5 were collected for use in the transmission assays. These fractions were pooled, diluted in buffered saline and pelleted by a final run in an SW41 rotor at 150.000 x g for 90 min at 4°C.
• the pellets were resuspended in 100 ⁇ l TNE buffer (50 M Tris pH 7.5, 100 mM NaCl, 1 mM EDTA). Usually, the pooled fractions were centrifuged-in 4 tubes, resulting in 400 ⁇ l of virus suspension which was added in 100 ⁇ l aliquots to 4 bottles prepared as described above. The suspensions were cultured and examined in an upside-down microscope (Leitz Wetzlar microscope) twice weekly and supernatant samples for PERT (see Example 5) were taken regularly. Transmission of the retrovirus is judged successful when RT activity is observed concomitantly with the major appearance of clonal growth; this happens usually after about one month. The corresponding controls eventually die out. These controls were parallel PHA-stimulated as well as untreated cultures of the recipient lymphocyte preparations for each transmission assay. Assays for reverse transcriptase (RT) activity were performed as described above.
• TNE buffer 50 M Tris pH 7.5, 100 mM NaCl, 1
• Retroviral particles from the supernatant of the established transmission cultures were purified by OptiPrep gradient ultracentrifugation as also described above and characterised by RT-PCR as follows: RT activity was measured for each gradient fraction and poly-A RNA was purified by lysing the 5-6 fractions with major RT activity directly in 5- 10 volumes of lysis-binding buffer (Dynal, Norway), adding 100 ⁇ l beads/sample (mRNA Direct kit, Dynal, Norway) and following the manufacturer's instruction. If not used directly, the RNA-coupled beads were stored in 80% EtOH at
• RNA sample was treated with amplification grade DNAse (Life Technologies) according to the manufacturer's instructions. After cooling on ice the DNAse was removed by exchanging the buffer containing the RNA-complexed paramagnetic beads with DEPC-ddH 2 O.
• RNA PCR (RT-PCR) was performed using the GeneAmp RNA PCR kit (Perkin Elmer) according to the manufacturer's suggestions, except that 1 u of Taq polymerase was used pr. reaction.
• First strand synthesis was primed with random hexamers, in conjunction with the bead- oligo-dT complexed RNA-template.
• the integrity of the RNA template was confirmed by cDNA synthesis, specifically primed with the downstream primer; in this case, control cDNA synthesis primed with the corresponding upstream primer was negative.
• Primer sets for PCR HERV-H/RGH-specific primer sets for the gag (1, 11) and immediate upstream env regions:
• the differences between presented donor and recipient sequences do not exclude transmission as the HERV-H/ RGH variants constitute a population of molecules (at least in the donor) rather than a single sequence.
• the absence of transformation of any of the PHA-stimulated control cultures and the particle production in the infected recipients are also indicative of transmission.
• RGH/HERV-H retrovirus particles Inter-species transmission of RGH/HERV-H retrovirus particles was demonstrated by cultivation of PHA-stimulated or non-stimulated cells from non-human species with purified retroviral particles from long-term cell cultures from peripheral blood from MS patients followed by assays for reverse transcriptase (RT) activity.
• RT reverse transcriptase
• Endogenous retroviruses are defined as being either xenotropic (i.e. do not infect cells of host, but are frequently able to infect other species), ecotropic (confined to host species, but may infect somatic cells within the species) or amphotropic (able to replicate in host and in distantly related species).
• endogenous retroviruses perse may or may not be infectious and, if infectious, may or may not be able to cross the species border.
• Many endogenous retroviruses are known to have spread to other species through horisontai transfer (Todaro, 1975) .
• the putatively infectious HERV-H/RGH associated retroviral particles from the long- term MS cell cultures might also be able to infect cells from other species.
• species that are evolutionarily distinct from the host species of the endogenous retrovirus (human in this case), i.e species that diversified from the human/primate line before the endogenous retrovirus entered the germ line.
• HERVs entered the primate lineage around or after the divergence of Old World and New World monkeys (see Anderssen et al., 1997). Accordingly, rodent cells or virus-permissive rodent cell lines appear to be an obvious possibility.
• rabbit lymphocytes were isolated from 50 ml heparinised blood samples by Ficoll-lsopaque (Amersham) density gradient centrifugation (900 x g for 30 min at room temperature), the cells were seeded and cultured as described in Example 5 (5x 10 6 cells/aliquot). Alternatively, the culture medium was supplemented with heat inactivated rabbit serum rather than human serum. 4-5 bottles were prepared of each, to which 2 ⁇ l PHA(Difco)/10 ml was added.
• the putatively infected rabbit cells were cultured and monitored essentially as outlined in Example 6.
• the cells were examined in the upside-down microscope (Leitz Wetzlar microscope) twice weekly, and supernatant samples for PERT were taken regularly. Transmission of the retrovirus was considered possible, when RT activity is observed, usually developing a transient peak in RT activity over time.
• the control cells remain RT negative.
• An example is shown in Fig. 7.
• transduction of the LacZ-construct (or a similar construct) as described in Example 5 would enable scoring of transmission by both RT-assays and by assays for induction of ⁇ -galactosidase activity.
• CD antigens Cluster of Differentiation
• Each CD antigen can bind monoclonal antibodies specific for that surface protein.
• FACS analysis fluorescence-activated cell sorting
• the long-term cell cultures from peripheral blood from MS patients were further characterised using this technique. These cultures were originally defined as "MS cell lines" and, using immunocytochemistry, shown to be B-lymphoblastoid in nature as outlined in Sommerlund et al., 1993. The more sensitive flow cytometric analyses described below have shown that, whereas the majority of cells in the culture are B cells (CD19+), a small fraction of the cells are actually T cells (CD3+). Thus, the cell cultures constitute a mixed population of cell-types that are co-existing. It is thus possible that an interaction within the population is necessary for the active production of retroviral particles; alternatively, either the B cells or the T cells produce the retrovirus.
• the cells were characterised as follows:
• anti-CD3 the cell receptor complex, T cells
• anti-CD4 accessory molecule in MHC II associated antigen recognition, subgroup of T cells, monocytes, macrophages
• anti-CD8 accessory molecule in MHC I associated antigen recognition, subgroup of T cells
• anti CD-10 common acute lymphoblastic leukaemia antigen (CALLA), haematopoietic stem cells
• anti-CD14 LPS complex receptor, B cells, monocytes, macrophages
• anti-CD19 regulation of proliferation, B cells
• anti-CD45 leukocyte common antigen
• anti-CD56 cell adhesion molecule, NK cells
• anti-HLA-DR tissue type
• anti-CD3, anti-CD4, anti-CD8, anti-CD19 were found relevant and important: anti-CD3, anti-CD4, anti-CD8, anti-CD19.
• the results were as follows: Most of the cells were identified as being CD 19+ cells, i.e. they are B-cells. A small fraction of all MS cell-cultures appeared to be CD3+ cells, i.e. are CD4- and CD8-. Examples of the precise percentages of the latter category are shown in the below table:
• T-cells in peripheral blood are either CD4+ or CD8+.
• CD3+ cells without these markers is an interesting observation.
• separation of the two cell types, and an extended panel of antibodies will be necessary.
• HERV-H (RGH) mRNA splicing variants in mononuclear cells from MS patients.
• spliced HERV-H mRNA sequence variants are expressed in vivo in cellular RNA from mononuclear cells from MS patients.
• the sequence variants were found specifically in cells from some of the blood samples from MS patients and autoimmune patients.
• the sequence variants were not found in mononulear cells from healthy controls.
• HERV-H virus family consists of about 1000 defective members containing large deletions and stop codons, whereas 100 copies are approximately full length, potentially encoding functional proteins (Mager and Freeman 1987, Hirose et a/., 1993, Wilkinson et al., 1993) .
• One env sequence with an open reading frame has been isolated and transcribed in vitro (Lindeskog and Blomberg, 1999).
• Expression of HERV-H RNA in vivo has not been very well characterised though there is accumulating evidence that transcription occurs in normal as well as in malignant cells (Hirose et al, 1993, Lindeskog et al, 1993, Lindeskog and Blomberg, 1997). Splicing of HERV-H subgenomic transcripts has been described, revealing a complex pattern of multiply spliced mRNA species (Wilkinson et a/., 1990, Lindeskog and Blomberg 1997).
• HERV-H env mRNA sequence variants were demonstrated by performing RT-PCR analysis on clinical specimens: blood samples from MS patients, patients with different autoimmune diseases and healthy controls. Mononuclear cells were isolated, total cellular RNA were extracted and assayed by RT-PCR. Amplicons were cloned and sequenced.
• RNA pellets were stored in 80% EtOH (in DEPC-treated H 2 0) at -80°C.
• RNA pellet was washed two times in 80% EtOH, air dried and suspended in DEPC-treated H 2 0, 50 ⁇ l per 10 7 cells. 7 ⁇ l were used for RT-PCR analyses.
• RNA aliqouts were stored at -80°C.
• RT-PCR was performed using the Superscript System (SuperscriptTM Preamplification System for First Strand cDNA Synthesis, Life Technologies). Before cDNA synthesis the RNA was treated with DNAsel (Deoxyribonuclease I, Amplification Grade, Life Technologies) according to the manufacturer ' s instructions, including Ribonuclease Inhibitor (Life Technologies), 0.01 U/ ⁇ l, at relevant steps . All reactions were performed in a Perkin Elmer DNA Thermocycler model 2400. Following inactivation of DNasel by treatment with EDTA at 65°C, the RNA was annealed, 70°C 10 min, to a primer designed from the RGH2 sequence (Hirose et al., 1993):
• cDNA synthesis was performed using the conditions prescribed in the protocol and RNA was digested with RNaseH (Ribonuclease H, Life Technologies) before purification of the cDNA using a QIAquick PCR purification kit (QIAGEN) according to the manufacturer ' s instructions.
• the cDNA was eluted in the EB buffer 2 x 25 ⁇ l and stored as aliquots at -80°C, unless used directly as template for PCR.
• PCR was performed in a Perkin Elmer DNA Thermocycler model 2400 using 50 ⁇ l reactions containing: 15 mM Tris-HCl, pH 8.0, 50 mM KCI, 2.5 mM MgCI 2 , 200 ⁇ M dNTP, 0.5 ⁇ M of each primer, 2.5 U AmpliTaq Gold polymerase (Perkin-Elmer) and 5 ⁇ l cDNA. Conditions for PCR were: 10 min 95°C 1 cycle, 94°C 20 sec, 59°C 20 sec, 72°C 30 sec 50 cycles and 72°C 2 min 1 cycle.
• Primers used for PCR amplification were designed from the assumed primer binding site (PBS) in the RGH2 sequence (Hirose et al., 1993) and from an HERV-H sequence containing an open env reading frame (Lindeskog et al.,1999):
• PCR products were analysed by agarose gel electrophoresis. Relevant bands were cut out, cloned in the pCR ® 2.1 vector (Original TA Cloning ® kit, Invitrogen) and sequenced in both directions using the Thermo Sequenase fluorescent labelled primer cycle sequencing kit (Amersham Life Science). Sequence reactions were analysed on the ALFexpress DNA automatic sequencer (Pharmacia Biotech) and sequence analyses were performed using fasta and blast programs (Wisconsin Package, Version 9.1, Genetics Computer Group (GCG)).
• GCG Genetics Computer Group
• primers for PCR were derived from the RTVLH2 (PBS) and RGH2 (env) sequences (Mager and Freeman 1987, Hirose et al., 1993) , probably explaining why alternatively spliced env mRNA sequences were also seen in healthy samples. It is our experience that PCR primers derived from the RGH2 sequence generally detects a broader group of HERV-H sequence variants and do not discriminate between samples from MS patients, other autoimmune patients or healthy controls (results not shown).
• the 850 bp sequences shared 90-95 % homology with the RGH2 leader/integrase and env region and 93-97% homology with each other. Most of the sequences contain 100 bp of an open env reading frame (5'of the env PCR primer).
• sequence variants containing the 104 bp protease encoding region are shown in Table 9.1.
• the sequences represent 10 clones (7 variants) isolated from 3 MS patients (MS1, MS2 and MS3) and 1 patient with diabetes mellitus (AU1).
• the sequence variants contain single base changes: substitutions and deletions, as compared to the clone MS1-1. This sequence was found in only one of the MS patients and was identical to the sequence isolated from a T-cell leukaemia cell line (u88896, Lindeskog and Blomberg, 1997).
• sequences of the inserted protease region is compared to the U88896 sequence (see above) with deviations highlighted.
• the material is the same as in Table 9.1 , i.e. 10 independent clonings of the 950 bp fragment from 4 individuals.
• the region analyzed in Table 9.2 starts at the splice donor site (position 1) and ends at the splice acceptor site (position 104) in the u88896 sequence (reference sequence). This region is designated PSR in the following.
• the single PSR sequence cloned from patient MS1 is identical to the reference sequence, while the single PSR sequence from patient MS2 shows two substitutions at positions 82 and 97. These two substitutions are shared by three individuals (MS2, MS3, and AU1). This may indicate that positions 82 and 97 are markers of RGH alleles at the same genetic locus in the human genome or markers of closely related RGH genes at different genetic loci. Similarly, the single-base deletion at PSR position 53 which is shared by two individuals (MS2 and AU1) may represent a further such marker.
• the single-base changes at position 13 in one PSR-region from patient MS2 and at position 75 in two PSR-regions from AU1 may represent additional genetic markers as described above or may be attributed to errors introduced into proviral DNA during reverse transcription.
• Table 9.1 Alignment of seguence variants from the protease encoding region in the alternatively spliced env mRNA from three MS patients and one patient with an autoimmune disease: diabetes mellitus.
• Boiler K Janssen O, Schuldes H, Tonjes RR, Kurth R. Characterization of the antibody response specific for the human endogenous retrovirus HTDV/HERV-K. J Virol, 1997, 71:4581-4588.
• HERV-K is the endogenous retrovirus sequence that codes for the human teratocarcinoma derived retrovirus HTDV. Virology, 1993, 196:349-354.
• Conrad B Weissmahr RN, Boni J, Arcari R, Sch ⁇ pbach J, Mach B. A human endogenous retroviral superantigen as candidate autoimmune gene in type 1 diabetes. Cell, 1997, 90:303-313.
• Kelleher CA Wilkinson DA, Freeman JD, Mager DL, Gelfand EW. Expression of novel transposon-containing mRNAs in human T cells. J Gen Virol, 1996, 77:1101- 1109. Klenerman P, Hengartner H, Zinkernagel RM. A non-retroviral RNA virus persists in DNA form. Nature, 1997, 390:298-301.
• Morozov VA Lagaye S, llyinski PO, Cherepovsky DN, llyin K. Analyses of retroviral proteins by two-step DNA, zinc binding and modified South-Western blotting. Intervirology, 1992, 34:117-123.
• Type C retrovirus inactivation by human complement is determined by both the viral genome and the producer cell. J Virol, 1994, 68:8001-8007.

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