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WO1996030398A1 - Nouvel auto-antigene - Google Patents

Nouvel auto-antigene Download PDF

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Publication number
WO1996030398A1
WO1996030398A1 PCT/AU1996/000166 AU9600166W WO9630398A1 WO 1996030398 A1 WO1996030398 A1 WO 1996030398A1 AU 9600166 W AU9600166 W AU 9600166W WO 9630398 A1 WO9630398 A1 WO 9630398A1
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Prior art keywords
seq
polypeptide
peptide
dna sequence
sequence
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PCT/AU1996/000166
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English (en)
Inventor
Mohammad Hossein Sanati
Patrick Robert Carnegie
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Murdoch University
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Publication date
Priority claimed from AUPN1974A external-priority patent/AUPN197495A0/en
Priority claimed from AUPN7205A external-priority patent/AUPN720595A0/en
Application filed by Murdoch University filed Critical Murdoch University
Priority to AU49995/96A priority Critical patent/AU4999596A/en
Publication of WO1996030398A1 publication Critical patent/WO1996030398A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens

Definitions

  • This invention relates to polypeptides or peptides or analogues thereof, the amino acid sequences of which encode antigenic segments which immunologically react with multiple sclerosis induced antibodies.
  • These polypeptides or peptides or analogues thereof are useful as diagnostic reagents for detecting the presence of antibodies or T cells from patients with multiple sclerosis and may also be useful as immunogens, in compositions and methods, to illicit anti-idiotypic antibodies against the abnormal autoimmune responses involved in the pathogenesis of multiple sclerosis.
  • MS Multiple sclerosis
  • MS Diagnosis of MS currently depends upon the demonstration of physical signs that imply the presence of plaques in multiple areas of the CNS of MS patients. When this cannot be determined clinically, electrical survey of the nervous system, using evoked potential testing, may be useful. Generally, the diagnosis of MS tends to be clinical; if laboratory support is not forthcoming, observation of the patients course over subsequent months or years may allow it to be made with confidence. There is now general agreement in the scientific community that abnormal autoimmune responses are involved in the pathogenesis of MS but despite intensive work over the last thirty years there is no consensus as to the importance of the various antigens which have been shown to react with antibodies or T cells from patients with MS.
  • the present invention provides peptides and polypeptides or fragments thereof which are immunologically reactive with antibodies obtained from the serum and cerebrospinal fluid of patients with MS. Such peptides and polypeptides or fragments thereof may be employed in diagnostic assays for MS and in the preparation of anti-idiotypic antibodies which may be used in the treatment of the disease.
  • the present invention resides in a DNA sequence encoding at least a peptide that is immunologically reactive with MS induced antibodies wherein that DNA sequence is derived from the following nucleotide sequence:
  • the present invention also resides in an amino acid sequence which corresponds to the above nucleotide sequence, that sequence being:
  • the above amino acid sequence is written as it would be expressed in mitochondria.
  • the methionines at positions 3, 29 and 42 are replaced by isoleucine.
  • the mitochondrial sequence has 2 tryptophan residues.
  • the codons for these mitochondrial tryptophans are recognised not as amino acids but as stop codons.
  • the present invention encompasses the product of the above nucleotide sequence as expressed in mitochondria as well as the expression of such a sequence in procaryotic and eucaryotic cells.
  • Polypeptide sequences described herein will be identified according to their origin of expression (ie mitochondrially expressed, procaryotically expressed etc). Where an amino acid sequence is not identified by an origin of expression, that sequence does not contain any amino acids which would be altered by an alternate origin of expression.
  • the invention further encompasses fragments of the above amino acid sequence(s) which fragments are immunologically reactive with MS induced antibodies.
  • MS IgG and IgA fragments within the scope of the invention include, but are not limited to:-
  • a polypeptide or peptide fragment written as expressed in mitochondria also encompasses polypeptide or peptide fragments expressed by procaryotes or eucaryotes and vice versa.
  • the above sequences are of the general formulae X-Y-Z wherein: X and Z each represent individually of each other a hydrogen atom, or another amino acid, a protected amino acid, another sequence of the general formulae X-Y-Z, a peptide, a polypeptide, an amino group, a carboxyl group or an adjuvant; and Y represents one of the above amino acid sequences.
  • Such sequences may be glycosylated or non-glycosylated.
  • At least one of X and Z may comprise at least one of the above sequences which may be the same or different from Y.
  • the invention also encompasses functionally equivalent analogues of the above polypeptide or peptide sequences, which have at least a immunological property in common with the immunological properties of the aforementioned sequences.
  • Analogues as used herein refers generally to amino acid sequences which are functionally equivalent to the polypeptide or peptide sequences of the invention but which contain substitutions, deletions or additions made to those sequences.
  • the present invention encompasses oligo-peptides prepared from the amino acid sequences set out above or fragments, analogues thereof which include linear (continuous) epitopes, conformational epitopes or both which are immunologically reactive with MS antibodies.
  • amino acid sequences of the invention are useful, alone or in combination, uncoupled or coupled to other molecules, in diagnostic methods for detecting MS, in treating MS, and in the production of polyclonal and monoclonal antibodies.
  • monoclonal or polyclonal antibodies may be produced which may find use in diagnostic assays or as immunogens for use in the treatment of MS.
  • Nucleotide sequences of the present invention may be employed in a process for the production of a peptide or polypeptide of the invention or a fragment or analogue thereof comprising the steps of: culturing a host organism contain an expression vector containing a gene which encodes a polypeptide or peptide of the invention or a fragment, or analogue thereof under suitable conditions to permit expression of that peptide, polypeptide fragment, or analogue; and recovering the expressed peptide, polypeptide, fragment, or analogue.
  • ND4 is one of forty one components of Complex I (NADH dehydrogenase or NADH:ubiquinone oxidoreductase). ND4 is one of the key components of the respiratory chain and is encoded by mitochondrial DNA.
  • Complex I is one of the components of the electron-transport chain which is involved in electron transport across mitochondrial membranes. Electrons from the reduced coenzyme NADH are passed to co-enzyme Q. The electrons then pass through a series of further complexes to oxygen. By coupling the transfer of electrons with a directional pumping of protons, some of the free energy released in the transport process is stored as an electrochemical potential that is in turn used to drive ATP synthesis in mitochondria.
  • LHON Leber's Hereditary Optic Neuropathy
  • DNA sequences described herein which encode peptides and polypeptides which are specific for MS antibodies are conspicuously valuable for the information which they provide concerning the mode of action of MS.
  • the DNA sequences are also valuable as products useful in effecting the large scale microbial synthesis of antigens related to MS by a variety of recombinant and synthetic techniques.
  • DNA sequences provided by the invention are useful in generating new and useful DNA vectors, new and useful transformed and transfected microbial procaryotic and eucaryotic host cells (including bacterial and yeast cells and mammalian cells grown in culture).
  • Peptides and polypeptides of the present invention embrace analogues and homologues.
  • modifications of cDNA and mitochondrial genes may be readily accomplished by well-known site-directed mutagenesis techniques and employed to generate analogues of the specified peptides.
  • Such analogues should possess at least one of the immunological properties of the sequences specified herein but may differ in others.
  • Amino acids residues that are functionally equivalent and that can be substituted in the same position for other amino acids to produce analogues without substantially effecting the conformational arrangement of an antibody are known to the art. For example, exchange of the positively charged amino acids arginine and lysine is considered to be a conservative substitution. Similarly substitution of the hydrophobic amino acids valine, leucine, isoleucine and methionine, the hydroxy amino acids threonine and serine and the acidic amino acids glutamic acid and aspartic acid are considered to be conserved.
  • Immunogenic peptides or polypeptides as described above may be used to produce either monoclonal or polyclonal antibodies.
  • a selected mammal eg mouse, rat, sheep, monkey
  • an immunogenic polypeptide bearing an MS specific epitope eg mouse, rat, sheep, monkey
  • an MS specific epitope eg. the diphtheria toxoid immunogen discussed herein.
  • Serum from the immunised animal is then collected and treated according to known procedures (Linthicum etal 1981) which will depend on the ultimate use to which the polyclonal antibody will be employed. If serum containing polyclonal antibodies to the MS specific peptides and polypeptides contain contamination antibodies (ie. generated against other antigens) the antibody sera can be purified by for example immunoaffinity chromatography.
  • polyclonal antibodies can be isolated from a patient suffering from MS. A full discussion of how such antibodies may be isolated is discussed below.
  • Monoclonal antibodies generated against the MS related peptides and polypeptides can also be readily produced by one skilled in the art.
  • the general methodology for making monoclonal antibodies by hybridomas is well known (Campbell, 1984).
  • Immortal antibody-producing cell lines can be created by cell fusion or by direct transformation of B lymphocytes with oncogenic DNA or by transfection with Epstein Barr Virus.
  • Antibodies either monoclonal or polyclonal, which are directed against MS related peptides or polypeptides are useful in diagnosis. Further monoclonal antibodies, may be used to raise anti-idiotypic antibodies (ie which carry an internal-image of the antigen of the MS specific peptides or polypeptides). Such antibodies may be used in the treatment of MS (Thornton and Griggs, 1994). Techniques for raising anti-idiotypic antibodies are known in the art see, for example Dreesman etal (1985) (Poskitt etal, 1991) and (Linthicum and Farid, 1988).
  • Peptides and polypeptides which react immunologically with serum containing MS immunoglobulins and the immunoglobulins raised against the MS specific epitopes are useful in immunoassays to detect the presence of MS immunoglobulins in biological samples, including cerebrospinal fluid, blood and saliva.
  • Such detection kits include, but are not limited to homogeneous and heterologous binding immunoassays, such as enzyme linked immunoabsorbant assays (ELISA), radioimmunoassays (RIA), Western Blot analysis, and enzyme inhibition assays.
  • ELISA enzyme linked immunoabsorbant assays
  • RIA radioimmunoassays
  • Western Blot analysis and enzyme inhibition assays.
  • Peptides and polypeptides of the invention may be labelled or unlabelled depending on the type of assay used.
  • Labels which may be coupled to the peptides are those known in the art and include but are not limited to enzymes, radionucleotides, fluourogenic and chromogenic substrates, cof actors and, biotin-avidin, colloidal gold and magnetic particles.
  • the peptides and polypeptides can also be coupled to other peptides or polypeptides, solids supports and carrier polypeptides by any means known in the art.
  • solid supports include for example polystyrene or polyvinyl microtitre plates, glass tubes, or glass beads and chromatographic supports such as paper, cellulose and cellulose derivates and silica.
  • Carrier polypeptides include for example bovine serum albumen (BSA) and Keyhole Limpet hemocyanin (KLH).
  • Techniques especially useful for large scale clinical screening of patients sera, cerebrospinal fluid or saliva include ELISA and agglutination assays. Such techniques are preferred for their speed, and their ability to test numerous samples simultaneously and ease of automation.
  • Protocols upon which ELISA assays may be based include for example competition assays, direct reaction assays and sandwich type assays.
  • samples including for example body fluids and tissue samples may be added to a peptide coated wells in for example a microtitre tray where an immunological complex forms if MS antibodies are present in the sample.
  • a signal generating means may be added to detect complex formation.
  • a detectable signal is produced if MS specific antibodies are present in the sample.
  • Peptides or polypeptides of the invention are conveniently bound to the inside of microtitre wells. Peptides may be directly bound by hydrophobic interaction with the microtitre wells or attached covalently to a carrier polypeptide by means known in the art. The resulting conjugant being used to coat the wells.
  • an assay which may be used to detect the presence of MS antibodies is an agglutination assay.
  • Such assays utilise latex support (eg beads) to which are bound at least one of the MS specific peptides.
  • the coated latex beads are mixed with a small volume of patient sera and examined for agglutination. If MS specific antibodies are present in the patient's serum agglutination (clumping) of the latex beads will be observed. While this assay is not as specific as an ELISA, latex assays are quick and easy to perform and would be suitable for medical practitioners or naturopaths as initial screening.
  • the ELISA is a far more sensitive and quantifiable assay than the latex agglutination assay. Preliminary screening studies using MS specific peptides and polypeptides have shown that MS antibodies are found in low levels in patient's sera. Thus it is expected that the ELISA test would be at least a method of choice for the detection of MS antibodies.
  • a more specific assay for detecting the presence of MS utilises an enzyme inhibition assay.
  • NADH ubiquinone reductase activity is measured in the presence of sample material suspected of containing MS antibodies.
  • NADH ubiquinone reductase enzymic activity is substantially reduced indicating the presence of MS in a patient.
  • the invention also provides a method of treating a patient suffering from MS wherein that treatment involves administration of an effective amount of suitable quinones to MS patients to restore the function of the damaged oligodendrocytes.
  • the invention provides a method of treating a patient suffering from MS wherein that treatment involves administration of a humanised anti-idiotypic monoclonal antibody which will negate the effects of MS antibodies.
  • Figure 1 shows the electrophoretic pattern of products of amplification by PCR of the DNA clones isolated with MS IgG. ⁇ gtl 1 primers were used for the amplification.
  • the marker DNA in lane 1 was a mixture of puc 19 DNA/MpA II and ⁇ DNA Eco Rl/Hind III.
  • Lanes 3 and 4 represent the products from clones M62 and M63 respectively.
  • Figure 2 shows the consensus sequence from analysis of nucleotides in clones M62 and M63 which were found to express peptides and polypeptides specific for multiple sclerosis antibodies. This sequence was found to be 98% identical to part of the human mitochondrial gene (HUMMTCG in GenBank release number 80) from nucleotide 11699 to 11960.
  • FIG. 3 shows that the consensus nucleotide sequence in M62/63 is over 95% identical to six depositions of human mitochondrial DNA in GenBank
  • Figure 4 shows amino acid sequences predicted from the nucleotide sequences.
  • Figure 4a shows the predicted amino acid sequence for the protein expressed in clone M62 which reacted with antibodies from patients with multiple sclerosis based upon codon usage in E. coli.
  • Figure 4b shows the predicted amino acid sequence for human mitochondrial ND4 protein from amino acids 315 to 367.
  • the amino acid sequences presented in figures 4a and 4b are represented in single letter amino acid coding form, in accordance with the following table:
  • Figure 5 shows an epitope prediction analysis of the sequence in Figure 4a and the peptide sequences synthesised.
  • Figure 5a represents the antigenic index analysis by MacVector 3.5.
  • Figure 5b represents the conjugate prepared for immunisation of a rabbit and as an antigen (diphtheria toxoid -S-MS immunoreactive peptide) for testing MS IgG and
  • Figure 5c represents the peptide prepared for use as an antigen in peptide ELISA.
  • Figure 6 shows the reactivity of rabbit sera to diphtheria toxoid and diphtheria toxoid -S-MS immunoreactive peptide.
  • the diphtheria toxoid was used to coat the wells 1 -5 and the conjugate was used in well 6.
  • Figure 7 shows the reactivity of rabbit sera to the 20 amino acid peptide presented in Figure 5c.
  • Figure 8 shows the reactivity of human MS IgG to diphtheria toxoid and diphtheria toxoid -S-MS immunoreactive peptide. Diphtheria toxoid was placed in wells 1 -3 and the peptide conjugate in well 4. Sequential transfer of the supernatant was done as described for Figure 6.
  • Figure 9 shows the reactivity of IgA in human saliva to the diphtheria toxoid
  • Sample numbers 1 to 12 were normal saliva, 13 to 31 were from people with MS.
  • Figure 10 shows the reactivity of human IgG to the 20 amino acid sequence in Figure 5c. IgG from individual MS patients (1 to 10) was reacted at an equivalent of 1/200 dilution of original sera with the 20 amino acid peptide. Pooled normal human IgG is shown as 0.
  • FIG 11 shows the activity of Complex I from chicken brain submitochondrial particles (SMP) with additions of human IgG.
  • SMP serum submitochondrial particles
  • the SMP were 10 ⁇ l,(2 mg total protein/ml) were incubated for 90 min with the 10 ⁇ l IgG (0.4 mg/ml) before the addition of NADH and decylubiquinone in K phosphate 20 mM buffer to 1ml total volume with final concentrations of substrates of 0.15 mM and 0.1 mM respectively.
  • Figure 12 shows the effect of time of incubation on inhibition of Complex I by MS IgG conditions as per Figure 11 except for time of incubation. Inhibition of enzyme activity was determined between 2 and 10 minutes.
  • Figure 13 shows inhibition of Complex I by increasing concentrations of MS IgG conditions as per Figure 11 except for the concentration of IgG.
  • Figure 14 shows the reactivity of rabbit serum to the diphtheria toxoid -S-
  • Figure 15 shows the reactivity of IgG in human sera to the diphtheria toxoid-S-MS immunoreactive peptide after subtracting residual activity to diphtheria toxoid from 3 patients with MS who were bled at various intervals. The numbers show the weeks between investigations.
  • Figure 16 shows the reactivity of IgG in human sera to the diphtheria toxoid-S-MS immunoreactive peptide after subtracting residual activity to diphtheria toxoid from 54 patients with MS and 12 other people.
  • Figure 17 shows the reactivity of IgG in human sera to the diphtheria toxoid-S-MS immunoreactive peptide after subtracting residual activity to diphtheria toxoid from 8 patients with Leber's Hereditary Optic Neuropathy and 4 carriers of the LHON mutation (Mr/VD4*LHON11778A).
  • a ⁇ gt11 library was obtained from Dr C. Campagnoni, Mental Retardation Research Center, University of California, Los Angeles. It was made from poly A + RNA from spinal cords at 14-16 weeks gestation. The cDNAs were cloned into ⁇ gtl 1 using Eco R1 linkers. The base was 1.5 x 10 9 pfu per ml.
  • the column was connected to a peristaltic pump, UV detector (280nm) and recorder (Bio-Rad, model 1326). The column was washed by pumping with PBS until the detector had a stable reading.
  • the bound IgG was washed off with elution buffer (0.1 M acetic acid in 0.15 M NaCI, pH 2.8), neutralised by adding 100 ⁇ l of 1M Tris buffe H 8.9) per each ml eluted and dialysed (Spectraopor membrane MW cut off 6-8000) against 11 PBS(pH 7.4) overnight at 4°C (with stirring and 3 changes).
  • the total yield of IgG was estimated by reading the optical density (OD) at 280nm and calculating as follows before storing at -20°C at a concentration of approximately 0.4 mg/ml for the MS samples.
  • IgG solutions were preabsorbed to remove the anti-E. coli and anti-beta- galactosidase activity before use in screening of the ⁇ gtl 1 HFSC cDNA library.
  • a non-recombinant ⁇ gt11 phage was isolated from HFSC cDNA library using white/blue colour selection. Ten thousand phage from the library were plated on one 150mm Petri dish according to the procedure in the following section except that the top layer of agar was supplemented with 5-bromo-4-chloro-3-indolyl- ⁇ -D- galactoside (X-gal) 40mg/ml final concentration. One of the blue plaques was picked up after an overnight incubation at 37°C. The plaque was added to 300ml SM (Promega Protocols and Application Guide, Second Edition 1991) buffer containing one drop of chloroform, vortexed for 30 sec and incubated at least for 2 h at 4°C.
  • SM Promega Protocols and Application Guide, Second Edition 1991
  • the phage solution was replated three times in small plates until there were 100% blue plaques.
  • One of non-recombinant blue plaques was added to 300ml of SM buffer plus 1 drop of chloroform before incubation for 2 h at 4°C.
  • the phage solution (top layer) was added to 100ml of late phase culture of E. coli Y1090 and 900ml of LB media (Sambrook et al. 1989) was added to the culture and incubated for 2 h at 37°C with 250 rpm shaking.
  • the non-recombinant ⁇ gt11 phage was induced by raising the temperature to 45°C for 15 min. Isopropyl thiogalactoside (IPTG) was added to 10 mM final concentration and incubation continued for a further 2 h at 37°C with 250 rpm shaking. The bacteria was harvested by centrifugation at 500g for 15 min, resuspended in 50ml of PBS and then lysed by three freeze-thaw cycles using liquid nitrogen and 37°C water bath respectively. The bacterial lysate solution was then sonicated 3x30 sec bursts at maximum power to reduce the viscosity and stored at -20°C until used.
  • IPTG Isopropyl thiogalactoside
  • a 137mm nitrocellulose disc (Hybond-c extra, Amersham, England) was soaked in the bacterial lysate solution for 30 min before incubation for 1 h in blocking solution (TBS-T containing 5% BSA). The filter was washed with TBST twice for 1 min, soaked in 50ml IgG solution (diluted 1 :10 with PBS) and incubated overnight at 4°C with slow shaking. This procedure was repeated 3 or 4 times to make sure that all anti-bacteria and anti-beta-galactosidase activity was absorbed. The IgG solution was tested with a dot blot procedure to make sure that there was no reactivity with the E. coli lysate proteins.
  • the IgG solution was mixed with the equal volume of bacterial lysate, incubated overnight at 4°C with rotation and then centrifuged at 10O.OOOrpm, 15 m at 4°C. The supernatant was stored in 1 ml Ependorph tubes at -20°C until required.
  • the bacterial parallel culturing method (Sambrook et al. 1989) was adapted for screening phage preparation production of duplicate filters carrying the expressed recombinant protein.
  • An overnight culture of E.coli Y1090 was made in 10ml LB
  • Parallel lifts were obtained by incubating the phage plates at 37°C for 4-6 h until the plaques became visible.
  • One filter was placed on the first plate, marked for later orientation and transferred to a second plate after 1 min.
  • the second plate had only a bacterial lawn, prepared at the same time as the phage was plated.
  • the second duplicate filter was then placed on the first plate and marked. Both plates were incubated at 37°C for at least 4 h to overnight. The treatment of both filters was the same from this stage onwards.
  • ECL Enhanced chemiluminescence
  • Amersham's ECL Western blotting kit (Amersham Life Science, England), a sensitive non-radioactive method for detection of immobilised antigens, was adapted for screening the expressed proteins from a cDNA library.
  • the filters from above were washed with TBS-T and immersed in blocking solution (5% BSA in TBS-T) for 1 h at room temperature with low speed shaking to block the non- specific binding sites. Washing the filters with TBS-T was similar for all stages of this procedure; two washes of 1 min, one of 15 min and a further two of 5 min at room temperature with slow shaking, except for the last washing which had two extra 5 min washings.
  • the filters were washed before and after incubation in IgG solution and the secondary antibody (anti-human Ig, horseradish peroxidase linked F[Ab]2, Amersham, England). Incubation in each antibody was 1 h at room temperature with low speed shaking. Finally, the filters were immersed in ECL detection reagents for 1 min and exposed to the radiography film for 15 min according the manufacturer's procedure.
  • the positive clones were isolated from the HFS cDNA library by probing with a pool of IgG prepared from the individual sera. About 2 million recombinant phages from HFSC cDNA library were plated out on 150mm Petri dishes (40,000 in each) as described above. Expressed proteins were transferred to nitrocellulose discs as described above. Positive plaques which produced signals in duplicate filters were picked after alignment with the original plate by stabbing a sterile Pasteur pipette through both the top overlay and underlying agar. The plaque was transferred to a 1.5 ml Ependorph tube containing 300 ⁇ l of SM buffer, vortexed for 30 sec after adding one drop of chloroform and incubated at 4°C overnight. Positive clones were rescreened three times in small plates (90mm, about 100 plaques each) until all plaques produced positive signals in duplicate filters.
  • Each phage solution was titrated by making 10 "3 , 10 "5 , 10 “7 and 10 "9 dilutions in SM buffer. 10 and 100 ⁇ l of each phage dilution was added to 100 ⁇ l of LE392 cells and incubated at 37°C for 20 min for transformation. The infected cells were then mixed with 4ml top layer agar (prewarmed to 60°C and supplemented with 10mM MgS0 4 ) and plated out on agar in 90mm Petri dishes. The plates were incubated at 37°C overnight upside down and then the plaques were counted to determine phage titres.
  • Each phage solution was transferred to an Ependorph tube and stored at 4°C with 2% (v/v) chloroform or at -70°C, for long term storage, after adding 7% (v/v) DMSO and snap freezing in liquid nitrogen.
  • Solid NaCI was added to each large scale phage culture to a final concentration of 1 M, dissolved and left in ice for 1 h to dissociate the phage particles from bacterial particles.
  • the clear phage solutions were poured, after removing the bacterial debris by centrifugation at 11 ,000g for 10 min at 4°C.
  • the bacteriophage particles were precipitated by adding solid polyethylene glycol (PEG 8000) to a final concentration of 10% w/v, incubated in ice for 1 h and centrifuged at 11 ,000g for 10 min at 4°C.
  • Each phage pellet was resuspended in 8ml SM buffer and then extracted with an equal volume of chloroform vortexed for 30 sec and centrifugation at 3000g for 15 min at 4°C.
  • Solid CsCI was added to the top aqueous phase (0.5g / ml supernatant) and dissolved by gentle mixing.
  • a CsCI step gradient was prepared by layering 1.45, 1.50 and 1.70 g/ml (CsCI dissolved in SM buffer) respectively and finally overlayed by CsCI bacteriophage solution and centrifuged at 22,000 rpm for 2 h at 4°C in a Beckman SW41 or SW28 rotor.
  • PCR technique was employed to amplify the inserted DNA from all isolated positive clones using the materials purchased from Biotech International, Perth Western Australia. Lambda gt11 forward and reverse primers (20ng each) were mixed with dNTP (1ml of 2mM), MgCI2 (1ml of 25mM), 0.7 unit of Tth plus DNA polymerase and 10 x PCR reaction buffer (Biotech International, Western Australia) in a 10ml total volume.
  • DNA template 40ng of phage DNA, 0.5 ⁇ l of phage solution or a touch a fresh plaque by tip of a pipette and left in PCR solution for 1 min was used.
  • the PCR product was extracted from low melt agarose gel and used for sequencing.
  • Low melt agarose Bio-RAD, California
  • 0.5 x TBE buffer 1% w/v final concentration
  • the gel was stained by immersing in ethidium bromide (EtBr) solution (1 Omg/ml in TBE buffer) for 2 min.
  • EtBr ethidium bromide
  • the DNA samples were loaded after mixing with 6 x gel loading buffer.
  • the electrophoresis was then conducted in 0.5 x TBE buffer in a horizontal apparatus (Bio-RAD) at 60 V until the blue dye passed 2/3 of the gel length.
  • the DNA band was monitored under the UV light.
  • the desired DNA bands were excised from agarose gel using a clean, sterile razor blade and spun down in a calibrated microcentrifuge for 30 sec to estimate its volume.
  • the gel was melted by incubation for 10 min at 70°C after adding the ⁇ h volume of TE buffer and 1/10 volume of 1 M NaCI solution.
  • the mixture was cooled to room temperature before adding an equal volume of phenol vortex for 30 sec and spun at 4000g for 10 min.
  • the upper aqueous phase was transferred to another tube and re-extracted once with phenol/chloroform and once with chloroform.
  • the sample was incubated at -80°C for 30 min after adding Na acetate solution (3 M, pH 5) to a final concentration of 0.3 M and 2 volume of cold 100% ethanol. DNA was then precipitated with centrifugation (14000 rpm at 4°C in a microcentrifuge), washed with 70% ethanol, dried in a vacuum centrifuge for 2 min and resuspended in sterile distilled water or TE buffer. The samples were stored at -20°C until used.
  • Sequencing reactions were preformed by the dideoxynucleotide chain termination method using a PRISM Ready Reaction DyeDeoxy Terminator cycle sequencing kit (Applied Biosystems Foster City, USA).
  • a PRISM Ready Reaction DyeDeoxy Terminator cycle sequencing kit (Applied Biosystems Foster City, USA).
  • To 1mg of each DNA template (purified PCR product) was added 3.2 pmol of ⁇ gtl 1 forward (or reverse) primer, 9.5 ⁇ l of terminator premixture and sterile distilled water to 20 ⁇ l final volume. The mixture was overlayed with one drop of mineral oil to avoid evaporation before the thermal cycling. A total of 25 cycles were employed in a Perkin-Elemer Cetus thermocycler (Model 480).
  • Each cycle included denaturing at 96°C for 30 sec, annealing at 50°C for 15 sec and polymerization at 60°C for 4 min.
  • the cycling program had a rapid thermal ramp to the desired temperature before each section and final soaking at 4°C.
  • the mixture was briefly centrifuged before adding 80 ⁇ l of water and then purified using the phenol/chloroform extraction method.
  • the mineral oil was dissolved in 10O ⁇ l chloroform and removed with a pipette.
  • the terminators were extracted twice with adding 100 ⁇ l of phenol:H 2 0:chloroform (68:18:14) reagent was added, vortexed and centrifuged.
  • the extracted products were precipitated by adding 15 ⁇ l of 2 M Na acetate, pH 4.5 and 300 ⁇ l of 100% ethanol before incubation at -80°C for 20 mh and then centrifuged (14,000 m at 4°C). The pellet was washed with 70% ethanol and dried in a vacuum centrifuge for 2 min. The samples were applied on 6% acrylamide gel and the sequences were analysed using the Applied Biosystem sequencing system, model 373A.
  • clone M62 Three of the clones M62, M63 and M64 had a very high degree of similarity over the first 300 nucleotides.
  • a concensus sequence was determined from that in M62 and M63 (Fig 2).
  • the consensus nucleotide sequence for clone M62 was compared to all sequences in the GeneBank database. It was 99% identical over 260 nucleotides to a known human sequence HUMMTCG (Fig 2). It is clear from this comparison that clone M62 contained an insert which had originated from part of the human mitochondrial DNA sequence (Fig 3). This is believed to be the first autoantibody discovered to any protein encoded by mitochondrial DNA.
  • ND4 is one of the 41 components which comprise the enzyme known as Complex I (NADH:ubiquinone reductase). ND4 has a total of 459 amino acids. Because of differences in codon usage between mitochondria and E. coli in the synthesis of proteins there are important implications with respect to the amino acid sequence recognised by the MS IgG.
  • the predicted amino acid sequence which has been synthesized by E. coli in the ⁇ gtl 1 library is shown in Fig 4. The main differences between the mitochondrial amino acid sequence and the cloned sequence are at positions 3, 29 and 42 where the methionine is replaced by an isoleucine in the E. coli sequence.
  • the MacVector 3.5 (IBI, New Haven, USA) program was used to predict likely epitopes in the amino acid sequence.
  • the region which had a positive antigenic index was used in the design of peptides chosen for synthesis ( Figure 5a).
  • Fig 5 shows a peak between amino acids 18 and 24 which suggests that this region could be an epitope.
  • Two peptides were synthesized. The first peptide CysLeuAlaAsnSerAsnTyrGluArgThrHisSerArg was linked to diphtheria toxoid as a carrier through a maleimido-thiol bond on its C-terminal cysteine. This peptide was used an immunogen to generate antibody in a rabbit and as an antigen in ELISA assays.
  • the second peptide had the sequence LeuAlaAsnSerAsnTyrGluArgThrHisSer ArglleMetlleLeuSerGlnGlyLeu was used as an antigen in ELISA assays.
  • Enzyme linked immunosorbant assay (ELISA) technique Linthicum et al 1981 was used to screen the activity of antibody against the synthetic peptides or conjugated proteins. Assays were carried out in 96-well micro-titer plates (NUNC ). A Trtertech multi-channel pipette (Flow Laboratories ) was used to dispense all the buffers or solutions into the wells. The volume of the used solutions was 10O ⁇ l per well, except for the blocking buffer which was 300 ⁇ l. Washing of the wells was carried out with phosphate buffered saline (PBS, 0.01 M Na phosphate in 0.15 M NaCI, pH 7.2) containing 0.1% Tween 20. The washing step that was repeated four times. The excess buffer was removed from the wells after the final washing by vigorously "slapping" the plate, well down, on a benchtop covered with paper towels. Incubation was preformed with gentle shaking on a rotary shaker at room temperature.
  • PBS phosphate buffered sa
  • the plates were left exposed to the air at 37°C overnight to allow the solution (1 OO ⁇ l of 5 ⁇ g / ml protein in water) to evaporated to dryness.
  • the wells were pre-coated with glutaraldehyde (0.2% v/v) for 45 min at room temperature. The wells were washed twice before dispensing the peptide solution (2 ⁇ g/100 ⁇ l PBS) into wells and incubating overnight at 4°C. The plate was washed once and blocked by adding the 200 ⁇ l of a solution containing 3% w v BSA in PBS or 1 % BSA plus 0.1 M glycine in PBS. The plates were incubated for 2 h at room temperature to block any remaining unblocked attachment sites on the wells.
  • the purified IgG was added after washing the plates and incubated 30 min to 2 h at room temperature. To find the optimum reactivity between antigen and antibody, different dilution of IgG and antisera prepared in blocking solution were tested. Each dilution was in duplicate or triplicate. Only blocking solution was added to some of the wells as a blank. The wells were washed before adding the secondary enzyme linked antibody and incubated 60 min at room temperature. The secondary antibody which was conjugated to horseradish peroxidase (same as above) was used in 1 :1000 dilution in blocking solution. The wells were washed and then finally washed with PBS without Tween before the substrate was added.
  • Fig 6 shows the reactivity of the rabbit antibody with the peptide after absorption of the antibody activity to the diphtheria toxoid carrier.
  • the rabbit antipeptide antibody was also used to compare the coating of the ELISA plates with the 20 amino acid peptide to ensure that the wells were evenly coated with the peptide (Fig 7).
  • Mitochondria were purified from heart and brain tissue using the procedure of Beckman et al. (1993). For example three brains from chickens (4 weeks old) were placed in ice just after slaughtering All subsequent procedures were carried out at 4°C. Fat and connective tissues were trimmed before cutting into the approximately 5mm cubes. Chilled homogenization buffer (100mM Tris-HCI, pH 7.4, 250mM sucrose, 10mM EDTA) was added to the samples (3-4 g weight each) in a ratio of 1 :5 (w/v) tissue to buffer. The mixtures were homogenized using a Polytron homogenizer equipped with basic unit K and hand unit PM10S for 1 min at 600 rpm. Nuclei and cellular debris were removed by centrifugation at 18,500 g for 10 min at 4°C. The supernatant was transferred to a fresh tube and centrifuged at 10,000 g for 10 min at 4°C to sediment the mitochondria.
  • Chilled homogenization buffer 100mM Tris-HC
  • Sub mitochondrial particles were prepared from mitochondrial pellets according the procedure of Estornell (1993). The pellets were resuspended in a solution 0.15 M in KCI, 0.01 M in Tris-HCI pH 7.5 and 5 mM in EDTA to a final concentration of 30 mg/ml total protein. The pH was adjusted to 7.5 with 1 M KOH and subjected to sonic treatment for 1 min in an ultrasonic, XL-series sonicator, Mixonix at sonicator. The temperature was maintained below 5°C using an ice bucket with NaCI powder added to the top of the ice during the sonication. The pH of the sonicated suspension was adjusted to 7.5 with 1 M/KOH.
  • the mixture was centrifuged at 50,000 rpm a Beckman ultracentrifuge model L8-M (50TI rotor) for 90 min at 4°C to precipitate the SMP.
  • the SMP pellets, containing the Complex I were resuspended in cold Tris-HCI buffer (25 mM, pH 8.0 ) supplemented with histidine and sucrose to the final concentration of 0.5 mM and 0.33 mM respectively (TSH buffer).
  • TSH buffer cold Tris-HCI buffer
  • the concentration of the samples were adjusted to 0.2 mg/ml of total protein using the cold TSH buffer and stored at -20°C until used.
  • NADH-ubiquinone reductase (Complex I) activity of each SMP sample was measured at 340 nm and 30°C according the procedure Hatefi (1978) using the Beckman spectrophotometer model DU-50 equipped with thermostable water circulation system.
  • To each 1-ml quartz cuvettes were added 20 ⁇ l of K phosphate buffer (1 M, pH 8.0), 20 ⁇ l of Na azide (0.1 M), 100 ⁇ l of decylubiquinone (Sigma, Sydney), 10 ⁇ l of 15 mM NADH (Boehringer Mannheim, Germany) and water to a final volume of 1 ml. Decylubiquinone was dissolved in ethanol and then added to distilled water to make 1 mM, 10% ethanol final concentration.
  • the cuvettes were placed in the spectrophotometer and the absorbance was measured for 1 min duration time adding 10 ⁇ l of enzyme solution (SMP fraction) for total 30 min.
  • SMP fraction enzyme solution
  • samples were mixed with the IgG solution and incubated at room temperature for 90 min before adding to the cuvette. The rate was calculated between 0 and 10 min and the % inhibition calculated.
  • Fig 11 shows the effect of pooled normal IgG and pooled MS IgG on the activity of Complex I in chicken brain submitochondrial particles.
  • Chicken was chosen because of the close similarity between the amino acid sequence in the peptide of Figure 5b from human and chicken ND4. Similar results were obtained with horse heart SMP.
  • Horse heart SMP was chosen because of their higher activity and stability (Degli Esposti, Personal Communication).
  • the incubation time was studied for optimising the time required for interaction of the antibody with Complex I. From the data in Fig 12, 90 min was chosen as the time for incubation of the IgG with the enzyme prior to the addition of the substrate.
  • the PCR product from M62 was cloned into Sma 1 site of the PGEX-1N expression vector to produce the recombinant protein.
  • the Sma 1 site is located in glutathione S-transferase (GST) gene that can be activated to express the protein by induction with isopropyl thiogalactoside (IPTG) (Amrad, Australia).
  • GST glutathione S-transferase
  • IPTG isopropyl thiogalactoside
  • the method used was adopted from Lorens (1991) and Liu and Schwartz (1992).
  • the 3' A overhang PCR products were purified using the GENECLEAN II kit (Bresatec Ltd., South Australia), filled in by Klenow enzyme and phosphorylated with T4 polymerase kinase.
  • the Klenow /kinase reaction was set up by adding the dNTPs (0.2 mM final concentration), 4 units of kinase and 5 units of Klenow to KK buffer.
  • the 10xKK buffer was Tris-HCI (300 mM, pH 7.8 ) containing MgCI2 (100 mM), DDT (100 mM) and 5 mM ATP 300 mM.
  • the purified PCR products 500 ng to 1 mg was added to the mixture and the volume was adjusted to 25 ⁇ l by adding the sterile distilled water. The tube was then incubated at 25°C for 2 h to complete the reaction.
  • the blunt ended product was purified by the GENECLEAN kit.
  • the vector was digested by Sma 1 and dephosphorylated by calf intestine phosphatase before using in the ligation. Dephosphorylation of the 5' end of the cut vector was carried out to prevent re-ligation of the linear plasmid.
  • the purified digested DNA (approximately 1 ⁇ g in 34 ⁇ l volume) was dephosphorylated using 2 units of calf intestine phosphatase (CIP) enzyme.
  • CIP calf intestine phosphatase
  • the CIP buffer was added before incubation at 37°C for 30 min. A further 2 ⁇ l of enzyme was added to the mixture and the incubation was continued for 30 min more.
  • the reaction was terminated by the addition of 8 ⁇ l TNE buffer, 2 ⁇ l of 20% SDS and 30 ⁇ l of sterile distilled water.
  • the product was stored at -20°C after purification with GENECLEAN and dissolved in sterile distilled water to a final concentration of 40 ng per ⁇ l.
  • the blunt ended PCR product was ligated into the cut and dephosphorylated PGEX-1N plasmid using the T4 DNA ligase (Progen, New South Wales, Australia).
  • T4 DNA ligase Progen, New South Wales, Australia.
  • Three different reaction mixtures were set up by mixing the insert and vector in a molar ratio of 1 :1 , 1 :3 and 3:1. Two additional mixtures were set up, one without insert to test the vector self-ligation and another one without insert and vector as negative control.
  • the T4 DNA ligase (2 units) was added to each tube as well as 10xligation buffer and sterile distilled water to 25 ⁇ l final volume. The mixtures were incubated overnight (12 to 16 h) at 22°C in a thermocycler.
  • the host strain E.coli JM83 was made competent by calcium chloride method (Sambrook et al. 1989). The competent cells made by this method were used directly or preserved at -70°C for future work. A single colony was picked from a fresh grown plate and transferred into 100 ml of LB broth medium in a 11 flask.
  • the culture was then incubated at 37°C with maximum vigorous shaking for about
  • the supernatant was totally removed and the pellet was resuspended in 10 ml of ice-cold 0.1 M CaCI 2 before storing on ice for 10 min.
  • the cells were recovered by centrifugation at 4,000 rpm for 10 min at 4°C. in a Sorvall centrifuge.
  • the cells were resuspended in 4 ml of ice-cold 0.1 M CaCI 2 (2ml for each 50 ml of original culture). These competent cells have been directly used for transformation or frozen at -70°C until used.
  • DMSO dimethyl sulfoxide
  • the ratio of vector to insert in tube 5 was 1 :1 ; 6, 1 :3; 7, 3:1.
  • the transformed competent cells (200 ml per 90mm plate) were spread over the surface of the agar plate containing 20 mM MgS04. The plates were left at room temperature for 15 min to absorb the liquid before incubating overnight at 37°C in invert position.
  • Colony hybridisation method (as described above) was used to confirm the success of cloning the isolate.
  • the bacterial colonies were transferred onto nitrocellulose filers and probed with 32 P labelled DNA from M62. About 50% of the total bacterial colonies showed the signal in duplicate filters.
  • the recombinant positive colonies (about 10) were picked and used for PRC testing and protein production. PCR amplification of these bacterial colonies (bacterial colony as DNA template and ⁇ gt11 primers), showed the correct size of insert.
  • a non-recombinant colony (containing PGEX-1 N) was used in all of these experiments as a negative control.
  • the 20 recombinant bacterial colonies (plus one non-recombinant) were grown in LB media where the GST fusion protein was induced by adding IPTG.
  • the bacterial cultures were sonicated before electrophoresis with 12.5% acrylamide followed by Coomassie blue staining.
  • a band of GST protein at 26Kd was visible.
  • the predicted (Fig 4) amino acid sequence from ND4 (5Kd) together with the GST (26Kd) would give rise to a 31 Kd band, three clones producing the 31 Kd band were chosen for production of recombinant.
  • the bacterial lysate mixture from this clone was diluted 1 :10 with PBS (pH 7.4) and used for testing with the ELISA method. Each well of a microlitre plate was coated with 100 ⁇ l of the bacterial lysate. Bacterial lysate from non-recombinant clone was used as negative control. Probing the plate with the anti-ND4 antibody from the rabbit showed a significant increase for recombinant clones compared to the non-recombinant clones. Pre-immune sera from the rabbit had a low activity against the recombinant protein compared to the immune sera (Fig 14).
  • Serum (100 ⁇ l, 1/100 in PBS) was added to the first well and incubated for 1 h before moving the sample to the second well and so on with 1 h incubation in each before it was moved into the sixth well which contained the peptide conjugated to diphtheria toxoid.
  • This procedure removes non-specific binding of IgG to diphtheria toxoid, BSA and plastic.
  • the bound human IgG was determined with alkaline phosphatase conjugated to goat antihuman IgG (Sigma, Sydney) and the optical density measured at 405nm. The presence of antibody to the peptide was determined by the difference in optical density between well six and well five for each sample which was analysed in triplicate.
  • Figure 16 shows the detection of antibody to the diphtheria toxoid-S-MS immunoreactive peptide to MS and other sera.
  • Samples of MS sera were obtained from Professor D.A.S. Compston, Cambridge 44; Professor C.A.A. Bernard, Melbourne 5; Dr W. Carroll, Perth 2; Dr R. Edis, Perth 1 ; and Dr W.W. Tourtellotte 2.
  • Other sera were from 9 people with no obvious disease (2 from Professor D.A.S. Compston, 4 from Professor C.A.A. Bernard, and 3 from Dr R.M. Chalmers, London), 2 with Guillain-Barre syndrome from Dr Pollard, Sydney, and 1 with breast cancer from Murdoch University.
  • Antibody to the peptide was detected in 11/54 samples of MS sera but in none of the Other" samples. This result shows that a subgroup of MS patients can be detected using an ELISA assay to the peptide. Because the peptide contains an arginine which is mutated to histidine in the commonest form of Leber * s Hereditary Optic Neuropathy (MTND4*LHON11778A) and because it has been speculated that patients with the LHON mutation do not develop clinical disease until an autoimmune reaction occurs, 11 sera were obtained from Dr R.M. Chalmers, London; and 1 sample was from Dr R.D. Simmons, Australia. Antibody was detected in 7/8 patients with clinical LHON but not in 3 carriers of the MT ⁇ /D4*LHON11778A mutation who had no disease nor in 1 carrier who had MS rather than LHON (Fig 17).
  • ND4 peptide CysLeuAlaAsnSerAsnTyrGluArgThrHisSerArg was used to search GenPeptide with the BLOSUM50 matrix and FastA for similarities between the peptide and sequences in bacteria and viruses.
  • the ELISA assay to the MS immunoreactive peptide could be useful in identifying a subgroup of MS patients who are infected with spirochaetes. Such patients could be treated with appropriate antibiotic therapy. There are reports that a proportion of randomly selected MS patients do improve with such a treatment.
  • the autoantibody to ND4 is not just a marker for tissue damage in MS and LHON but is involved in the pathogenesis then it might be possible to generate an antiidiotypic antibody to block the autoantibody.
  • CTTCTAGCAA GCCTCGCTAA CCTCGCCTTA CCCCCCACTA 200

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Abstract

Cette invention concerne des polypeptides, des peptides ou des analogues de ces derniers dont les séquences d'acides aminés codent des segments antigéniques qui réagissent immunologiquement avec des anticorps induits par la sclérose en plaques. Ces polypeptides, peptides ou analogues de ces derniers sont utiles comme réactifs de diagnostic pour détecter la présence d'anticorps ou de cellules T chez des patients atteints de sclérose en plaques, et peuvent être également utilisés en tant qu'immunogènes dans des compositions et des procédés pour des anticorps anti-idiotypiques illicites dirigés contre les réponses autoimmunes anormales impliquées dans la pathogénèse de la sclérose en plaques.
PCT/AU1996/000166 1995-03-24 1996-03-25 Nouvel auto-antigene WO1996030398A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU49995/96A AU4999596A (en) 1995-03-24 1996-03-25 Novel auto antigen

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
AUPN1974A AUPN197495A0 (en) 1995-03-24 1995-03-24 Novel auto antigen
AUPN1974 1995-03-24
AUPN7205 1995-12-18
AUPN7205A AUPN720595A0 (en) 1995-12-18 1995-12-18 Novel auto antigen

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WO1996030398A1 true WO1996030398A1 (fr) 1996-10-03

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Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
ACTA NEUROL. SCAND., Supplement 6, 1995, D.A.S. COMPSTON et al., "Genes and Susceptibility to Multiple Sclerosis", pages 43-51. *
ACTA NEUROPATHOLOGICA, Vol. 90, No. 3, 1995, L. CLAUDIO et al., "Evidence of Persistent Blood-Brain Barrier Abnormalities in Chronic-Progressive Multiple Sclerosis", pages 228-238. *
BIOCHIMICA ET BIOPHYSICA ACTA, Vol. 1234, No. 1, 1995, H.A.C.M. BENTLAGE et al., "Multiple Deficiencies of Mitochondrial DNA and Nuclear Encoded Subunits of Respiratory NADH Dehydrogenase Detected with Peptide - and Subunit - Specific Antibodies in Mitochondrial Myopathies", pages 63-73. *
J. NEUROL., Volume 242, (1995), R.M. CHALMERS et al., "Sequence of the Human Homologue of a Mitochondrially Encoded Murine Transplantation Antigen in Patients With Multiple Sclerosis", pages 332-334. *
JAPAN JOUR. OPHTHALMOL., Vol. 36, 1992, M. NAKAMURA et al., "High Frequency of Mitochondrial ND4 Gene Mutation in Japanese Pedigrees with Leber Hereditary Optic Neuropathy", pages 56-61. *
JOURNAL OF NEUROLOGY, Vol. 241, 1994, L. BET et al., "Multiple Sclerosis and Mitochondrial Myopathy: an Unusual Combination of Diseases". *

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