WO1991011718A1 - Assays and treatments for autoimmune diseases - Google Patents
Assays and treatments for autoimmune diseases Download PDFInfo
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- WO1991011718A1 WO1991011718A1 PCT/US1991/000682 US9100682W WO9111718A1 WO 1991011718 A1 WO1991011718 A1 WO 1991011718A1 US 9100682 W US9100682 W US 9100682W WO 9111718 A1 WO9111718 A1 WO 9111718A1
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- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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- 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
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
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- G—PHYSICS
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/56983—Viruses
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- C12N2760/00011—Details
- C12N2760/20011—Rhabdoviridae
- C12N2760/20211—Vesiculovirus, e.g. vesicular stomatitis Indiana virus
- C12N2760/20222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- G—PHYSICS
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Definitions
- This invention is in the area of the prevention, diagnosis and treatment of autoimmune diseases, especially systemic lupus erythematosus.
- SLE Systemic lupus erythematosus
- SLE can be grouped with those diseases that commonly have autoantibodies present but for whom a central role of autoantibody in pathogenesis leading to clinical expression has yet to be fully established or accepted.
- Other such diseases include Sjogren's syndrome, rheumatoid arthritis, juvenile onset diabetes mellitus, primary biliary cirrhosis, Wegener's granulomatosis, inflammatory bowel disease, and many others.
- autoimmune diseases present with a wide array of symptoms and clinical signs.
- the production of circulating autoantibodies to ribonucleoprotein complexes (RNPs) is a unifying characteristic of some of the rheumatic autoimmune diseases.
- the most common antigens in SLE and closely related disorders include: Ro/SSA, La/SSB, nRNP and Sm. Initially, these antibodies were found using double immunodiffusion, but more recently sensitive solid phase assays have been developed to quantitate the autoantibodies.
- the Ro/SSA RNA-protein particle has been found to be a constituent of all human cells evaluated to date.
- Anti-Ro/SSA has been found to be specifically concentrated in a parotid gland of a patient with Sjogren's syndrome and primary biliary cirrhosis (Penner, E. and Reichlin, M. Arthritis Rheum. 25:1250-1253 (1982)).
- the concentrations of anti-Ro/SSA autoantibody achieved by patients can be extraordinary, and is commonly higher than 1 mg/ml of specific anti-Ro/SSA immunoglobulin (K.K. Gaither and J.B. Harley, Prot. Biol. Fluids Proc. Colloq. 33:413-416 (1985); J.B. Harley, et al., Arthritis Rhuem. 29:196-206 (1986)).
- the immune system derangement leading to this specific overproduction of anti-Ro/SSA is not apparent but is likely to reflect a fundamental mechanism related to the immunopathogenesis of the related diseases.
- the Ro/SSA family of proteins has now been shown to have several molecular forms which are operationally defined by the molecular weight of the antigen identified.
- a major form has an apparent molecular weight of 60 kiloDaltons (kD). This protein is associated with one of four hY RNAs.
- kD kiloDaltons
- M.D. Rader et al.
- J. Clin. Invest. 83:1556-1562 (1989)
- molecular weights 52 kD and 54 kD.
- a 48 kD protein, calmodulin has been identified as being bound by anti-Ro/SSA sera (McCauliffe, et al., Clin. Invest.
- La/SSB protein a 48 kD peptide, as described by J.C. Chambers and J.D. Keene, Proc. Natl. Acad. Sci. USA 82:2115-2119 (1985), is also a member of this group of autoantibodies, and binds small RNAs with a polyuridine terminus, as reported by J.E. Stephano, Cell 36:145-154 (1984).
- La/SSB is bound by a third of the anti-Ro/SSA precipitin positive sera. Anti-Ro/SSA autoimmune sera and heteroimmune rabbit sera have been used to demonstrate different specificities for lymphocyte Ro/SSA and red blood cell Ro/SSA.
- autoimmune disorders including such diverse diseases as Sjogren's syndrome, rheumatoid arthritis, juvenile onset diabetes mellitus, primary biliary cirrhosis, Wegener's granulomatosis, inflammatory bowel disease, potentially autoimmune disorders, or any other disease having immune manifestations.
- a specific method has been developed to identify the etiologic or antigenic agent responsible for the production of autoantibodies characteristic of a particular disorder.
- the antigen is first isolated, using, for example, autoantibodies isolated from one or more patients.
- the antigen is then divided into overlapping short amino acid sequences, preferably twenty amino acids or less, octapeptides in the example described below for Ro/SSA and La/SSB.
- the sequences having the greatest reactivity with the autoantibodies are identified. These sequences are then compared with all known amino acids sequences using the available computer data bases.
- the protein having the maximum number or proportion of sequences homologous to the sequences of greatest reactivity with the autoantibodies is among the likeliest candidate of the known sequenced proteins for the etiological agent or immunogen.
- the etiological agent for the production of several autoantibodies including the Ro/SSA antigen, characteristic of numerous autoimmune diseases such as SLE, appears to be a virus homologous to the Indiana strain of the vesicular stomatitis virus.
- the nucleocapsid protein of this virus contains at least . six areas identically homologous to the 60 kD Ro/SSA sequence (three pentapeptides, and three quadrapeptides).
- Figure 1 is a schematic of the properties of the 119 amino acid sequence at the carboxyl terminus of the human 60 kD Ro/SSA protein.
- Panel A Predictions of physical characteristics based upon the amino acid sequence from the Wisconsin Genetics Systems software (Devereaux, et al., Sequence analysis software of the genetics computer group, pp. 233-239, University of Wisconsin, Madison, WI (1987); Jameson and Wolff CABIOS 4:181-187 (1988)).
- Panel B Solid phase immunoassay of 119 overlapping octamers at each position of the sequence.
- Bars represent activity in a solid phase ELISA of an anti-Ro/SSA reference serum that also binds the carboxyl terminal 13 kD peptide of V8 protease digested bovine Ro/SSA.
- the background activity in this assay is from a normal control serum.
- the mean values from two experiments are presented.
- the octapeptide number is the sequence position of the amino terminal amino acid of the octapeptide.
- Figure 2 is an analysis of binding of a human anti-Ro/SSA reference serum to overlapping octapeptides of Ro/SSA. Every possible octapeptide from the human 60-kDa Ro/SSA amino acid sequence has been synthesized onto a solid-phase support. An enzyme-linked immunosorbent assay has been used to detect binding by human antibody. Octapeptide numbers indicate the sequence position of their amino-terminal amino acid and (A) Binding of a normal control serum. (B) Binding of the reference anti-Ro/SSA serum. (C) Central antigenic octapeptides are identified by broken lines.
- Figure 3 is a schematic of the binding of a human anti-Ro/SSA reference serum to overlapping octapeptides of Ro/SSA. Every possible octapeptide was synthesized from the human 60 kD Ro/SSA amino acid sequence onto a solid phase support. The solid phase was blocked with albumin and then incubated with the anti-Ro/SSA reference serum or other Ro. Binding of immunoglobulin to the solid phase octapeptide was revealed with an anti-human gamma chain specific IgG from goat conjugated to alkaline phosphatase following standard procedure for an ELISA assay.
- Figure 4 shows the homologous regions of four or more amino acids between the human 60 kD Ro/SSA protein and the nucleocapsid (N) protein of the Indiana strain of vesicular stomatitis virus. Homologous regions are labeled in their order on the Ro/SSA sequence. Sequence positions of the homologous regions are demonstrated on Ro/SSA and the N protein. The standard single letter code is presented for amino acids in the homologous regions.
- Figure 5 shows the areas of homology of at least four sequential amino acids between 60 kD Ro/SSA and rhabdoviral N proteins. Shared small peptides between Ro/SSA and N proteins which overlap central antigenic peptides of Ro/SSA or which may be extended by single amino acid gaps (*) in the Ro/SSA sequence.
- Vesicular stomatitis virus Indiana serotype VSV- IND#1 (Gallione, et al., J. Virol. 39:529-535 (1981)
- VSV-Indiana strain 2 VSV-IND#2
- DePolo et al., J. Virol.
- Figure 6 shows two examples of juxtaposition of sequence from different proteins which may have immune and autoimmune regulatory consequences.
- Figure 6A shows that the 60 kD Ro/SSA sequence (Ro) between amino acid residues 217 and 226 exactly matches the combined portions of the sequences of the 60S PO protein (PO) between its amino acid sequence residues 250 and 255 followed by residues 76 through 79.
- a second E K L L sequence is found in Ro/SSA between residues 311 and 314.
- Figure 6B shows HLA-B27 amino acid sequence between residues 69 and 77 is exactly matched by the combined regions between residues 51 and 54 in addition to 188 and 193 of the Klebsiella neuramidase protein (KLEB).
- the present invention is the discovery of a method for identifying an etiologic or antigenic agent for an autoimmune disorder such as SLE, non- self nucleic or amino acid sequences eliciting an immune type response, diagnostics and therapeutics for the prevention and treatment of these disorders.
- the method is specifically applied to determine the etiologic and/or antigenic and/or immunogenic agent eliciting an autoimmune response in SLE patients.
- the agent a virus homologous to vesicular stomatitis virus- Indiana strain (VSV-Ind), was characterized by identity of sequence at areas of antigenicity in the major antigens for autoantibodies characteristic of SLE.
- autoimmune diseases are diseases that are primarily autoimmune, as well as diseases which do not appear to be primarily autoimmune but have immune manifestations involving immunoglobulins, antigen specific B cell surface receptors, or antigen-specific T cell receptors.
- diseases which fall into these categories are SLE, Sjogren's syndrome, rheumatoid arthritis, juvenile onset diabetes mellitus, Wegener's granulomatosis, inflammatory bowel disease, polymyositis, dermatomyositis, multiple endocrine failure, Schmidt's syndrome, autoimmune uveitis, Addison's disease, adrenalitis, Graves' disease, thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis, presenile dementia, demyelinating diseases, multiple sclerosis, subacute cutaneous lupus erythemato
- Immunization is any procedure occurring leading to a humoral or cellular immune response to a specific substance.
- An autoantigen is any protein, or portion of a protein specifically recognized by and bound to an autoantibody.
- An etiologic or antigenic agent is any agent eliciting production of autoantibodies, including infectious agents such as bacteria, viruses, viroids, Rickettsia, and fungi, or environmental agents, including foods or chemicals.
- An autoantibody is any immunoglobulin, antigen specific B cell surface receptor (surface immunoglobulin), or antigen specific T cell receptor directed against a self-antigen, such as a protein or a nucleic acid.
- An antibody is any immunoglobulin, antigen specific B cell surface receptor (surface immunoglobulin), or antigen specific T cell receptor directed against a antigen.
- the methodology which can be applied to determine the antigenic or etiologic agent for any autoimmune disorder, potentially autoimmune disease, or disease having immune manifestations, was specifically applied to the determination of an etiologic and/or antigenic agent eliciting autoantibodies such as Ro/SSA in patients having systemic lupus erythematosus (SLE).
- SLE systemic lupus erythematosus
- homology between two peptide sequences is defined as having at least three identical or structurally similar contiguous amino acids in common between the two peptides.
- Contiguous means covalently bound amino acids or amino acids presenting a contiguous sequence to an antibody (i.e., This would also include structures such as the beta pleated sheet where every other amino acid may bind and, hence, every other amino acid of a structure would be identical or structurally similar).
- Proteins defined as having regions of homology and antigenicity are proteins having at least two contiguous sequences of three or more amino acids in common, based on identity or structural similarity, which react with the same source of antibody.
- Peptides reacting with anti-Ro/SSA antibodies Isolation of a major immunoreactive fragment of an autoantigen.
- Overlapping octapeptides of the carboxyl terminal portion of Ro/SSA were synthesized which included the 13 kD peptide.
- the surrounding overlapping peptides were also antigenic but less reactive in ELISA.
- Two separate antigenic octapeptides with less reactivity and with ho overlapping antigenic peptides were also identified.
- Antisera to bovine Ro/SSA has been prepared as described by Mamula, et al., J. Exp. Med. 86:1889-1901 (1986).
- New Zealand White rabbits are immunized with 200 mg purified Ro/SSA in complete Freund's adjuvant both intramuscularly and subcutaneously. They have been boosted at 2 weeks and 4 weeks with intravenous injections and bled 10 days later. All human sera used have been obtained from rheumatic disease patients or normal laboratory personnel.
- Optimal conditions utilized a 1:15 enzyme (V-8 protease) to protein (Ro/SSA) ratio, with 0.1% sodium dodecyl sulfate and RNAase at 50 ⁇ g/ml, and digestion at 37°C for 4 hrs in a gyratory water bath.
- Protein samples were then electroblotted onto nitrocellulose or polyvinyladine difluride (PVDF) membranes using the procedure of P. Matsudaira, J. Biol. Chem. 262:10035-10038 (1987) in a TransBlot apparatus (BioRad Labs, Richmond, CA). Transfers were done overnight at 200 mAmps in 0.025 M Tris, 0.192 M glycine, and 20% methanol, pH 8.3. Nitrocellulose blots were then stained with Fastgreen (0.1 %) to validate molecular weight by simultaneously processed standards. After blocking, serum samples containing autoantibodies were diluted and allowed to incubate with gentle agitation for 4 hrs.
- PVDF polyvinyladine difluride
- Staphylococcal V-8 protease digests of Ro/SSA (20 ⁇ g) were electrophoresed, electroblotted and probed with heteroimmune rabbit sera raised against purified 60 kD Ro/SSA at dilutions of 1:1000. A number of immunoreactive peptides were generated. Strongly immunoreactive peptides were found with molecular weights of 51 kD, 40 kD, 35 kD, 28 Kd and 13 kD, with minor bands at 56 kD, 45 kD, and 22 kD.
- Overlapping octamers differing by one sequential amino acid were assembled for the 119 amino acids of the carboxy terminal peptide.
- the predicted amino acid sequence for the human 60 kD Ro/SSA gene product reported by Academicr, et al., Proc. Natl. Acad. Sci. 85:9479-9483 (1988), was used as the template to determine the appropriate amino acid sequence.
- F-moc, t-butyl amino acid solutions (30 mM) were prepared in N,N-dimethylformamide (DMF) which had 1-hydroxy-benzotriazole added to final concentration of 30 mM and dispensed into the wells of a microliter plate as dictated by the known sequence of the human Ro/SSA cDNA. After 18 hrs the pins were then bathed sequentially, once in N,N-dimethylformamide for 5 min, four times in methanol for 2 min each, and once in final N,N-dimethylformamide for 5 min. The Fmoc protecting group was then removed from the amino acid added last by a 20% piperidine/DMF bath for 30 minutes. This procedure was repeated until the desired peptides were assembled.
- DMF N,N-dimethylformamide
- the amino terminal group of each peptide was acetylated by placing the pins in a 5:2:1 (v/v/v) mixture of N,N dimethylformamide:acetic anhydride: triethylamine for 90 min at room temperature. Subsequently, the pins were bathed in N,N-dimethylformamide for 2 min, four times in methanol for 2 min and then air-dried for 10 min. Finally, side chain amino protecting groups were removed by a 95:2.5:2.5 (v/w/v) of trifluoracetic acid:phenol:ethanedithiol.
- Pins were then washed with methylene chloride for 2 min, two times in 5% diisopropylethylene in methylene chloride for 5 min, and repeated methylene chloride for 5 min. After drying for 10 min, pins were placed in distilled H,O for 2 min, transferred to a methanol bath for 18 hrs and dried under vacuum for 18 hrs.
- Solid phase anti-peptide assays were conducted carrying out all steps by lowering the pin blocks into microliter plate wells. First, the pins were blocked with 1% bovine serum albumin (BSA) in PBS with 0.05% Tween (PBS/Tween) and then incubated with sera at 1:100 dilution in 1 % BSA in PBS overnight at 4°C in a humidified closed container. The pin blocks were then washed four times with PBS/Tween for 10 min with vigorous agitation.
- BSA bovine serum albumin
- PBS/Tween 0.05% Tween
- each pin was incubated with a Staphylococcal protein A-alkaline phosphatase conjugate or an anti-human gamma chain specific IgG from goat also conjugated to alkaline phosphatase (Sigma Chemical Co., St. Louis, MO) diluted 1:300 at 4°C for 18 hrs. Washing was repeated as above prior to incubation of the pins in para-nitrophenyl phosphate disodium. Plates were read at 405 nm with a MicroELISA Reader (Dynatech, Alexandria, VA). Classification of octapeptides into groups based on immunoreactivity with anti-autoantigen sera.
- the solid phase assay with the octapeptides covalently bound to the solid phase was used to screen for immunoreactivity against normal human sera, against anti-Ro/SSA serum identifying the 13 kD peptide and against anti-Ro/SSA serum not identifying the 13 kD peptide.
- the reference sera bound one group of six consecutive octamers spanning the amino acid residue positions 480-494 which had marked reactivity above that of both normal serum and other surrounding octamers, as shown in Figure 1.
- the corresponding sequence A I A L R E Y R K K M D I P A is predicted to contain an antigenic site and to have a high likelihood of being on the surface of the Ro/SSA 60 kD molecule.
- Major reactivity also resided in these and neighboring amino acids in seven additional anti-Ro/SSA positive sera identifying the 13 kD peptide in Western .
- blots Figure 2
- This region has the second highest antigenic index in the 119 amino acid sequence, as determined by the algorithm of Jameson and Wolff. CABIOS 4:181-187 (1988)( Figure 1).
- Figure 2 presents the log average with respect to binding carboxyl terminal 112 overlapping octapeptides of eight anti-Ro/SSA patients who are positive in Western blot for binding to the 13 kDd peptide of Ro/SSA in Panel A.
- Panel B presents patients who are negative for binding the 13 kD peptide.
- Group A was identified by three, Group B by seven, and Group C by four of the eight 13 kD binding anti-Ro/SSA sera tested. Panel C shows the maximum reactivity of the eight sera which bound the 13 kD peptide (shaded bars) and the two sera which did not bind the 13 kD peptide but for which an argument could be made for binding Group B octapeptides (opened bars).
- Table I Inhibition of binding toEYKKMDIby preincubation of an anti-Ro/SSA serum with Ro/SSA antigen or with soluble synthesized peptide.
- Example 2 Relationship between the 60 kD Ro/SSA peptide and the nucleocapsid protein of the Indiana strain of vesicular stomatitis virus (VSV).
- VSV vesicular stomatitis virus
- a search of the National Biomedical Research Foundation (NBRF) protein sequence bank found the sequence EYRKKLMD in the nucleocapsid (N) protein of the Indiana strain of vesicular stomatitis virus which with the inclusion of a gap (*) in the Ro/SSA sequence of EYRKK*MD has very close homology.
- NBRF National Biomedical Research Foundation
- One central antigenic octapeptide was chosen from amongst multiple neighboring octapeptides with an absorbance greater than 1.0 when all octapeptides showed at least quadrapeptide homology. Octapeptides developing an absorbance less than 1.0 were taken into account only when an otherwise equivalent choice was required between candidates for the central antigenic octapeptide. Then, the octapeptide with the greatest near neighbor octapeptide binding by the anti-Ro/SSA serum was chosen.
- the octapeptides of the Ro/SSA molecule were organized into groups based upon the presence or absence of homology with N. A threshold of at least quadrapeptide homology was required for an individual octapeptide to be included in a group of Ro/SSA octapeptides all with this level of homology to N. By these criteria, there were six groups of Ro/SSA octapeptides with homology to N. Each member of these octapeptide groups had quadrapeptide homology between N and Ro/SSA and are identified by the solid areas in Figure 3. The remainder of the molecule was arbitrarily divided into groups of octapeptides of essentially the same average size. No Ro/SSA octapeptide member of any of these latter groups had quadrapeptide homology with N.
- a contingency table was developed by assigning each group of Ro/SSA octapeptides to one of four categories based upon the presence or absence of homology between N and Ro/SSA and upon whether or not the group contained an octapeptide that had previously been identified as a central antigenic octapeptide.
- five of the six octapeptide groups of Ro/SSA which shared quadrapeptide homology with N also contained a central antigenic peptide.
- only nine of the ninety octapeptide groups which had no quadrapeptide homology with N contained a central antigenic peptide.
- the Antigenic Alignment Probability for the epitopes of Ro/SSA and its homology with N is 0.00017; meaning that such a relationship would be expected only once every 5,900 such comparisons.
- the odds ratio of 45 with a 95% confidence interval of 4.7 to 426 also supports there being a strong relationship. There is, therefore, a powerful association between the autoimmunity of Ro/SSA and the structure of the N protein of vesicular stomatitis virus. These are the first data suggesting that different areas of sequential antigenicity may be related to multiple areas of short sequence homology between an autoantigen and a foreign protein.
- the central issue is whether or not antigenicity of Ro/SSA is more closely related to homologies between Ro/SSA and another sequence in the data bank rather than that already identified between Ro/SSA and N.
- the simulation experiments have been extended to calculate the Antigenicity Alignment Probability for all 12,476 protein sequences in the NBRF data base as shown in Table II. A number of sequences are shown to have a potentially important association between homology and antigenicity; sixteen have Antigenicity Alignment Probabilities less than 0.01 (Table II). For every one thousand tested, 1.3 sequences achieve this level of probability. Nevertheless, of the 12,476 protein sequences, the N protein of vesicular stomatitis virus remains the most closely related to the autoantigenicity of Ro/SSA. Comparisons are made in Table II. Table II: Protein Sequences with Homology to 60 kD Ro/SSA that is
- Nucleoprotein (vesicular stomatitis virus-Indiana)-vhv ⁇ m Nucleoprotein (vesicular stomatitis virus-Indiana)-v vnv4 Ribonucleoside diphosphate reductase (E. c ⁇ / -s00926 Thymidylate kinase (S. cerevisiae)- kibyt ⁇ Thymidylate kinase (S. cerevisiae)- a26127 Probable glycoprotein H (H. zoster)- vgbe37 Interferon-induced 56 kD protein
- the Antigenicity Alignment Probability is calculated from a one-tailed Fisher's exact test of the categorical distribution of the central antigenic octapeptides upon the groups of octapeptides of the Ro/SSA sequence organized according to the presence or absence of quadrapeptide homology.
- the Odds Ratio for AAP is the odds ratio of the resulting contingency table.
- the Homology Score is a measure of overlapping quadrapeptide similarity between Ro/SSA and the query protein.
- Vesicular stomatitis virus is the prototype of the Vesiculovirus genera, and both New Jersey and the Indiana strains are in common laboratory use.
- the N proteins from the two strains are 80% homologous at the amino acid level, as reported by Gallione, C.J., et al., J. Virol. 39:529- 535 (1981); and Banerjee, A.K., et al., Virology 137:432-438 (1984). The differences are sufficient, however, to dramatically alter the alignment of homology with antigenicity. Only three areas of quadrapeptide homology exist between the New Jersey N protein and Ro/SSA and of these, only one identifies an antigenic octapeptide of Ro/SSA.
- the Antigenicity Alignment Probability, calculated as in Table ⁇ , for homology of Ro/SSA with N protein of the New Jersey strain being associated with Ro/SSA autoantigenicity is 0.95. This is dramatically different than the result obtained with the Indiana strain N protein. The New Jersey strain is, therefore, not likely to be the most closely related to the candidate viral agent.
- the amino acid sequence of the Indiana strain in the near neighborhood of identical homology with Ro/SSA provides additional evidence that this strain must be very closely related to the candidate virus.
- the inclusion of single amino acid gaps in the Ro/SSA sequence increases the homology between Indiana N and Ro/SSA as seen in Figure 5. In each instance, the gap is found in the Ro/SSA sequence on the carboxy side of an area of quadrapeptide or pentapeptide homology with Ro/SSA.
- Each of the three regions with near neighborhood homology with the Indiana strain are antigenic.
- the rabies virus N protein has no such near neighbor homology. Such similarity is present in the New Jersey strain homologies, but neither of the two regions is antigenic.
- vesicular stomatitis virus leader sequence binds La/SSB, and the earliest known effect of vesicular stomatitis virus infection upon cellular metabolism is the inhibition of U particle assembly, as reported by Kurilla, M.G., et al., Cell 34:837-845 (1983); Fresco, L.D., et al., Mol. Cell Biol. 7:1148-1155 (1987); and Crone, D.E., et al., J. Virol. 63:4172- 4180 (1989).
- La/SSB and the U particles are common autoantigens in systemic lupus erythematosus, as reported by Harley, J.B., et al., Rheumatic Disease Clinics of North America: Systemic Lupus Erythematosus 14(1): 43- 56 (1988).
- Vesicular stomatitis virus infects cells through nonspecific glycoproteins. Consequently, the virus has a tremendous host range, even with isolates from plants growing well in mammalian tissue culture cells, as described by Superti, F., et al., J. Gen. Virol. 68:387-399 (1987).
- Vesicular stomatitis virus has improved survival in complement deficient serum which is consistent with the observation that many complement component deficiency states predispose to SLE, reviewed by Beebe, D.P., et al., J. Immunol. 126:1562-1568 (1981); Agnello, V., in Systemic Lupus Erythematosus. pp. 565-589 (John Wiley and Sons, New York 1987). Patients with SLE produce very high levels of interferon which induces latency of vesicular stomatitis virus.
- viral antigens are expressed on cell surfaces, which may constitute a source of continuing antigenic stimulation.
- N protein and vesicular stomatitis virus would accordingly only be related to anti-Ro-SSA autoantibodies.
- Other viruses or foreign antigens would serve as stimuli or immunogens for other observed autoantibodies and other autoimmune disease related phenomena within the context of the general defect.
- the Antigenic Alignment Probability was revised for the case of tripeptide homology between N and Ro/SSA.
- Antigenicity was defined by central antigenic octapeptide binding by the reference anti-Ro/SSA serum as presented in Figure 3. Eight of the twenty-seven areas in Ro/SSA which had at least - tripeptide homology with N were antigenic while only six of the fifty-two areas with no tripeptide homology were antigenic. Therefore, the Antigenic Alignment was 0.048 with an odds ratio of 3.2. This is a much weaker association than was found at the quadrapeptide level but nevertheless remains significant. Consequently, these data support a specific autoimmune relationship between N and Ro/SSA even at the level of tripeptide homology where the number of antigenically irrelevant homologies may be increased. Other Anti-Ro/SSA Sera.
- Anti-Ro/SSA autoantibodies have been associated with other autoantibodies including an anti-La/SSB.
- the gene for La/SSB has been cloned and the protein sequenced, as reported by Chambers and Keene, Biol. Chem. 263:18043-18051 (1988).
- La/SSB against which autoantibodies are made in approximately a third of the anti-Ro/SSA precipitin positive patients, binds the vesicular stomatitis virus leader sequence.
- the VSV matrix (M) protein contained short sequence homology to La/SSB in three positions which included residues 315-318(L K K I), 337-342(K G K G K G), and 351-354(G K G K).
- the Ul nRNP 68KD antigen has also been compared to the proteins of the vesicular stomatitis virus, both Indiana and New Jersey strain.
- the Ul 68KD protein is the major autoantigen to which the anti-nRNP autoantibodies are directed.
- Anti-nRNP antibodies are found in about 40% of patients with SLE. This autoantigen has been found to have homology with the M protein of the vesicular stomatitis virus, Indiana strain. This is the same viral protein which shares homologous regions with La SSB; however, no individual sequence has homology of four or greater amino acids shared among all three proteins.
- Overlapping oligopeptides spanning the Ul 68KD protein sequence will be synthesized and a search against the known protein sequences will be conducted as has been done for Ro/SSB.
- the data that these autoantigens share multiple short homologies with a vesicular stomatitis virus protein reinforces the idea of vesicular stomatitis virus as a potential etiologic agent in autoimmune diseases.
- data such as that obtained for Ro/SSA and La/SSB concerning the coincidence of antigenicity and multiple site molecular mimicry will be of interest.
- Example 4 Testing of antigenic agents homologous to Ro/SSA as an etiologic agent of autoimmune disease.
- Anti-Ro/SSA autoantibodies are not only common in systemic lupus erythematosus and Sjogren's syndrome (SS), but may also have a pathogenic role in congenital complete heart block and neonatal lupus dermatitis. Mothers of young children, less than ten years old, and mothers of male lupus children have unexpectedly elevated levels of anti-Ro/SSA compared to the other primary relatives, as reported by T.J.A. Lehman, et al., Arthritis Rheum. 32, 1414-1420 (1989). These observations along with immunochemical evidence showing an antigenic relationship of Ro/SSA to a viral structural protein (discussed in example 3) led to the hypothesis that a specific viral infection in an appropriately susceptible host leads to anti- Ro/SSA autoantibodies.
- the rabies virus N protein is the most distant from the Indiana strain virus and the two N proteins have only 40 percent homology at the amino acid level.
- the rabies virus N protein sequence has three quadrapeptides homologous with 60 D Ro/SSA ( Figure 5), none of which were found in an antigenic region of Ro/SSA.
- the New Jersey strain is serologically distinct from the Indiana strain of vesicular stomatitis virus, as reported by Frazier and Shope, Serologic relationships of animal rhabdoviruses, as reported by Bishop, D.H.L., Rhabdoviruses pp. 43-64 (CRC Press, Boca Raton, FL 1979), but the two viruses are closely related.
- the two N proteins have 60 percent homology at the amino acid level. These changes, however, are sufficient to substantially alter the homologies with 60 kD Ro/SSA ( Figure 5).
- the relationship between antigenicity of Ro/SSA and the homologies with the New Jersey strain N protein are substantially weaker than those found with the Indiana
- the overlapping octapeptides of 60 kD Ro/SSA and the vesicular stomatitis N protein can be constructed and tested for antibody in childhood systemic lupus erythematosus (SLE) proband and controls. From the hypothesis, one would predict that antibodies to areas of structural similarity between Ro/SSA and the N protein might predominate in the early anti-Ro/SSA autoimmune response and the antibodies to the other regions of N protein would have the greatest likelihood of still being present in the early anti-Ro/SSA autoimmune response.
- SLE systemic lupus erythematosus
- these same antigenic properties of the N protein may be found in the mothers of younger children and male children with lupus, consistent with the possibility of a congenital or childhood infection being an important factor in the subsequent disease of these patients.
- various rhabdovirus strains can be tested for antigenicity. Those which have proteins reactive with anti-Ro/SSA autoantisera in addition to the N protein can be tested in the pedigrees available by Western immunoblot.
- CMV cytomegalovirus
- EBV Epstein-Barr virus
- retroviruses have been most often implicated in the pathogenesis of both SLE and Sjogren's syndrome. Seroepidemiologic evidence for an association of EBV or retrovirus with SLE or SS has been presented, but other studies have been reported which have not found evidence implicating these viruses in rheumatic diseases.
- Vesicular stomatitis virus is sensitive to interferon in that lytic infection with formation of intact virions is inhibited in vitro and the virus infection becomes latent.
- Vesicular stomatitis virus has been documented to be latent in vivo for up to a year.
- Rhabdoviruses have a nonspecific mechanism for cellular attachment which means that they can infect many different tissues, perhaps contributing to the clinical heterogenicity of SLE.
- Vesicular stomatitis virus does infect humans (Sekellick, M.J. and Marcus, P.I. Persistent infections of rhabdoviruses: in Rhabdoviruses. Vol. 1, Bishop, D.H.L., ed.pp. 67-98 (CRC Press, Boca Raton, 1980); Tesh, R.B., et al., Am. J. Epidemiol. 90:255-261 (1969); Tesh, R., et al., Am. J. Trop. Med. 26(2): 299-306 (1977)). In areas where the virus is known to be epizoic, up to 90% of the human population shows serologic evidence of past infection.
- Epizoic areas include most of the Western hemisphere and parts of the Middle East; however, since vesicular stomatitis virus is not believed to have been found in Europe, it is unlikely that the Indiana strain of vesicular stomatitis virus itself is responsible for loss of tolerance to Ro/SSA. Nonetheless, as the prototype rhabdovirus, the Indiana strain of vesicular stomatitis virus has been well studied. There are approximately 30 rhabdoviruses known to infect humans, most of which have not been characterized. One of these or a virus yet to be isolated must be seriously considered as important etiologic agents leading to autoimmunity to Ro/SSA and the associated disease states.
- the pathogenic agent may be separable from the factor that predisposes to or causes SLE. Accordingly, the anti-Ro/SSA negative SLE patients may have no immunologic evidence of prior exposure to a virus like vesicular stomatitis virus and one may not expect to find much in the way of Ro/SSA octapeptide binding from the Ro/SSA negative sera.
- the second general model is that the rhabdovirus being sought is the etiologic agent for SLE. If so, then all SLE patients may, but are not required to, have evidence of N protein antigenicity demonstrated by octapeptide binding.
- the explanation for the presence of not only anti-nRNP and anti-Sm autoantibodies but also the myriad of other known autoantibody specificities in SLE would be the major enigma requiring explanation. Possibilities would include a similar relationship between the U peptides and a rhabdovirus protein as is herein described between Ro/SSA and N.
- High-titer virus can be grown and purified as previously described by Clark, H.F., Rhabdoviruses pp. 23-42 (1980).
- Cloned vesicular stomatitis virus is added to monolayers of BHK-21, or other appropriate cell line. With an infectious innoculum, a general cytopathic effect is noted at about 24 hours. Supernatant is then harvested and subjected to sequential centrifugation at 1,000 x g for 10 minutes, then 10,000 x g for 10 minutes.
- the virus is pelleted through a 50% glycerol cushion at 85,000 x g for 90 minutes.
- the pellet is then suspended in a 10 mM Tris, 0.1 M NaCl, and 0.001 M EDTA (TNE) solution, and sonicated at 40 watts for 10 seconds.
- TNE EDTA
- the virus is then layered on a 10% to 40% sucrose gradient and subjected to centrifugation at 50,000 x g for 90 minutes.
- the visible band obtained is diluted in TNE solution and centrifugated again at 50,000 x g for 90 minutes.
- the now purified virus is dialyzed against a Tris buffer overnight at 4°C and can be stored at -70°C.
- Antibody binding can be assessed by standard Western blotting strategy against a panel of sera.
- the virus isolate from which the N protein binds at least the anti-Ro/SSA precipitin positive patients and any of the four other rhabdovirus proteins in any SLE or appropriate control serum will be of great interest.
- This strategy exploits the potential for the extremely broad host range of rhabdoviruses to be relevant. For example, even the yellow lettuce virus is a rhabdovirus which infects and grows in mammalian cells.
- Example 5 Diagnostic Reagents and assays based on a VSV-like rhabodovirus.
- diagnostic reagents there are several embodiments of diagnostic reagents according to the present invention that can be used for diagnosis of autoimmune disorders, exposure to, or infection by, an etiologic or immunogenic agent, and vesicular stomatitis virus infection.
- anti-viral antibodies could be used to detect viral antigens in cell extracts or serum and bodily fluids.
- antibody assays include assays such as sandwich ELISA assays, Western immunoblot, radioimmunoassays, and immunodiffusion assays. Techniques for preparing these reagents and method for use thereof are obvious to one skilled in the art.
- vesicular stomatitis virus persists as a latent infection in some patients.
- immunologic reagents could be used to detect the presence of particular viral antigens in tissue sections or on isolated cells using methodology familiar to one skilled in the art.
- Anti-viral antibody can be detected by using the anti-viral agents to trap viral antigens, as in a sandwich ELISA, using methods known to those skilled in the art.
- Nucleotide or amino acid probes can be prepared based on the antigenic sequences identified in Figures 3 and 4 for either the Ro/SSA or the rhabdoviruses tested as described in example 5, such as the Indiana strain of VSV. These are labelled using dyes, or enzymatic, fluorescent, chemiluminescent, or radioactive labels which are largely commercially available. These probes can be used to screen sera or tissue samples from suspected patients or animals suspected of being reservoirs of the virus believed to be the etiological agent. For example, the appropriate viral sequences (or their complementary nucleic acid) could be marketed for use in en situ hybridization as a method to detect infection in specific tissues or peripheral blood cells.
- nucleic acid primers could be prepared which with reverse transcriptase and the polymerase chain reaction could be used to expand viral sequences. Detection of the viral sequences may follow with specific nucleic acid probes, with electrophoresis of a fragment with a predicted size or with electrophoresis and restriction digestion with a particular restriction endonuclease.
- Example 6 Methods and compositions for treating an autoimmune disorder having a VSV-like rhabdovirus as the etiological agent. Treatment with anti-viral agents alone or in combination with immunosuppressants.
- Immunosuppressants which are currently in use include glucocorticoids, methotrexate, azathioprine, cyclophosphamide, non-steroidal antiinflammatory agents, antimalarials, and other non-specific therapeutics such as sun screens. Usage and dosage of these drugs is dictated by the disease manifestations.
- Glucocorticoids for example, are used in high dosages to treat some neurologic complications of SLE. Both azathioprine and cyclophosphamide are used as an attempt to halt or reverse renal damage. Limiting side effects are common for all of the immunosuppressants.
- cyclophosphamide is generally regulated, and limited, by toxicity to hematopoesis.
- autoimmune disorders such as SLE are initiated by infection with a VSV-like rhabdovirus. Accordingly, patients could be treated not only by the immunosuppressants mentioned above but also by immunosuppressant compounds having non ⁇ specific antiviral activity, such as interferon, or inducers of interferon including fluorene, dibenzofuran, tilorone HC1, or bacterial lipopolysaccharide, or compounds having specific activity against vesicular stomatitis virus, alone or in combination with the immunosuppressants.
- immunosuppressants having non ⁇ specific antiviral activity, such as interferon, or inducers of interferon including fluorene, dibenzofuran, tilorone HC1, or bacterial lipopolysaccharide, or compounds having specific activity against vesicular stomatitis virus, alone or in combination with the immunosuppressants.
- Immunosuppressants would also have the potential to be specific and be biological.
- a protein containing the appropriate composition based upon the homology of N protein with Ro/SSA and antigenicity of Ro/SSA and N could act as an immunosuppressant for the anti-Ro/SSA response.
- This or another compound, for example, based upon complementary considerations may stimulate the anti-N protein (or other viral antigen) response and act to clear the virus infection from the affected patient.
- anti-sense DNA or RNA or derivatives thereof containing nucleic acid sequence related to VS V may inhibit the production of virus antigens and potentially eliminate viral infection.
- Therapeutic compounds are generally administered in a pharmaceutically acceptable carrier.
- Pharmaceutical carriers are known to those skilled in the art and include encapsulation of compounds for oral administration, for example, in an enteric coating or in combination with a binder such as stearate or lactose, or in solution. Acceptable solutions include sterile water, saline, and buffered solutions at physiological pH.
- Vaccines can be administered orally, intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.
- a pharmaceutical carrier is usually inert by itself but may have biological activity.
- a vaccine may consist of immunogenic peptides or proteins in combination with an adjuvant.
- the agents used for treatment might include peptides homologous to the identified antigenic, molecular mimicry sequences. These peptides, either free or bound to a carrier, could be delivered to a patient in order to decrease the amount of circulating antibody with a particular specificity.
- knowledge of the cross-reacting epitopes between a foreign antigen and an autoantigen, such as N and Ro/SSA may allow for re- induction of tolerance. It is well known in experimental models of the immune response that the response can be suppressed and tolerance induced by treatment with the antigen.
- Peptide therapy with the cross-reacting sequences may be a potential therapy in autoimmune diseases. Production of vaccines to prevent development of autoimmune disease based on the isolated etiologic or antigenic agent.
- Vaccination programs have been very successful in man for selected diseases (e.g. polio). Such a vaccine could be an attenuated or killed virus.
- a vaccine product could also be manipulated by recombinant DNA techniques so that the regions of antigenicity and homology in the autoantigen are altered, deleted or destroyed in the expressed immunogen.
- stimulating the immune response to portions of the immunogen the immune system may be able to eliminate the infection or reinduce an immune state of tolerance in the affected patient.
- the engrafted scid mouse model will be extremely useful in assessing the consequences of these biologies upon the patient's immune system.
- DNA primers based upon the N protein nucleic acid sequence and constructed from the areas of peptide homology between N and Ro/SSA could be used to expand rhabdovirus sequences from patient and normal specimens.
- Northern blots of mRNA from lymphocytes using available or constructed cDNA viral probes may reveal additional evidence of infection and provide the basis by which to recover and clone viral sequences.
- viral peptides either produced by cleaving cultured virus or synthetically, using methods known to those skilled in the art, to create vaccines for that portion of the population at risk, to serve as neutralizing agents for the circulating autoantibodies, or to induce tolerance to epitope(s) of a foreign antigen or an autoantigen.
- the sequences preferably used for the vaccine are those portions of the viral proteins which are antigenic but not homologous to regions of the autoantigen. For example, the multiple homologous regions between the rhabdovirus proteins and self proteins that become autoantigens could be eliminated by recombinant DNA technology.
- a vaccine administered before the infection with the virus or at least before onset of the disease could prevent autoimmunity. Using this strategy, an individual would develop immune competence against the etiologic agent after vaccination with the manipulated virus. When challenged with the etiologic agent, an infection and the autoimmune consequences would be prevented.
- amino acid sequences can also be used to make agents for neutralizing circulating antibodies or immobilized on substrates in extracorporeal devices for specific removal of autoantibodies, using methodology known to those skilled in the art.
- Example 7 Juxtaposition and lesser degrees of homology shuffling between autoantigens and other proteins.
- the human 60S ribosomal phosphoprotein PO (access code NW: A27125) is an autoantigen whose autoantibodies are related to psychiatric symptoms in SLE.
- This protein has specific homology with 60 kD Ro/SSA.
- In reviewing the homologous regions there is a 10 amino acid stretch of Ro/SSA which is composed of two regions of PO ( Figure 6A).
- the E K L L sequence is found in this and a second region of Ro/SSA.
- An animal model was developed which represents not only the successful induction of the production of human autoantibodies in an animal, but may provide a means for screening of compounds which inhibit the production by these cells of the autoantibodies or neutralization in vivo of the effects of the autoantibodies. It also serves as a tool in the study of the production of these antibodies, allowing experiments which cannot be performed in humans due to the unacceptable risk involved.
- these animals can be injected with antigen to the autoantibodies to determine if the antigen elicits a still greater response or if the antigen can be used to neutralize circulating antibody.
- These animals can be infected with viruses. They can also be used to determine the requirements which perpetuate the immune disorder.
- mice Severe Combined Immunodeficient Mice.
- Adult retired breeder mice C.B-17 SOD homozygous for SCID mutation
- All SCID mice were 24 weeks of age or older at the time of injection.
- Mice were removed from the sterile environment and injected with human peripheral blood mononuclear cells. Mice were subsequently fed standard non-sterile mouse chow and kept in isolation laminar flow cubicles (BioClean, Inc.). SCID mice were bled by tail vein.
- Engrafted mouse sera were screened for autoantibodies using a standard anti-Ro/SSA ELISA which relied upon highly purified Ro/SSA as described by Mamula, et al., J. Exp. Med. 86:1889 (1986).
- Indirect immunofluorescent anti-nuclear antibodies SLE, primary biliary cirrhosis, normal human sera, and engrafted mouse sera were analyzed for anti-nuclear antibodies on Hep-2 cells using a NOVA Lite ANA (INOVA DIAGNOSTICS, Inc., San Diego, CA). Serum samples were diluted and 50-75 ⁇ l were applied to each substrate slide. After 30 min incubation in a moist chamber at room temperature, the slides were thoroughly washed with PBS. Goat anti-human IgG gamma chain specific FITC conjugate (Sigma Chemical Co., St. Louis, MO) is added at 1:7500 dilution and incubated for 30 min at room temperature. Slides were subsequently washed with PBS and immunofluorescent staining visualized under a fluorescence microscope.
- Goat anti-human IgG gamma chain specific FITC conjugate Sigma Chemical Co., St. Louis, MO
- C.B-17 SCID mice were injected intraperitoneally with sterile human peripheral blood mononuclear cells at various concentrations.
- Peripheral blood mononuclear cells were isolated from a reference patient who had the clinical diagnosis of systemic lupus erythematosus (SLE) and had precipitating antibodies to Ro(SSA) and La(SSB). Previous clinical studies of this patient had shown a positive ANA with a speckled nuclear pattern at titers of 1:3240. A normal sex and age matched control was identified and an identical number of peripheral blood mononuclear cells were injected intraperitoneally into SCID mice.
- mice which received intraperitoneal human mononuclear cells
- engrafted mice were bled 14 weeks after injection and their sera screened using ELISA for production of human IgG.
- Two of the four mice injected with normal human mononuclear cells were positive in the ELISA screen for IgG, the ones injected with 50 x 10 6 cells.
- the two mice injected with 5 x 10 6 and 1 x 10 6 mononuclear cells showed no human IgG.
- two of the four SLE mice 50 x 10 6
- a single SCID mouse serum reconstituted with 50 x 10 6 cells from a patient with primary biliary cirrhosis was also tested for anti-nuclear antibodies.
- This serum demonstrated a diffuse cytoplasmic staining pattern without staining nuclear material which appeared identical to that of the patient.
- ELISA were performed testing for the presence of anti-Ro/SSA and anti-La/SSB.
- the SLE engrafted mouse (50 x 10 6 ) demonstrated positive immunoreactivity in ELISA at serum dilutions of 10 "2 . None of the other SLE engrafted mice or normal volunteer engrafted mice, pre-immune mouse sera, or sham treated mice showed reactivity.
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Cited By (15)
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WO1993021223A1 (en) * | 1992-04-13 | 1993-10-28 | Board Of Regents Of The University Of Oklahoma | Methods and reagents for diagnosis of autoantibodies |
US5272256A (en) * | 1992-02-03 | 1993-12-21 | The General Hospital Corporation | Nuclear autoantigen |
US5637454A (en) * | 1990-01-31 | 1997-06-10 | Oklahoma Medical Research Foundation | Assays and treatments of autoimmune diseases |
WO1999025380A2 (en) * | 1997-11-17 | 1999-05-27 | The University Of Kentucky Research Foundation | Methods and compositions for the treatment of psoriasis |
FR2773078A1 (en) * | 1997-12-30 | 1999-07-02 | Univ Toulouse | USE OF CITRULLIN PEPTIDES DERIVED FROM FILAGGRIN FOR THE TREATMENT OF RHUMATOID POLYARTHRITIS |
US6232522B1 (en) | 1990-01-31 | 2001-05-15 | Oklahoma Medical Research Foundation | Non-human animal model for systemic lupus erythematosis |
WO2001088128A1 (en) * | 2000-05-17 | 2001-11-22 | Istac | Ssa-56kda polypeptide and its fragments and polynucleotides encoding said polypeptides and therapeutic uses |
US7192715B2 (en) | 1993-11-30 | 2007-03-20 | Oklahoma Medical Research Foundation | Diagnostics and therapy of epstein-barr virus in autoimmune disorders |
US7273613B1 (en) | 1997-01-13 | 2007-09-25 | The Board of Regents, The University of Oklahoma | Diagnostics and therapy of Epstein-Barr virus in autoimmune disorders |
US7485708B2 (en) | 1996-09-23 | 2009-02-03 | University Of Arkansas | Nucleic acids encoding ara h 3 polypeptides |
WO2008148563A3 (en) * | 2007-06-08 | 2009-05-07 | Univ Ghent | Viral latency model |
US7785819B2 (en) | 2001-04-10 | 2010-08-31 | The Board Of Trustees Of The Leland Stanford Junior University | Therapeutic and diagnostic uses of antibody specificity profiles |
US7879977B2 (en) | 1998-01-31 | 2011-02-01 | University Of Arkansas | Methods and reagents for decreasing clinical reaction to allergy |
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AUPQ347199A0 (en) * | 1999-10-15 | 1999-11-11 | Csl Limited | Novel polypeptide fragments |
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US6897287B1 (en) | 1990-01-31 | 2005-05-24 | Oklahoma Medical Research Foundation | Ro/SSA peptide reagents for diagnosis of autoantibodies |
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US5272256A (en) * | 1992-02-03 | 1993-12-21 | The General Hospital Corporation | Nuclear autoantigen |
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WO2008148563A3 (en) * | 2007-06-08 | 2009-05-07 | Univ Ghent | Viral latency model |
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