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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K39/00—Medicinal preparations containing antigens or antibodies
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  • A61K2039/6031—Proteins
  • A61K2039/6075—Viral proteins

Definitions

• IL-l ⁇ is well characterized as a primary mediator of inflammation and its role in inflammatory related disease has been suggested in several animal models.
• Human IgG autoantibodies (aAb) against interleukin (IL)-Ia have been detected with a relatively high frequency in the general population. In fact, it has been reported that more than 20% of ostensibly healthy persons have highly specific IL-l ⁇ aAb.
• FIG. 1 Anti-IL-l ⁇ autoantibody formation on day 56 in C57BL/6 mice after three subcutaneous injections with IL-l ⁇ -PPD conjugate in alum ( ⁇ ). Control mice immunized with PPD in alum only ( ⁇ ).
• FIG. 2 Antibody-dependent complement-mediated killing of EL-4 cells.
• EL-4 cells were incubated with serial dilutions of mouse anti-mouseIL-l ⁇ polyclonal antiserum. The ratio of killed cells to viable cells is proportional to the serum concentration.
• a human anti-mouseIL-l ⁇ monoclonal antibody was used as a positive control.
• Incubation with na ⁇ ve murine serum or with culture medium alone served as the two negative controls.
• the ApoE-/- mice have an engineered lipid transport defect that results in rapid progression of atherosclerosis-like plaques in major arteries. These mice are considered the most compelling model for human atherosclerosis, because they are hypercholesterolemic and spontaneously develop arterial lesions. The ApoE-/- mice have consequently been extensively used as a model system for studying atherosclerosis and treatments.
• the invention provides an animal model for antibody neutralization of IL-Ia which can be obtained, e.g., by immunizing ApoE-/- mice against IL- l ⁇ . All immunized animals develop IgG aAb to IL- l ⁇ , which persists at high levels.
• the IL- l ⁇ aAb from sera of immunized mice inhibits binding of IL-I ⁇ to NOB-I, an IL- l ⁇ responsive murine T cell line, and neutralizes IL-l ⁇ (but not IL-l ⁇ -induced IL-6) in vivo.
• ApoE-/- mice are well protected against atherosclerosis-related disorders (e.g., peripheral ischemic heart disease, coronary artery disease, cerebrovascular disease, peripheral arterial disease) by the presence of endogenous IL-l ⁇ autoantibody generated through immunization.
• the invention therefore provides an elegant animal model that supports our earlier clinical observations that men with natural IL-l ⁇ aAb have a reduced incidence of atherosclerosis-related heart disease compared to men who do not have neutralizing IL-l ⁇ aAb.
• the invention also provides a method of treating individuals, including humans, at risk for the development of atherosclerosis-related disorders (e.g., peripheral ischemic heart disease, coronary artery disease, cerebrovascular disease, peripheral arterial disease) by inducing protective IL- l ⁇ auto-antibodies against the disease.
• atherosclerosis-related disorders e.g., peripheral ischemic heart disease, coronary artery disease, cerebrovascular disease, peripheral arterial disease
• mice The ApoE -/- mice are obtained from Jackson Laboratory, Bar Harbor, ME. Only male animals are used to avoid possible influence of gender on the development of vascular lesions; moreover, clinical studies observing a protective role for IL- l ⁇ aAb in progression of atherosclerosis have been made, to this point, only in men.
• Ten week-old mice are used and fed a diet with high cholesterol content (1.25% cholesterol, 0% cholate; Research Diets, New Brunswick, NJ). The mice are fed the diet for 10 weeks and then sacrificed. Blood is sampled and aortas are perfused, cut into parts, and either fixed or frozen according to standard methods.
• mice are immunized with murine IL- l ⁇ conjugated to purified protein derivative of tuberculin (PPD) at a ratio of 0.41 (w/w) according to the method described by Svenson et al, J Immunol Methods. 2000 Mar 6;236(l-2):l-8. Mice are inoculated with subcutaneous injections in the base of the tail. Inoculations are repeated three times, three weeks apart. To analyze IL- l ⁇ aAb, mice are bled from the retroorbital plexus 2 weeks after each injection. Control animals receive identical inoculation schedule with a PPD solution containing no IL- l ⁇ . EXAMPLE 3
• Mouse IgG responses to IL-l ⁇ are determined as described by Svenson et al, 2000. Saturation binding analysis of IL-l ⁇ to IgG is performed as described (Svenson et al, J Clin Invest. 1993 Nov;92(5):2533-9). Identical samples are run in parallel on the protein G Sepharose columns and columns containing Sephadex G-75 superfine (Svenson et al., Cytokine. 1992 Mar;4(2):125-33) to compare the 125 I-IL-Ia bound to serum IgG with the total binding to serum.
• Cellular receptor assays are performed using the NOB-I murine T cell line as described in Svenson et al, 2000.
• IL-l ⁇ RIAs and IL-6 ELISAs also are performed as described in Svenson et al, 2000.
• Sera are collected 2 weeks and 6 weeks after vaccination of the positive mice are tested. No difference is seen between total IL- l ⁇ binding to serum and binding to IgG.
• the IQs range from 0.1 nM to 1.3 nM (e.g., 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3 nM).
• IL- l ⁇ aAb are tested using an RIA.
• the antisera function similarly to those disclosed in ' Svenson et al., 2000.
• the binding of 125 I-IL-Ia to the murine cell-line NOB-I is suppressed by at least 10% ⁇ e.g., at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95%) by all aAb-positive sera collected two weeks after vaccination and tested as described in Svenson et al., 2000. aAb-negative controls are negative.
• mice are sacrificed at different time points, and the extent of atherosclerosis is evaluated. Plaque deposition and atherosclerotic lesions are assessed in aortic roots and thoracoabdominal aortas and quantified according to standardized methods (e.g., Trogan et al, Proc Natl Acad Sci U S A. 2002 Feb 19;99(4):2234-9; Chaabane et al, Invest Radiol. 2003 Aug;38(8):532-8).
• Aortic root atherosclerotic lesion areas in IL-l ⁇ - immunized ApoE -/- mice are significantly decreased as compared to ApoE -/- control mice (e.g., by at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95%).
• Atherosclerotic lesion development is also examined in preparations of the descending aorta stained with Sudan IV.
• Luminal area of coronary arteries are significantly diminished in control ApoE-/- mice (e.g., by at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95%) compared to control mice. Histological analysis of aortic roots demonstrates the presence of CD68-positive cells in the neointima in ApoE -/- controls but not in IL- l ⁇ immunized animals.
• HRP horseradish peroxidise
• the coloring reaction is made with ABTS buffer.
• ABTS buffer (3-ethylbenzthiazoline-6-sulfonic acid, Sigma Cat. No. A-1888, 150 mg, 0.1 M citric acid, Fisher anhydrous, Cat. No. A-940, in 500 ml; the pH is adjusted to 4.35 with NaOH pellets, and 11 ml aliquots are stored at -20 0 C, 40% SDS (80 g SDS in 200 ml dd H 2 O), with the addition of 200 ml DMF (N,N-dimethyl formamide)).
• SDS 80 g SDS in 200 ml dd H 2 O
• DMF N,N-dimethyl formamide
• ApoE-/- mice were obtained from Jackson Laboratory (Bar Harbor, Maine, strain B6.129P2-Apoe tmlUn 7J). Mice homozygous for the Apoe tmlUno mutation show a marked increase in total plasma cholesterol levels that are unaffected by age or sex. Fatty streaks in the proximal aorta are found at 3 months of age. The lesions increase with age and progress to lesions with less lipid but more elongated cells, typical of a more advanced stage of pre-atherosclerotic lesion. Moderately increased triglyceride levels have been reported in mice with this mutation on a mixed C57BL/6 x 129 genetic background.
• mice Aged ApoE deficient mice (>17 months) have been shown to develop xanthomatous lesions in the brain consisting mostly of crystalline cholesterol clefts, lipid globules, and foam cells. Smaller xanthomas were seen in the choroid plexus and ventral fornix. Recent studies indicate that ApoE deficient mice have altered responses to stress, impaired spatial learning and memory, altered long term potentiation, and synaptic damage. C57BL/6 and SCID mice were obtained from Harlan (Horst, the Netherlands).
• Al(OH) 3 Rehydragel; Reheis Chemical, Dublin, Ireland
• mice [30] Therefore, we vaccinated mice with an IL-l ⁇ -PPD conjugate in alum to ensure effective T-cell help for the IL-l ⁇ -specific B-cells.
• Antibody titers were determined by ELISA. Groups of 5 mice received subcutaneous immunizations with 15 ⁇ g of recombinant IL-l ⁇ conjugated to 10 ⁇ g-PPD using an incubation step with glutaraldehyde. The IL-l ⁇ -PPD conjugate is then absorbed to alum. Mice received three such subcutaneous immunizations with 2 weeks time interval.
• ApoE knock out mice (age 6 weeks) were actively immunized with 15 ⁇ g murine IL-l ⁇ conjugated with 10 ⁇ g PPD (purified protein derivate from M. tuberculosis) in aluminium hydroxide on days 0, 14 and 28 by subcutaneous administration in the neck region.
• the injection volume was 100 ⁇ l, and the amount of aluminium hydroxide was approximately 1 mg.
• Control mice were treated similarly but with a preparation that contained the same amount of PPD and aluminium hydroxide but that did not contain IL- l ⁇ .
• Blood was sampled from the tail vain on days 0, 28, 42, and 56 for measuring the anti-ILl ⁇ antibody response by ELISA.
• mice Four weeks after the first immunization, mice were started on an atherogenic diet with food pellets containing 16% fat, 1.16% cholesterol and 0.5 % cholic acid, a diet known to accelerate atherosclerosis. Mice were then euthanized at 18 weeks of age. Their aorta was removed for macroscopic and microscopic analysis. Histology slides were stained using Haematoxylin and Eosin (HE), as well as Sudan.
• HE Haematoxylin and Eosin
• C57BL/6 mice were actively immunized against IL- l ⁇ with 3 subcutaneous injections of IL-l ⁇ -PPD conjugate in alum. After 56 days their serum was collected and generation of anti-IL-l ⁇ autoantibody titers were confirmed by ELISA. 200 ⁇ l of such serum was passively transferred to 6 weeks old ApoE knock out mice. These passive serum transfers were repeated every week. Control ApoE-/- mice received passive weekly passive transfers of serum from na ⁇ ve C57BL/6 mice. Starting with these passive serum transfers, the ApoE-/- mice were fed an atherogenic diet with food pellets containing 16% fat, 1.16% cholesterol and 0.5 % cholic acid, in order to accelerate the formation of atherosclerosis.
• Control ApoE-/- mice were passively transferred 200 ml of serum from na ⁇ ve C57BL/6 mice in weekly intervals. Mice were euthanized on after 6 weeks for macroscopic and histological analysis of the aorta. Histological analysis included haematoxylin and eosin staining of cross sections, as well as Sudan stains. [34] After these 6 weeks, inspection of the cut open aorta under a binocular microscope showed a marked reduction of atherosclerotic plaques in ApoE-/- mice passively transferred anti-IL-l ⁇ antiserum, but not in ApoE-/- control mice receiving na ⁇ ve serum.
• ApoE knock out mice (age 6 weeks) were actively immunized with 15 ⁇ g murine IL- l ⁇ conjugated with 10 ⁇ g PPD (purified protein derivate from M. tuberculosis) in aluminium hydroxide on days 0, 14 and 28 by subcutaneous administration in the neck region.
• the injection volume was 100 ⁇ l, and the amount of aluminium hydroxide was approximately 1 mg.
• Control mice were treated similarly but with a preparation that contained the same amount of PPD and aluminium hydroxide but that did not contain IL- l ⁇ .
• Blood was sampled from the tail vain on days 0, 28, 42, and 56 for measuring the anti-ILl ⁇ antibody response by ELISA.
• mice Four weeks after the first immunization, mice were started on an atherogenic diet with food pellets containing 16% fat, 1.16% cholesterol and 0.5 % cholic acid, a diet known to accelerate atherosclerosis. Mice were then euthanized at 18 weeks of age. Their aorta was removed for macroscopic and microscopic analysis. Histology slides were stained using Haematoxylin and Eosin (HE), as well as Sudan.
• HE Haematoxylin and Eosin
• C57BL/6 mice were actively immunized against IL-l ⁇ with 3 subcutaneous injections of IL-l ⁇ -PPD conjugate in alum. After 56 days their serum was collected and generation of anti-IL-l ⁇ autoantibody titers were confirmed by ELISA. Two hundred ⁇ l of such serum was passively transferred to 6 weeks old ApoE knock out mice. These passive serum transfers were repeated every week. Control ApoE-/- mice received 200 ⁇ l serum transfers from na ⁇ ve C57BL/6 mice. Starting with these passive serum transfers, the ApoE-/- mice were fed an atherogenic diet with food pellets containing 16% fat, 1.16% cholesterol and 0.5 % cholic acid, in order to accelerate the formation of atherosclerosis.
• Control ApoE-/- mice were passively transferred 200 ml of serum from na ⁇ ve C57BL/6 mice in weekly intervals. Mice were euthanized after 6 weeks for macroscopic and histological analysis of the aorta. Histological analysis included haematoxylin and eosin staining of cross sections, as well as Sudan stains.
• ADCK Antibody dependent complement mediated killing
• C57BL/6 mice were actively immunized against IL-Ia with 3 subcutaneous injections of IL-l ⁇ -PPD conjugate in alum. After 56 days their serum was collected and generation of anti-IL-l ⁇ autoantibody titers were confirmed by ELISA. Sera were heat inactivated. 50 ⁇ l of an EL-4 cell suspensions were plated into 96 well plates. To each of these wells 15 ⁇ l of 1 :2 serial dilutions of the heat inactivated serum was added. Plates were then incubated for 20 minutes at 37 0 C. Then 25ml of murine serum were added to each well. After another 5h incubation at 37 0 C wells are photographed and then the cells counted in a counting chamber using trypan blue to distinguish dead from alive cells.

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