Data consistent with autoimmune disease being caused by Epstein-Barr virus are shown. Based on this evidence, an effective vaccine would prevent the autoimmune disease in those vaccinated, modified or administered so that the vaccine is not itself capable of inducing autoimmune disease. In the case of anti-Sm, structures to be avoided in an Epstein-Barr virus-derived vaccine have been identified. Differences have been identified in the immune responses to Epstein-Barr infection between individuals who develop a specific autoimmune disease and those who do not. These differences are used to distinguish those who are at greater risk for developing specific autoimmune diseases from those who are a lesser risk. Assuming Epstein-Barr virus causes autoimmune disease and that Epstein-Barr virus remains latent in the patient for life, reactivation of the virus from the latent state is important in generating or maintaining the autoimmune response that culminates in autoimmune disease. Cells infected with latent virus may also encourage autoimmunity. Based on the understanding that reactivation or latency are important to produce or sustain autoimmunity, then therapies directed against Epstein-Barr virus will also be effective therapies for the autoimmune manifestations of disease for which Epstein-Barr virus is responsible.

SUMMARY OF THE INVENTION

Data consistent with autoimmune disease being caused by Epstein-Barr virus are shown. Some of the features of the mechanism in the specific example of the anti-Sm autoantibody response were found in systemic lupus erythematosus. Based on this evidence, an effective vaccine would prevent the autoimmune disease in those vaccinated, modified or administered so that the vaccine is not itself capable of inducing autoimmune disease. In the case of anti-Sm, structures to be avoided in an Epstein-Barr virus-derived vaccine have been identified.

Differences have been identified in the immune responses to Epstein-Barr infection between individuals who develop a specific autoimmune disease and those who do not. These differences are used to distinguish those who are at greater risk for developing specific autoimmune diseases from those who are a lesser risk.

Assuming Epstein-Barr virus causes autoimmune disease and that Epstein-Barr virus remains latent in the patient for life, reactivation of the virus from the latent state is important in generating or maintaining the autoimmune response that culminates in autoimmune disease. Cells infected with latent virus may also encourage autoimmunity. Based on the understanding that reactivation or latency are important to produce or sustain autoimmunity, then therapies directed against Epstein-Barr virus will also be effective therapies for the autoimmune manifestations of disease for which Epstein-Barr virus is responsible.

DETAILED DESCRIPTION OF THE INVENTION

In the United States, about 95% of the adult population has been, and continues to be, infected with Epstein-Barr virus. Observations described herein are consistent with a small proportion of these developing autoimmune disease, related to this virus. Other factors are also likely to be important in the development of autoimmune disease, but are not essential to understand in order to develop therapeutics and diagnostics for use in diagnosing, treating and preventing or ameliorating autoimmune diseases involving Epstein-Barr virus as the etiologic agent. Epstein-Barr virus is the probable etiologic agent for nearly all cases of lupus, which serves as an example of autoimmune disease.

Diagnostics and therapeutics derived from the discovery that Epstein-Barr virus causes autoimmune disease as applied to the prevention, diagnosis and treatment of autoimmune disease are described herein. Systemic lupus erythematosus (lupus) is the particular autoimmune disease evaluated and for which data have been obtained. Within lupus, the work on a molecular understanding of the relationship between anti-Sm and systemic lupus erythematosus and the relationship of anti-Sm autoantibodies to Epstein-Barr virus is the best illustration of the data supporting these diagnostics and therapeutics.

The experiments described herein to address Epstein-Barr virus in lupus were guided by the results of immunochemical studies, not by the previous studies. These data pointed toward a curious mechanism in the anti-Sm autoantibody system in lupus which could involve Epstein-Barr virus.

The technology applied to the problem is very important in two ways. First, the assays for anti-Epstein-Barr virus antibodies have been dramatically improved. The classic method is to evaluate antibody binding to an Epstein-Barr virus infected cell line by immunofluorescence. This assay is dependent upon the expression of different Epstein-Barr virus proteins, depending upon whether the cell line is producing virus or the virus is latent. The autoantibodies of lupus often render these assays uninterpretable, making their use in lupus especially problematic.

Consequently, solid phase assays using enriched preparations of the surface antigen constitute a major improvement. The surface antigen is largely composed of a glycoprotein called gp340/220 or the Viral Capsid Antigen. These preparations of surface antigens have had many interfering substances removed. Epstein-Barr virus infection in man virtually always generates antibodies against this surface antigen. The assay is much simpler than is the cell line immunofluorescence assay and subject to much less variation in interpretation.

Second, molecular methods have been designed and developed to detect Epstein-Barr virus which appear to be at least as reliable as the serologic methods, and may even be superior. In normal adults Epstein-Barr virus infects only about one in every 20,000 to 500,000 B cells (Miyashita, E. M. et al. Cell 80:593 601 (1995)). B cells usually constitute only about 8% of the peripheral blood mononuclear cells. The vanishingly small quantity of Epstein-Barr DNA is lost in a relative ocean of genomic human DNA and is very difficult to detect. The improved sensitivity and specificity of detection of Epstein-Barr virus DNA improves the measurement made and leads to more accurate interpretation of the data. The older methods detected Epstein-Barr virus DNA in the peripheral blood in about 70% of individuals who had serologically converted, while the method described herein appears to detect more than 95% of those who have seroconverted and a few who have not serovonverted.

Reliable assays were used to address the prevalence of seroconversion and infection in the cases and controls in a way that took optimal advantage of the known properties of the viral infection. Others have selected sub-optimal study populations, have poorly chosen their controls, or have focused upon the quantitative level of antibody rather than qualitative evidence for infection, in addition to the technical problems outlined above in reliably detecting anti-Epstein-Barr virus seroconversion and Epstein-Barr viral DNA.

The data in the anti-Sm autoantibody system, discussed below, are used as a model in which the antigen presenting capacity of the B cell is important in generating autoimmunity. For example, the PPPGRRP (SEQ ID NO:1) structure is found in Epstein-Barr virus Nuclear Antigen-1. This sequence induced autoimmunity against the Sm B/B' of a rabbit after immunization. This autoimmunity not only included the related PPPGMRPP (SEQ ID NO:4) of Sm B/B', but also many other structures of B/B'. When the B cell generates a receptor that binds PPPGRRP (SEQ ID NO: 1) and PPPGMRPP (SEQ ID NO:4) then this B cell is capable of presenting the spliceosome to the immune system. Of course, once this cross reacting autoantibody is produced, then it may facilitate spliceosomal autoimmunity. Epstein-Barr virus is important because the immune control of the infected B cell is altered by the infection, rendering autoimmunity more likely. This mechanism can be directly extended to other antigens to generate other immune responses (both cellular and humoral) which lead to a variety of autoimmune diseases. Also, double infection with Epstein-Barr virus and another virus would extend the immune regulatory abnormalities to the antigens of the second virus.

Autoimmune Diseases

There are a large number of disorders in man that are thought to be autoimmune. These include systemic lupus erythematosus, autoimmune thyroid disease (Graves' disease or Hashimoto's thyroiditis), autoimmune beta islet disease of the pancreas (more commonly referred to as juvenile or Type 1 diabetes mellitus), primary biliary cirrhosis and many others. The particular disorders listed above are thought to involve antibodies produced in the host (the patient, in this instance) which bind to constituents of self. These antibodies are called autoantibodies. The particular constituent of self bound by the autoantibodies is associated with the different disorders. For example, anti-mitochondrial autoantibodies are associated with primary biliary cirrhosis. Anti-acetylcholine receptor autoantibodies are associated with myasthenia gravis. The list of such autoantibodies is quite long and often only one or a few autoantigens are bound by autoantibodies in each particular disorder. Systemic lupus erythematosus (or abbreviated as lupus herein) is an exception to this tendency, since many autoantibodies may be found in the disease and since patients do not necessarily share any particular autoantibody specificity. Anti-Sm (which is an anti-spliceosomal autoantibody specificity) is one of the autoantibodies closely associated with systemic lupus erythematosus, but even this autoantibody is found in only a minor fraction of patients with systemic lupus erythematosus. (Please refer to a review of this area (Harley, J. B. and Gaither, K. K. Rheum. Dis. Clin. N. Amer. 14:43 56 (1992)).

It is not sufficient just to produce autoantibodies. There must be some consequence of their presence in order to develop pathology which culminates in clinical disease. There are many instances of detecting autoantibodies in the absence of any detectable clinical illness. Autoantibodies may realize their pathologic potential by binding their antigen in the circulation. They then become part of circulating immune complexes. They may deposit in tissues, induce an inflammatory response, and cause tissue injury, as appears to occur in lupus. Autoantibodies may interfere with the functioning of receptors or otherwise activate cells as may happen in Wegener's granulomatosis or Graves' ophthalmopathy. Autoantibodies may simply block normal functioning of a protein, as happens to the acetylcholine receptor in myasthenia gravis. No doubt there are other mechanisms by which autoantibodies encourage clinical illness.

These mechanisms involve humoral autoimmunity; that is, autoimmunity that is mediated by autoantibodies. There is another form of autoimmunity mediated by cells, in particular T cells. Multiple sclerosis is thought by some to be an example of a disease that is mediated by autoimmune T cells. Although the methods and compositions described herein are particularly concerned with humoral autoimmunity, it is expected that cellular autoimmune processes are also involved with producing autoimmunity as a consequence of Epstein-Barr virus infection. As in many situations, one skilled in the art would expect cellular immune mechanisms to dominate in some individuals and humoral mechanisms to dominate in others. This situation would be expected to give rise to different clinical expression of disease. Tuberculous and lepromatous leprosy are examples where differences in the dominant form of the immune response lead to profound differences in the clinical illness, despite being caused by the same organism.

The traditional distinction between humoral and cellular immune mechanisms are being reevaluated under a new paradigm. T cells appear to have the capacity to respond in at least two ways. These cells are called Th1 and Th2, for T helper cells, Type 1 and 2. Characteristic cytokine production profiles are often used to distinguish these different responses. Th1 responses tend to be the more traditionally appreciated cellular immune responses. Th2 responses lead, among other consequences, to more of an antibody response and are more aligned with the classic humoral response. However, these boundaries do not appear to operate strictly since some types of antibody are more likely found in Th1 responses and the Th2 response clearly has its cellular component. Most autoimmune diseases probably have important components of both humoral and cellular autoimmunity.

Therapeutic and Diagnostic Compositions Vaccines

Immunity against a viral infection can be induced using either peptides, viral proteins or other components of the virus such as carbohydrate components, substances which imitate structures of the virus, or the virus. In the preferred embodiment, the vaccine is based on the viral proteins wherein the epitopes cross-reactive with the splicesomal proteins are deleted. In other embodiments, the vaccine is based on viral proteins where epitopes cross-reactive with antibodies to other known autoantigens are deleted or altered to decrease their immunoreactivity with autoantibodies. In still other embodiments, the vaccine includes the virus, either live, modified or inactivated, or components thereof, in a vehicle which can be administered in a dosage form and over a schedule eliciting a strong immune response to kill the virus, but which does not elicit an autoimmune response.

An unmodified vaccine may be useful in the prevention and treatment of autoimmune disease related to Epstein-Barr virus. The dose, schedule of doses and route of administration may be varied, whether oral, nasal, vaginal, rectal, extraocular, intramuscular, intracutaneous, subcutaneous or intravenous, to avoid autoimmunity and, yet, to achieve immunity from Epstein-Barr virus infection. The response to the unmodified vaccine may be further influenced by its composition. The particular adjuvant employed (its concentration, dose and physical state), concentration of the virus in the vaccine, and treatment of the unmodified vaccine with physical environmental changes, for example, temperature and pressure, the particular buffer, and the particular preservative(s) (if any) will be selected to reduce the liklihood of developing an autoimmune disorder, for example using the animal strains discussed below. This same or different vaccine may be useful in reducing or eliminating the effect of an existing latent or active Epstein-Barr virus infection upon autoimmunity.

Peptides of up to about forty amino acids, more preferably between four and twenty-five amino acids, most preferably eight amino acids, can be synthesized using any one of the methods known to those skilled in the art. In general, an epitope of a protein is composed of between three or four and eight amino acids (see Watson et al., "Certain Properties Make Substances Antigenic," in Molecular Biology of the Gene, Fourth Edition, page 836, paragraph 3, (The Benjamin/Cummings Publishing Company, Menlo Park, 1987). As used herein, the peptides can contain the entire native epitope, or portions thereof sufficient to react with autoantibody.

Although described in the literature with reference to specific sequences encoding viral proteins or the autoantigens, a number of substitutions using natural or synthetic amino acids can be made in the peptides to yield an peptide acting as a linear epitope that is functionally equivalent to the disclosed sequences, for example, as demonstrated by James and Harley, J. Immunology 148:2074 2079 (April 1992). Accordingly, the term linear epitope as defined by a specific sequence is used herein to include peptides having substitutions yielding a peptide bound in an equivalent manner or extent by an antibody or autoantibody. For example, using monoclonal antibodies against peptide determinants of Sm B/B', substitution studies demonstrated that A, G, and S can substitute for R in PPPGMRPP (SEQ ID NO:4) in the binding of one antibody, KSm3. Analogously, F, H, T, V and Y can substitute for I in PPPGIRGP (SEQ ID NO:5) in the binding of KSm3.

Solid phase binding of autoantibodies to peptides has proven useful for examining sequential linear epitopes, also referred to as "linear" or "sequential". These have been useful to define important residues in epitope structure. This approach may or may not be less useful in defining conformational epitopes or regions where two or more linear, but not sequential, epitopes are brought together by the tertiary structure. The particular structural relationships between the autoantigens and autoantibody with particular regard to the particular conformation assumed by the peptide determines what can be learned by this approach. In addition, although many peptides may assume conformations in solution that are not found in the native protein structure, true epitopes may still be delineated by this method. Those peptides that tend to have a structure similar to that found in the native molecule are expected to usually be bound by a larger proportion of the autoantibodies that bind the analogous sequence on the native protein and/or may be bound with greater affinity.

It is believed that naturally arising human lupus follows a progression similar to that induced in the rabbit model described in the examples. Using this model, an immune response to a peptide, one very similar to a region of EBNA-1, is the seminal, initiating event for the subsequent autoimmunity and disease manifestations of lupus, where they occur. A structure, such as a peptide, that is capable of inducing autoimmunity is not necessarily identical to the structure found in the autoantigen. Indeed, it is possible that these structures would commonly be at least slightly different, when comparing the substance that induces the autoimmune response and the analogous structure in the autoantigen. On the other hand, there must be a basis for the non-autoantigen substance to induce autoimmunity. This is best identified as a cross-reaction wherein the immune recognition molecule binds, though not necessarily equally, to both the non-autoantigen substance as well as to the autoantigen.

The proposed mechanism is as follows. An immune response against a non-autoantigenic substance occurs. Some fraction of the antibodies thereby produced recognize the autoantigen and hence are autoantibodies. These autoantibodies facilitate the processing and antigen presentation of the autoantigen via the B cell surface immunoglobulin which serve as receptors, in this case for autoantigen, or via immunoglobulin cell surface receptors that are found on a variety of cells capable of antigen presentation. Once this occurs the immune response expands to other structures of the autoantigen and a full-blown, complete autoimmune response against the autoantigen ensues, which can result in clinical illness.

Based on this mechanism, the autoimmune responses progress from one or a few initial antigenic structure(s) to a much more complex response focused upon the autoantigen. Elucidating the pattern of progression and understanding the relationship of autoimmune serologic findings to clinical manifestations places the physician in a strong position to accurately prognosticate and prepare patients and their families for the more likely outcomes.

In the case of systemic lupus erythematosus and the anti-Sm response, one method is to repeat the assays determining autoantibody binding to peptides over time. The effect of the peptide in vitro on cells from patients can also be measured. Proliferation, secretion of cytokines, interferons and other substances, expression of cell surface molecules and activation are typically useful diagnostic indicators.

This strategy to generate autoimmunity can also be used to develop reagents that are useful in diagnosis or treatment of autoimmune disorders. Animal antibodies that compete with or otherwise facilitate the identification of particular fine specificities of binding can be important in evaluating prognosis. Moreover, the peptide binding pattern to the octapeptides from the nRNP A protein show two different patterns. It should be possible to correlate a particular pattern found in a patient to obtain an indication of the stage the disease currently is at as well as the clinical prognosis. Reagents developed as a consequence of immunizing animals with autoantigenic peptides could be used to identify these differences. Such reagents include antisera, T cell lines, subsets of antibodies, individual antibodies, subsets of cells bearing a subset of the T cell receptors, individual T cell receptors, and cytokines and other substances elaborated by cells from the animal. The antibodies and T cell receptors are construed to include recombinant antibodies or T cell receptors derived from a peptide-immunized animal.

The RNA-protein particles which are the major autoantigens may now be purchased commercially. The reagents made available by the animal model of autoimmunity described herein will be useful in the manufacturing and testing of autoantigens. Affinity purification using animal antiserum (absorbed or otherwise prepared) could be used for purification of the naturally occurring autoantigens.

Having a mechanism of disease provides the opportunity to apply new strategies for prevention of disease and for specific immunologic correction of the immune abnormalities that lead to disease, and therefore more accurately design the therapy.

For example, with the realization that the generation of autoimmune disease can be divided into phases comes the appreciation that the therapeutic opportunities will be similarly partitioned. As a specific example, the influence of vaccination with an analog of PPPGMRPP (SEQ ID NO:4) will be different depending not only upon the structure of the immunogen, but also upon the pharmaceutical carrier, upon the maturity of the autoimmune response against PPPGMRPP (SEQ ID NO:4) and Sm, and upon other therapeutics that may be administered concomitantly. Such therapeutics include drugs as well as biologics, such as cytokines, immunogloblins, and interferons, among others.

As a second specific example, mice strains exist which produce lupus autoimmunity, as well as strains which do not produce lupus autoimmunity, after immunization with PPPGMRPP-MAP.TM. (SEQ ID NO:4). The genetic, biochemical, and physiological differences between these strains can be used to develop diagnostics, including gentic markers and risk factors, and therapeutics for autoimmune disease, especially lupus, using techniques known to those skilled in the art.

As discussed above, based on the data confirming that Epstein-Barr virus is an inducing agent for lupus, it is possible to design therapeutics to prevent or inhibit further progression of lupus by vaccinating with Epstein-Barr virus, or components thereof, using standard vaccination procedures, most preferably after alteration or removal or masking of the sites which elicit the autoimmunity.

The peptides can be used therapeutically in combination with a pharmaceutically acceptable carrier. The peptides can be administered in a dosage effective to block autoantibodies or as a vaccine to block the production of autoantibodies, by eliciting a protective immune response against non-autoantigenic regions of the pathogen. The peptide acts as a functional antagonist by binding to antibody that does not stimulate or activate the immune cells and thereby block the immune response to the autoantigens.

Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of vaccines to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. Peptides used as vaccines are most preferably administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

Alternatively, the peptides used for treatment might include peptides homologous to an identified antigenic sequence. 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. In addition, knowledge of the cross-reacting epitopes between a foreign antigen and an autoantigen 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 useful therapy in autoimmune diseases.

The 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.

Diagnostics

Individuals who are not at as great a risk for developing autoimmune disease can be identified by reactivity to the various peptides, for example, as demonstrated in the examples where individuals who are not prone to develop lupus are characterized by antibodies to GAGAGAGAGAGAGAGAGAGAGAGA (SEQ ID NO:7). Other structures derived from Epstein-Barr virus can be used to predict who will develop autoimmune disease. For example, GAGAGAGAGAGAGAGAGAGAGAGA (SEQ IF NO: 7) is a commonly identified antigen from Eptein-Barr virus in normal individuals, while lupus patients do not tend to bind this structure. An individual identified at risk for the development of an autoimmune disease, but who does not yet manifest autoimmunity or symptoms of the disease, may require a special therapeutic approach. This is an opportunity to induce immune suppression before the process leading to autoimmune disease is initiated. Strategies such as intravenous administration of large amount of the initiating structure is known to induce tolerance. Small sub-immunogenic doses of the initial immunogen can also be used to induce tolerance.

There is a limited opportunity to interrupt or redirect an immune response that has been initiated against the first components of the autoantigen. Here again the induction of suppression by the use of the component peptides or analogs thereof with or without concomitant drugs or biologics has the potential to inhibit progression into an autoimmune disorder. Once autoimmunity against the autoantigen is established, the use of component peptides or their analogs with or without concomitant drugs or biologics may interrupt the course of the autoimmune response, thereby ameliorating the illness.

The animal model provides an opportunity to optimize ways of interrupting and reversing the autoimmune process. For example, it has been observed that one of the rabbits immunized with Map-PPPGMRPP (SEQ ID NO:4) seemed to improve clinically somewhat after developing the most severe manifestations of systemic autoimmunity. If this result is the effect of a particular antibody, then this antibody may have the capacity to influence the maturation of the immune response toward alleviating the disease in other species. For example, such an antibody could be isolated by biochemical methods, by recombinant DNA methods or by hybridoma monoclonal methods, humanized using standard technology and then administered to patients as a specific therapeutic agent for disease. T cell receptors or cytokines could be equally useful.
 

Claim 1 of 5 Claims

1. A method for determining that an individual has or may develop Epstein Barr Virus (EBV)-induced systemic lupus erythematosus (SLE) comprising screening antibodies obtained from individual's serum for reactivity with antigen epitopes of peptide molecules PPPGRRP (SEQ ID NO:1), GRGRGRGG (SEQ ID NO:2), RGRGREK (SEQ ID NO:3), PPPGMRPP (SEQ ID NO:4), PPPGIRGP (SEQ ID NO:5) and GAGAGAGAGAGAGAGAGAGAGAGA (SEQ ID NO:7), wherein the peptides are present either in free form or bound to a carrier molecule, wherein the presence of antibodies that bind only the antigen epitopes of SEQ ID NOS:2 5 indicates that the individual has or may develop EBV-induced SLE, and wherein the presence of antibodies that only bind the antigen epitope of SEQ ID NO:7 indicates that the individual does not have or may not develop EBV-induced SLE.

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