Methods for diagnosis and therapy of autoimmune disease,...

Details Methods for diagnosis and therapy of autoimmune disease,... Methods for diagnosis and therapy of autoimmune disease,...

2000-01-24

2004-10-05

Helms, Larry R. (Department: 1642)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Transferase other than ribonuclease

C435S320100, C536S023200

Reexamination Certificate

active

06800469

ABSTRACT:

The present invention relates to methods for the diagnosis of human autoimmune disease, for example Insulin Dependent Diabetes Mellitus (IDDM), and to methods for identifying substances which can be used in the therapy and prevention of such diseases. The invention further relates to novel human retroviruses involved in autoimmune disease and having superantigen activity, as well as to their expression products.
For some autoimmune diseases such as IDDM, Multiple Sclerosis, arthritis and others, it is known that a combination of genetic, environmental and possibly exogenous infectious factors may be important in precipitating disease. However, the precise roles of each these factors remains incompletely elucidated. For example, for IDDM, the Major Histocompatibility Complex (MHC) Class II genotype is one of the strongest genetic factors determining disease susceptibility (Vyse, T. J. and Todd J. A., 1996) although the respective roles of the different MHC Class II
+
cell types in promoting disease has not yet been clarified. Furthermore, IDDM shows temporal, epidemic-like variations and the clinical disease exhibits preferential seasonal onset (Karvonen et al., 1993). Recently, Conrad et al. (1994) provided evidence for superantigen involvement in IDDM aetiology and postulated that viruses may be the modifying agent responsible for the presence of superantigen on diabetic islets.
Genetic background also has an important influence in multiple sclerosis. In addition, Perron et al (Perron et al, 1997) have recently identified a retrovirus which can be isolated from cells of multiple sclerosis patients. Whether the retrovirus contributes as a causative agent of multiple sclerosis or as a link in the pathogenic process, or whether it is merely an epiphenomenon, has not been identified. No superantigen activity of the retrovirus has been identified.
It is an aim of the present invention to identify agents implicated in the pathogenesis of human autoimmune diseases, such as IDDM, and on the basis of these agents to provide reliable diagnostic procedures and therapeutic or prophylactic substances and compositions.
These objectives are met by the provision, according to the invention, of diagnostic procedures involving the detection of expressed retroviruses having superantigen (SAg) function, these retroviruses being directly involved in the pathogenesis of human autoimmune disease by activation of autoreactive T-cells. Compounds and compositions capable of blocking SAg function or production are also provided as therapeutic and prophylactic agents in the treatment of autoimmune disease.
The present invention is based on the discovery, by the present inventors that superantigens (SAgs) encoded by retroviruses, particularly endogenous retroviruses, play a major role in the pathogenesis of autoimmune disease, very likely by activating autoreactive T-cells.
Superantigens (SAgs) (Choi et al, 1989; White et al, 1989) are microbial proteins able to mediate Interactions between MHC Class II
+
—and polyclonal T-cells resulting in reciprocal activation (Acha-Orbea et al, 1991; Choi et al, 1991; Fleischer and Schrezenmeier, 1988). Their function is restricted by only two absolute requirements: the presence of MHC Class II on the surface of the presenting cells and the expression of one or more defined Variable (V)-&bgr; T cell receptor (TCR) chain(s) on T cells.
The potential role of SAgs in human diseases is ill-defined. Bacterial SAgs have been proposed to be associated with the pathogenesis of autoimmune disease (White et al, 1989). However, although pathogen disease associations have been described, none of these have as yet implicated a pathogen-encoded SAg (Howell et al, 1991; Paliard et al, 1991). A SAg-like activity resembling the one encoded by MMTV has been reported to be associated with herpesvirus infections (Dobrescu et al, 1995; Sutkowski et al, 1996). However, in none of these two systems has it been demonstrated that the SAg activity is actually encoded by the infectious agent. SAg activity has been reported in patients having Type I diabetes (Conrad et al 1994). However, the origin of the Sag activity is not identified.
In the framework of the present invention, the inventors have identified the source of SAg activity in IDDM patients as being a novel endogenous retrovirus, (HERV) designated IDDKK
1.2
-22. This retrovirus is related to, but distinct from mouse mammary tumor virus (MMTV). It is ubiquitous in the human genome but is only expressed in diabetic individuals, possibly in response to a particular environmental stimulus. The HERV encodes superantigen (SAg) activity within the env gene. Expression of the SAg gives rise to preferential expansion of V&bgr;-7 T-cell receptor positive T-cells, some of which are very likely to be autoreactive. Thus the expression of self-SAg leads to systemic activation of a sub-set of T-lymphocytes, among which autoreactive T-cells, will in turn give rise to organ-specific autoimmune disease.
The involvement of retroviral SAg, particularly endogenous retroviral SAg in autoimmune disease is unexpected. Indeed, endogenous retroviruses (HERV) form an integral part of the human genome. If expressed from birth, any autoreactive T-cells activated by expression of a retroviral SAg should be deleted as part of the normal development of the immune system (thymic deletion). However, in the case of autoimmune diseases such as diabetes, the expression of the retrovirus, and hence of the encoded SAg, occurs only later in life, leading to the proliferation of autoreactive T-cells.
To identify the microbial agent responsible for SAg activity in diabetes, the present inventors have developed a novel primer-extension technique. This method can be used to isolate and identify, in a sample polyadenylated RNA, any expressed, previously unidentified retroviral RNA, particularly retroviruses having SAg activity and being involved in human autoimmune disease. This strategy relies on the following three characteristic features of functional retroviruses. First, retroviral genomes contain a primer binding site (PBS) near their 5′ end. Cellular tRNAs anneal to the PBS and serve as primers for Reverse Transcriptase (reviewed by Whitcomb and Hughes, 1992). Second, the R (repeat) sequence is repeated at the 5′ and 3′ ends of the viral RNA (Temin, 19B1). Third, the RT-RNAse H region of the pol gene is the most conserved sequence among different retroelements (McClure et al., 1988; Xiong and Eickbusch, 1990). The method comprises the following steps:
i) isolation of the 5′ R-U5 ends of expressed putative retroviral genomes using nucleic acid amplification, the 3′ primer being complementary to known <primer binding sites> (pbs).
ii) isolation of the 3′ R-poly(A) ends corresponding to the 5′ R-U5 ends, by use of primers specific for the R regions isolated in step i).
iii) amplification of the conserved RT-RNase H region within the pol gene by using degenerate primers corresponding to the conserved region.
iv) amplification of the 5′ moiety of the putative retroviral genome by using primers specific for the different U5 regions isolated in step i) in conjunction with a primer specific for the 3′ end of the central pol region isolated in step iii).
v) amplification of the 3′ moiety of the putative retroviral genome using primers specific for the central pol region isolated in step iii) in conjunction with primers specific for the poly(A) signals present in the 3′ R-poly(A) sequences isolated in step ii).
vi) confirmation of the presence of an intact retroviral genome by amplification using primers specific for its predicted U5 and U3 regions.
Once an expressed retrovirus has been identified, its SAg activity can be tested by contacting a biological sample containing MHC Class II
+
cells expressing the putative Sag activity, with cells bearing one or more variable (V)-&bgr; T-cell receptor (TCR) chains and detecting preferential proliferation of a V&bgr; subset.
The techniques developed

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