Patent Application
20070249808
The present invention relates to a human endogenous retroviral superantigen associated with autoimmune disease, particularly multiple sclerosis. The invention also relates to derivatives of the superantigen, and to nucleic acid molecules encoding the derivatives. The invention further concerns methods for the diagnosis of autoimune disease, particularly multiple sclerosis, and methods for identifying substances which can be used in the therapy and prevention of these diseases.
For some autoimmune diseases such as Insulin Dependent Diabetes Mellitus (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 of these factors remains. incompletely elucidated.
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.
Perron et al (Perron et al, 1997) have recently identified a retrovirus, <<MRSV>>, 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. Using sequence homology with the pol gene of MRSV, Alliel et al. (1998) identified a full length endogenous provirus located on the long arm of human chromosome 7 (7q21-22). On the basis of the PBS t-RNA motif usually used for the classification of human endogenous retrovirus families (HERVs), this retrovirus has been designated <<HERV-W>> (W=tryptophan), although some authors use the designation <<HERV-7q>>. It has been postulated that HERV-W is involved in the pathogenesis of multiple sclerosis although no precise mechanism has been identified. To date, no superantigen activity of the retrovirus <<HERV-W>> has been reported.
Further endogenous retroviral sequences associated with autoimmune disease or with pregancy disorders have been reported in International Patent Application WO 99/02696.
It is an aim of the present invention to identify agents involved directly or indirectly in the pathogenesis of autoimmune disease, particularly Multiple Sclerosis (MS). On the basis of these agents reliable diagnostic procedures and therapeutic or prophylactic substances and compositions can be provided.
The present invention provides diagnostic procedures involving the detection of an expressed retrovirus having superantigen (SAg) function. It is thought that this retrovirus may be directly involved in the pathogenesis of MS by activation of autoreactive T-cells.
The present invention is based on the discovery, by the present inventors that the HERV-W (HERV-7q) retrovirus encodes superantigen (SAg) activity.
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)-β 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 neither 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). The retroviral origin of the Sag activity has also recently been identified (Conrad et al., 1997).
In the context of the present invention, the inventors have identified that the ENV domain of HERV-W encodes superantigen activity. Expression of the SAg gives rise to preferential expansion of Vβ6.7 and/or Vβ17 and/or Vβ21.3 T-cell receptor positive T-cells, some of which may be autoreactive. Thus it is postulated that 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 autoimmune disease.
In the context of the present invention, the following terms encompass the following meanings:
Such remnants include fragments of the typical structure, and have a minimal size of one LTR. Typically, the HERVs have at least one LTR, preferably two, and all or part of gag, pol or env.
In a first embodiment, the invention relates to proteins expressed by a human endogenous retrovirus having SAg activity and being associated with autoimmune disease, particularly MS.
More particularly, the invention relates to a protein or peptide having superantigen (SAg) activity, said protein or peptide comprising or consisting of the ENV protein of the human endogenous retrovirus HERV-W, the surface protein (SU) or transmembrane (TM) sub-units thereof, and fragments of HERV-W ENV and its subunits, particularly C-terminal fragments, which possess superantigen activity.
Preferably, the protein or peptide having superantigen (SAg) activity consists or comprises all or part of the Env protein of HERV-W (HERV-7q), illustrated as protein <<G>> or <<GT>> in
Specifically, said HERV-W SAg protein or peptide comprises:
In the context of the invention, the different portions of the Env protein are generally defined as follows, wherein the numbering of the amino acid positions corresponds to that shown in
According to the invention, the HERV-W SAg protein or peptide may consist exclusively of:
Particularly preferred HERV-W SAg proteins are those having between 95% and 99% identity, for example at least 98% identity with protein (i), (ii) or (iii), for example no more than a maximum of 9 or 10 amino acid differences over the whole length of the protein of reference or the sub-unit of reference, and preferably no more than 4 or 5 amino acid differences with respect to the whole length of the protein of reference or sub-unit of reference. Most preferably, the homologous sequences show no more than 4 or 5 amino acid differences with respect to the full length sequence <<G>> of
The protein or peptide of the invention may be a <<composite>> protein having SAg activity, and having the formula (I):
wherein
Such a composite protein has superantigen (SAg) activity. Component (b) is advantageously the SU or SU/TM regions of the <<G>> protein of
In Formula (I), when (b) consists of the SU or SU/TM regions of the <<G>> protein of
MALPYHIFLFTVLLPSFTLT, SEQ ID NO: 5
MGLPYHIFLCSVLSPCFTLT, SEQ ID NO: 6
MALPYHIFLFTVVSPSFTLT. SEQ ID NO: 7
According to a preferred embodiment, the protein or peptide of the invention comprises a protein having the formula (II):
wherein
Preferably, Formula (II) corresponds to a fragment of the full length SP-SU-TM HERV-W-ENV <<G>> protein as illustrated in
Most preferably, in Formula (II), the signal sequence (a), the SU portion (b) and the TM portion (c) are those illustrated for protein <<G>> or <<GT>> in
The present inventors have established that the SAg activity of the HERV-W ENV protein resides in the portion of the protein lying beyond the first N-terminal 120 amino acids, i.e. the first 120 amino acids are not essential for SAg activity. Thus, according to a preferred embodiment, the protein or peptide (b) in the above general formula (I) is a fragment consisting of a stretch of at least 50 and preferably at least 60, 70, 80, 90 or 100 consecutive amino acids comprised within amino acids 121 to 538 of the protein <<G>> illustrated in
Preferred examples of the protein (b) in Formula (I) are
When protein (b) is any one of the above-listed fragments consisting of a stretch of at least 50 consecutive amino acids comprised within amino acids 121 to 538 of the HERV-W ENV protein, the values of x and z in general formula (I) may be 0 or 1, for example, x may be equal to zero and consequently the fragment defined as (a) in the general formula is absent. In such a case, the N-terminus of the HERV-SAg protein is defined by amino acid 121 as illustrated in
In keeping with the above, component (b) in Formula (II) can be the full SU region as illustrated for protein <<G>> in
The proteins of the invention may be made by synthetic or recombinant techniques. If recombinant DNA technology is used, the HERV-W SAg protein can be obtained by the following method
The cells for the production of recombinant HERV-W SAg are preferably, but not necessarily, mammalian cells and may be MHC Class II+ or MHC Class II−. SAg activity can only be exhibited in cells which are MHC Class II+ (or which have been induced to become MHC Class II+), but expression of the SAg protein can be obtained in both MHC Class II+ and MHC Class II− cells. Typical MHC Class II+ cells are APCs such as B-lymphocytes, monocytes, macrophages or dendritic cells. Typical MHC Class II− include HeLA cells etc.
In accordance with this embodiment, a nucleic acid encoding the full length HERV-W protein depicted in
The SAg activity of the proteins or peptides according to the invention is specific for Vβ6.7- and/or Vβ17- and/or Vβ21.3-TCR chains. The inventors have established that the specificity of the HERV-W SAg activity with regard to Vβ expansion varies, within the specified spectrum, from individual to individual, reflecting the possible existence of polymorphic genetic factors and/or immunological tolerance to the SAg. At least one of Vβ6.7- and/or Vβ17- and/or Vβ21.3-TCR chains is stimulated. The most common pattern observed is the specific expansion of Vβ6.7+ and Vβ17+-T cells, although individuals showing other combinations such as Vβ21.3 and Vβ17 expansion have been identified.
In the context of the invention, the inventors have devised a highly sensitive bicistronic assay system which is particularly adapted for measuring expression levels of transfectants expressing HERV-W SAg proteins. The bicistronic constructs are illustrated in
The invention also relates to nucleic acid molecules encoding a HERV-W SAg protein as defined above.
The nucleic acid molecule encoding HERV-W SAg activity typically corresponds to the ENV open reading frame of the retrovirus. Preferably, the nucleic acid of the invention comprises or consists of all or part of the env gene (encoding the envelope glycoprotein) of an HERV associated with MS, such as HERV-W, illustrated in
The nucleic acid of the invention may be RNA, DNA or cDNA, for example proviral DNA, or retroviral genomic RNA. Proviral DNA is naturally found integrated into the human genome. Alternatively the nucleic acid may be synthetic.
More particularly, the nucleic acid molecules of the invention have the formula (III):
5′(A)x-(B)y-(C)z3′ (III)
wherein
<<x>>=0 or 1,
<<z>>=0 or 1,
<<y>>≧1.
Preferably, in the above formula (III) the oligonucleotide (A) does not encode a peptide comprising or consisting of any one of the signal sequences:
MALPYHIFLFTVLLPSFTLT, SEQ ID NO: 5
MGLPYHIFLCSVLSPCFTLT, SEQ ID NO: 6
MALPYHIFLFTVVSPSFTLT. SEQ ID NO: 7
Preferred nucleic acid molecules according to the invention comprise or consist of the sequence illustrated in
The nucleic acid molecules of the invention may comprise a chimeric gene wherein (A) and (C) as defined above include heterologous transcription regulatory regions operably linked to (B). By <<heterologous transcription regulatory sequences>> is meant regulatory sequences which are not those naturally used for transcription of the HERV ENV protein in the human genome.
Particularly preferred nucleic acid sequences are those encoding the proteins of Formulae (I) and (II) above, for example, encoding the following:
The nucleic acid molecules of the invention further comprise sequences which are complementary to a nucleic acid molecule as defined above, for example probes, primers, ribozymes or antisense molecules to the HERV-W ENV.
Nucleic acid molecules capable of hybridizing in stringent conditions with any of the above-defined nucleic acid molecules are also within the invention. Typical stringent conditions are those where the combination of temperature and salt concentration chosen to be approximately 12-20° C. below the Tm (melting temperature) of the hybrid under study. Such nucleic acid molecules may be labelled with conventional labelling means to act as probes or, alternatively, may be used as primers in nucleic acid amplification reactions.
The invention further relates to vector comprising any of the afore mentioned nucleic acid molecules.
The present invention involves, in a further embodiment, methods of diagnosis of autoimmune disease, particularly MS, based on the one hand on the specific presence in individuals susceptible to MS, of HERV-W SAg, and nucleic acids encoding the HERV-W SAg and on the other hand on the specific expression, in MS patients, of retroviruses having SAg activity.
The methods of diagnosis of the present invention are advantageous in so far as they are highly specific, distinguishing between different polymorphic forms of the MS-associated HERV, and further distinguishing between expressed and non-expressed viral nucleic acid. These methods can thus be reliably used even if the pathological agent is a ubiquitous endogenous retrovirus. They can be carried out on easily accessible biological samples (fluids or tissue), such as blood or plasma, without extensive pre-treatment. The diagnostic methods of the invention detect either disease-specific polymorphic forms of the retrovirus, and/or disease-specific expression of the retroviral superantigen. They can thus be applied before appearance of clinical symptoms, for example on genetically predisposed individuals. This allows suitable therapy to be initiated before autoimmune destruction occurs.
More particularly, in a first embodiment, the present invention relates to a process for the diagnosis of Multiple Sclerosis (MS) by detection of disease-specific retroviral polymorphic forms, comprising:
According to this embodiment, particularly preferred amplification primers are selected from sequences flanking the HERV-W retrovirus on chromosome 7 (7q21-22). In a preferred variant, the 3′ primer corresponds to approximately 100 bases or more, of the 3′ genomic sequence immediately flanking the HERV-W 3′ LTR on chromosome 7 (see Alliel et al, 1998), and the 5′ primer corresponds to a region of approximately 100 bases or more immediately upstream of the ATG translation initiation codon of HERV-W env. For example, the 5′ primer may be selected from any 100 base stretch, or longer, within the 5′ UTR of env (approximately nucleotides 1 to 760) as illustrated in
In a further embodiment, the present invention relates to a process for the diagnosis of Multiple Sclerosis (MS) by detection of disease-specific expression of SAg, comprising:
Specific detection of retroviral expressed mRNA is preferably carried out using nucleic acid amplification with viral specific primers which discriminate between proviral DNA and expressed RNA template. This is of particular importance since the MS associated retrovirus is an endogenous retrovirus. Indeed, it is thought that the proviral DNA is present in all human cells, whether or not the autoimmune disease is present. False positives could therefore be obtained if a detection method were used which does not distinguish between proviral DNA and transcribed mRNA.
The biological sample to be used for specific mRNA detection according to the invention may be any body fluid or tissue but is preferably plasma or blood. Normally, total RNA is extracted from the sample using conventional techniques. DNAse treatment may be carried out to reduce contaminating cellular DNA.
By performing the amplification on total RNA samples, the effects of contaminating DNA are reduced but not eliminated, even after treatment by DNAse. The method of the present invention allows selective amplification of expressed viral RNA transcripts using at least one m-RNA specific primer, for example a poly-A specific primer, even in the presence of contaminating viral DNA in the sample. The poly-A specific primer is specific for the poly-A signals present in the R-poly(A) sequences and the 3′ extremity of the retrovirus (see for example Alliel et al).
A poly-A-specific primer having from four to 25 T's for example 5 or 20 T's is particularly suitable for the purposes of the present invention.
The mRNA specific amplification requires a reverse transcriptase (RT) step, for which the poly A-specific primer is also be used.
The second primer in the mRNA-specific PCR step may be complementary to the U3 region, or other region of the retroviral genome, for example the 5′UTR of env. When the amplification product has a size of about 300 to 500 nucleotides, the conditions applied for the amplification (PCR) step are normally the following
The amplified material is subjected to gel electrophoresis and hybridised with suitable probes, for example generated from the U3 region.
By performing the mRNA specific detection of the invention, the presence of expressed MS retrovirus can be reliably determined in a biological sample. This can be detected well before the apparition of any clinical symptoms. The diagnosis of the invention can thus be used to detect onset of the disease process, enabling treatment to be administered before irreversible autoimmune attack occurs.
According to a particularly preferred embodiment, MS is diagnosed by a combination of the detection of the disease-specific polymorphic form, and the detection of the disease-specific SAg expression.
The invention also encompasses pro-viral specific detection of retroviral DNA, and simultaneous detection of both expressed retroviral m-RNA and proviral DNA. Specific proviral DNA detection can be used on healthy biological samples to confirm the endogenous nature of the retrovirus. the assay detecting both retroviral mRNA and proviral DNA can be used as an internal standard.
Multiple Sclerosis may also be diagnosed according to the invention by specifically detecting SAg protein expressed by the retrovirus. Preferably, the expressed protein is detected in the biological sample, such as blood or plasma, using antibodies, particularly monoclonal antibodies, specific for the said protein. A Western-like procedure is particularly preferred, but other antibody-based recognition assays may be used.
According to another embodiment of the invention, the autoimmune disease is diagnosed by detecting in a biological sample, antibodies specific for the SAg protein expressed by the MS-associated retrovirus.
Detection of antibodies specific for these proteins is normally carried out by use of the corresponding retroviral protein or fragments thereof having at least 6 amino-acids, preferably at least 10, for example 6-25 amino acids. The proteins are usually Env or fragments thereof and usually have superantigen activity. The retroviral proteins used in the detection of the specific antibodies may be recombinant proteins obtained by introducing viral DNA encoding the appropriate part of the retrovirus into eukaryotic cell and the conditions allowing the DNA to be expressed and recovering the said protein.
In the context of the present invention, the terms “antibodies specific for retroviral proteins” signifies that the antibodies show no significant cross reaction with any other proteins likely to occur in the biological sample. Generally, such antibodies specifically bind to an epitope which occurs exclusively on the retroviral protein in question. The antibodies may recognize the retroviral protein having HERV-W SAg activity as presented by the M.H.C class II molecule.
Detection of specific antibodies may be carried out using conventional techniques such as sandwich assays, etc. Western blotting or other antibody-based recognition system may be used.
According a further embodiment of the invention, the autoimmune disease is diagnosed by detecting, in a biological sample, HERV-W SAg activity specifically associated with the autoimmune disease, for example Vβ6.7 and/or Vβ 17 and/or Vβ21.3 specific proliferation. This is done by carrying out a functional assay in which a biological fluid sample containing MHC class II+ cells, for example Antigen Presenting Cells (APC) such as dendritic cells is contacted with cells bearing one or more variable β-T-receptor chains and detecting preferential proliferation of the Vβ6.7 and/or Vβ 17 and/or Vβ21.3 subset characteristic of HERV-W associated disease. This method of diagnosis may be combined with one or more of the other methods described above to maximize specificity.
The biological sample according to this variant of the invention is typically blood and necessarily contains MHC class II+ cells such as B-lymphocytes, monocytes,macrophages or dendritic cells which have the capacity to bind the superantigen and enable it to elicit its superantigen activity. MHC class II content of the biological sample may be boosted by addition of agents such as IFN-gamma.
The biological fluid sample is contacted with cells bearing the Vβ-T receptors belonging to a variety of different families or subsets in order to detect specific Vβ6.7 subset stimulation by the putative SAg, for example V-β2, 3, 5, 6.7, 7, 8, 9, 11, 12, 13, 17, 21, 22, 23. Within any one V-β family it is advantageous to use V-β chains having junctional diversity in order to confirm superantigen activity rather than nominal antigen activity.
The cells bearing the V-β receptor chains may be either an unselected population of T-cells or T-cell hybridoma. If unselected T-cells are used, the diagnostic process is normally carried out in the following manner: the biological sample containing MHC Class II+ cells is contacted with the T-cells for approximately 3 days. A growth factor such as Interleukin 2 (IL-2) which selectively amplifies activated T-cells is then added. Enrichment of a particular V-β family or families is measured using monoclonal antibodies against the TCR-β-chain. Only amplified cells are thus detected. The monoclonal antibodies are generally conjugated with a detectable marker such as a fluorochrome. The assay can be made T-cell specific by use of a second antibody, anti CD3, specifically recognizing the CD3-receptor.
T-cell hybridoma bearing defined T-cell receptor may also be used in the functional or cell-based assay for SAg activity. An example of commercially available cells of this type are given in B. Fleischer et al. Infect. Immun. 64, 987-994, 1996. Such cell-lines are available from Immunotech, Marseille, France. According to this variant, activation of a particular family of V-β hybridoma leads to release of IL-2. IL2 release is therefore measured as read-out using conventional techniques.
The invention also relates to antibodies capable of specifically recognizing a protein according to the invention. These antibodies are preferably monoclonal. Preferred antibodies are those which specifically recognize a retroviral protein having HERV-W SAg activity and which have the capacity to block HERV-W SAg activity, i.e. block Vβ6.7 and/or Vβ 17 and/or Vβ21.3 specific proliferation. The capacity of the antibody to block this SAg activity may be tested by introducing the antibody under test into an assay system comprising:
The steps described below involve the use of Sag-expressing transfectant cells such as those described in the examples, to inhibit the effect of Sag in vitro and in vivo.
Mabs directed against the HERV-W SAg protein (or portion of it) are generated by standard procedures used to generate antibodies against cell surface antigens. Mice are immunized with mouse cells expressing both Sag and MHC class II (such as a Sag-transfected mouse B cell line described in the examples below). After fusion with hybridoma cell lines, supernatants are screened for the presence of anti-Sag antibodies on microtiter plates for reactivity to Sag transfectants cells, with non-transfected cells as negative controls. Only Mabs with reactivity specific for Sag expressing cells are selected.
All such Mabs, either as culture supernatants or as ascites fluid, are then tested for their ability to block the Sag activity, as assayed by the T cell assay in the presence of Sag-expressing human MHC class II positive transfectants. A preferred version of this assay makes use of Vβ-specific hybridomas as T cell targets for read out. Controls are blocking of the same assay by anti-HLA-DR Mabs, which is known to inhibit the Sag effect on T cell activation. Mabs capable of efficiently blocking the Vβ-specific Sag effect, when tested at several dilutions, are selected as anti-Sag blocking Mabs.
Sufficient numbers of anti-Sag Mabs are screened in the functional assay to identify anti-Sag Mabs with optimal Sag blocking activity, in terms of T cell activation. Selected Sag blocking Mabs are then converted into their <<humanized>> counterpart by standard CDR grafting methodology. A humanized anti-Sag blocking Mab, directed against the HERV-W Sag, can then be tested clinically in patients.
The invention also relates to cells transfected with and expressing human endogenous retrovirus protein or peptide having HERV-SAG SAg activity. The cells may be preferably human cells other than the naturally occurring cells from auto-immune patients and may also include other type of eukaryotic cells such as monkey, mouse or other higher eukaryotes. The cells may be established cell-lines and are preferably MHC class II+, or MHC II+-inducible, such as β-lymphocytes and monocytes. Non-human higher eukaryotic cell-lines (e.g. mouse) stably transfected with the HERV-W Sags of the invention (as exemplified in the Examples below) have been found to specifically stimulate in vitro human Vβ6.7-T cells.
According to a particularly preferred embodiment, the cells of the invention are cells transfected with a chimeric gene encoding the HERV-W SAg as described above. Again, these cells are usually MHC Class II+ or MHC Class II-inducible, and have the capacity to exhibit SAg activity, specific for Vβ6.7 and/or Vβ17 and/or V21.3-TCR chains.
The invention also relates to a transgenic animal model for HERV-W-associated disease such as MS. The transgenic animal is made according to conventional techniques and includes in its genome, nucleic acid encoding the HERV-W Sags of the invention.
A further important aspect of the invention relates to the identification of substances capable of blocking or inhibiting HERV-W SAg activity. These substances are used in prophylactic and therapeutic treatment of HERV-W associated disorders such as MS.
The invention thus concerns methods for treating or preventing HERV-W associated disorders such as MS, by administering effective amounts of substances capable of blocking HERV-W Sag activity. The substances may be antibodies, proteins, peptides, derivatives of the HERV, derivatives of the Sag or small chemical molecules. The invention also relates to pharmaceutical compositions comprising these substances in association with physiological acceptable carriers, and to methods for the preparation of medicaments for use in therapy or prevention of autoimmune disease using these substances.
Further, this aspect of the invention includes a process for identifying substances capable of blocking or inhibiting HERV-W SAg activity of, comprising introducing the substance under test into an assay system comprising:
The cells bearing the β-T cell receptors and the MHC Class II+ cells may be those described earlier. Read-out is IL-2 release.
The substances tested for inhibition or blockage of Sag activity in such screening procedures may be proteins, peptides, antibodies, small molecules, synthetic or naturally occurring, derivatives of the retroviruses themselves, etc. . . . Small molecules may be tested in large amounts using combinatorial chemistry libraries.
The screening procedure may include an additional preliminary step for selecting substances capable of binding to retroviral protein having HERV-W SAg activity. This additional screening step comprises contacting the substances under test, optionally labelled with detectable marker with the retroviral protein having SAg activity and detecting binding.
The HERV-W Sags of the invention or a portion thereof may be used for the identification of low molecular weight inhibitor molecules as drug candidates.
The rationale is that because HERV encoded Sags are the product of ancient infectious agents, they are not indispensable to humans and can thus be inhibited without adverse side effects.
Inhibitors of HERV-W Sag, as potential drug candidates, are preferably identified by a two step process:
In the first step, compatible with large scale, high throughput, screening of collections (<<libraries>>) of small molecular weight molecules, the recombinant HERV-W Sag protein (as defined in Claims 1 to 5) is used in a screening assay for molecules capable of simply binding to the HERV-W Sag protein (=<<ligands>>). Such high throughput screening assays are routinely performed by companies such as Novalon Inc or Scriptgen Inc, and are based either on competition for binding of peptides to the target protein or on changes in protein conformation induced by binding of a ligand to the target protein. Such primary high throughput screening for high affinity ligands capable of binding to a target recombinant protein are available commercially. This screening method requires that the HERV-W SAg protein, be available.
In the second step, any low molecular weight molecule identified as described above as capable of binding to the Sag protein, is tested in the functional Sag assay consisting of human MHC class II positive Sag transfectants and responding Vβ-specific T cells (preferably hybridomas), as described herein. Positive control for Sag inhibition is an anti-HLA-DR Mab, known to inhibit the Sag effect. All candidate molecules are thus tested, at different concentrations, for a quantitative assessment their anti-Sag inhibitory efficacy.
Compounds exhibiting anti HERV-W-Sag inhibitory effects are then tested for obvious toxicity and pharmacokinetics assays, in order to determine if they represent valuable drug candidates.
Once a substance or a composition of substances has been identified which is capable of blocking or inhibiting SAg activity, its mode of action may be identified particularly its capacity to block transcription or translation of SAg encoding sequences. This capacity can be tested by carrying out a process comprising the following steps:
The antibodies used in such a detection process are of the type described earlier.
The invention also relates to a kit for screening substances capable of blocking HERV-W SAg activity of an endogenous retrovirus associated with an autoimmune disease, or of blocking transcription or translation of the retroviral SAg protein. The kit comprises:
According to a further important aspect of the invention, there is provided a protein or peptide derived from an autoimmune related retroviral SAg as previously defined wherein the protein is modified so as to be essentially devoid of SAg activity, thereby no longer being capable of significantly activating auto-reactive T-cells. Such modified proteins are however capable of generating an immune response against SAg, the immune response involving either antibodies and/or T-cells responses. The immunogenic properties of the modified proteins are thus conserved with respect with the authentic SAg.
Such modified immunogenic proteins may be obtained by a number of conventional treatments of the SAg protein, for example by denaturation, by truncation or by mutation involving deletion, insertion or replacement of amino acids. Modified SAg proteins being essentially devoid of SAg activity but capable of generating an immune response against SAg include the truncations of the SAg protein, either at the amino or carboxyterminal, and may involve truncations of about 5-30 amino acids at either terminal.
These proteins are used in the framework of the invention as vaccines, both prophylactic and therapeutic, against HERV-W associated disorders such as MS. The vaccines of the invention comprise an immunogenically effective amount of the immunogenic protein in association with a pharmaceutically acceptable carried and optionally an adjuvant. The use of these vaccine compositions is particularly advantageous in association with the early diagnosis of MS using the method of the invention. The invention also includes the use of the immunogenic proteins in the preparation of a medicament for prophylactic or therapeutic vaccination against MS.
The rationale behind this prospective immunization technique is that because HERV encoded Sags are the product of ancient infectious agents, they are not indispensable to humans and can thus be inhibited without adverse side effects.
Identification of suitable anti-sag vaccine proteins or peptides can be made in the following way. Modified forms of the original active HERV-W Sag protein, including truncated or mutated forms, or even specific peptides derived from the Sag protein, are first tested in the functional Sag assays described above to confirm that they have lost all Sag activity (in terms of T cell activation). These modified forms of Sag are then used to immunize mice (or humans) by standard procedures and with appropriate adjuvants. Extent and efficacy of immunization is measured, including circulating anti-Sag antibodies. In a preferred example, eliciting a B cell immune response, by selecting B cell epitopes from the Sag protein as immunogen, is deliberately aimed at.
Successfully immunized animals are then tested for the effect of Sag in vivo by a standard assay, namely the injection of MHC class II positive Sag transfectants (such as the transfectants described in the examples below), known to induce in vivo a Vβ-specific T cell activation. Successful immunization against a Sag protein is expected to result in a reduction or in a block of the in vivo Sag-induced T cell activation and proliferation in effectively immunized individuals. This procedure is referred to as anti-Sag vaccination.
The vaccines of the invention can be prepared as injectables, e.g. liquid solutions or suspensions. Solid forms for solution in, or suspension in, a liquid prior to injection also can be prepared. Optionally, the preparation also can be emulsified. The active antigenic ingredient or ingredients can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Examples of suitable excipients are water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vaccine can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants such as aluminium hydroxide or muramyl dipeptide or variations thereof. In the case of peptides, coupling to larger molecules (e.g. KLH or tetanus toxoid) sometimes enhances immunogenicity. The vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration includes suppositories and, in some cases, oral formulations.
The vaccines of the invention also include nucleic acid vaccines comprising nucleic acid molecules encoding the human retroviral Sag or modified forms of the SAg known to be immunogenic but no longer active as SAgs. The nucleic acid vaccines, particularly DNA vaccines, are usually administered in association with a pharmaceutically acceptable carrier as an intra-muscular injection.
The invention also relates to use of substances inhibiting either the retroviral function or the SAg function of the associated retroviruses, or Sag synthesis, in therapy for HERV-W associated disorders such as MS. These substances may be identified by the screening procedures described herein.
The invention further relates to methods for treatment or prevention of MS comprising administering an effective amount of a substance capable of inhibiting retroviral function or a substance capable of inhibiting SAg activity or synthesis.
Different aspects of the invention are illustrated in the figures:
legend: C=pCi (expression vector alone); W=pCi74 (expression vector containing pCl-HERV W-ENV); TT=Tetanus Toxoid; SEB=Staphylococcal enterotoxin B; open bars show 3H-thymidine incorporation; dark bars show IL-2 release.
1. Molecular Cloning
1.1 HERV Envelope Constructs
The molecular species containing the envelope coding sequence (HERV-W ENV, also designated HERV-7q ENV) has been described (Alliel et al., 1998).
An XbaI-NotI fragment from this species was subcloned into the NheI-NotI linearized expression vector pCI-neo (Promega) to give pCI-74. 3 individual molecular clones from pCI-74 were sequenced and selected for further analysis.
Generation of the minimal coding sequences for the complete envelope, SU-TM, and for the two proteolytical subunits, SU and TM respectively.
1.2 Construction of Bicistronic Expression Vectors:
Expression cassettes were generated by PCR and sequenced on both strands.
The bicistronic expression vectors were constructed based on pcDL-SRα296: SEQ ID NOs: 14-15 respectively
pcDL: a fragment containing the IRES-EYFP cassette was PCR amplified from pIRES-EYEP (Clontech) with the oligonucleotides 5′ ATT AAT ATC TCG AGA CTA CTG ATC ACG CGT CGA CTC TAG GGC GGC CAA TT 3′ and 5′ CGG GCC TCG AGT TAA TTA ATT ACT TGT ACA GCT CGT CC 3′. Subsequently, the fragment was digested with XhoI and subcloned into pcDL-SRα296, from which the 16S splice junction and the MCS had been previously removed.
Primer sequences used to clone the HERV-W envelope gene into the pCDL vectors (bicistronic expression cassettes): The vector pCi containing the complete HERV-W envelope gene, which was previously employed for SAg assays with monocistronic expression cassettes, was used as template for PCR:
For the construction of the HA tag SEQ ID NOs: 18-19 respectively pBS-SK-3xHA: 5 μg of each of the following oligonucleotides was resuspended in 100 μl of Tris pH 8.0. 5′ CTA GAG CCA CCA TGG TCG ACG GCT ACC CAT ACG ATG TTC CAG ATT ACG CTG GAT ATC CCT ATG ACG TGC CCG ACT ATG CCG GTT ACC CGT ACG ATG TCC CGG ACT ACG CCG GGC CGC GGT GAT TGA TTG AGC 3′; 5′ GGC CGC TCA ATC AAT CAC CGC GGC CCG GCG TAG TCC GGG ACA TCG TAC GGG TAA CCG GCA TAG TCG GGC ACG TCA TAG GGA TAT CCA GCG TAA TCT GGA ACA TCG TAT GGG TAG CCG TCG ACC ATG GTG GCT 3′. The oligonucleotides were denatured for 5′, annealed and subcloned into pBS-SK-.
2. Cell Lines and Cells
Cell lines were obtained from ATCC: the human B lymphoblast cell line TK6, CRL-8015 and the mouse lymphoma cell line A20 (gentic null background for HERVs), TIB-208. Peripheral blood lymphocytes were generated from blood samples of healthy donors obtained from the blood bank in Geneva by Ficoll Hypaque gradient centrifugation.
HtTA 4: HELA cells stably transfected with the tetoperator-CIITA construct have been previously described (Otten et al., (1998) Eur. J. Immunol. 28, 473-478.)
Transfection: Bulk transfectants of TK6 and A20 cells were generated by electroporation. Cells were split 24 h before transfection and then resuspended at 10×106 cells in 250 μl RPMI with 20 μl (1 μg/μl) linearized plasmid in TE pH 8.0.
Cotransfections: Linearized plasmids encoding either a fusion protein of the hygromycin resistance gene with EGFP or alternative resistance genes, such as blasticidin (BSD, Invitrogen), were cotransfected with the expression vector PBSK74SU-TM, at a molar ratio of 1:10 as compared to the expression vector.
Electroporation was performed at 960 mF, 300 V and infinite resistance, yielding time constants between 60-90 msec. Starting 24 h after transfection, cells were selected for resistance to G418 (50-400 μg/ml) or BSD (1-10 μg/ml) present on the cotransfected plasmid.
Transfection of HtTA4 cells with bicistronic cassettes was carried out with the FUGENE 6 transfection reagent (Roche). Briefly, 100000 cells per well were plated the day before transfection in 6 well plates. 1 μg plasmid DNA was used with 3 μl FUGENE 6 to transfect a 35 mm Patri dish. The percentage of cells transfected was analysed by flow cytometry for GFP expression. For stable transfection of the HtHA4, 1 μg linearized plasmid DNA and 100 ng of linearized blasticidin resistance plasmid were used.
Selection of clones: Bulk transfectants were maintained under continuous drug selection. Individual clones were generated by limiting dilution and selected for by function. Alternatively, bulk cultures of transfectants obtained with bicistronic expression vectors were selected for by FACS sorting for EGFP expression under limiting dilution conditions.
3. Functional Assays
It can be concluded from the above that the HERV-W (HERV-7q) superantigen expressed by TK6-11S gives rise to Vβ6.7 and Vβ17 specific enrichment.
v) Functional Assays with Bicistronic Expression Cassettes
A bicistronic expression cassette was generated with IRES driven expression of enhanced green fluorescent protein (EGFP) as indirect marker. First, this serves the purpose of facilitating the structure—function analysis of the SAg. Second, it allows the direct comparison of expression levels of individual constructs.
The following constructs (illustrated schematically in
i) Confirmation of Specific Vβ-6.7, Vβ-17 and, to a Lesser Degree, Vβ-21.3 Expansion Using Bicistronic Constructs:
ii) Vβ Specific T Cell Responses Vary Between Individuals.
iii) Localization of the SAg Activity in the C-Terminal Part of ENV:
iv ) Western Blot Analysis:
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10133036 | Apr 2002 | US |
| Child | 11811964 | Jun 2007 | US |
| Parent | PCT/EP00/10659 | Oct 2000 | US |
| Child | 10133036 | Apr 2002 | US |
