Patent Application
20240301040
The present invention relates to a newly identified group of patients previously diagnosed with a psychotic disease, in particular Schizophrenia or Bipolar disorder, in whom a high level of a pro-inflammatory cytokine in a body fluid sample, makes them eligible to a targeted treatment against HERV-W ENV.
Psychotic disorders are genetically- and environmentally-mediated mental illnesses that involve impairments in perception, mood, and social interactions (1). Until now, diagnoses of psychotic disorders are clinically-defined and are only treated by chemical compounds (26, 27, 28).
Although the pathogenic mechanisms underpinning these disorders are still unclear, it recently emerged that immune-related genes and mobile genetic elements constitute major actors in the etiopathogenetic chain (1-3). Mobile genetic elements, including human endogenous retroviruses (HERVs), are remnants of infections that took place several million years ago and embody around 8% of the human genome (4). Beside their initial classification as “junk” DNA, endogenous retroviruses capacity to control gene regulatory networks during human brain evolution and development (5-8), and their growing association to major neurological and psychiatric disorders (9, 10) provide new conceptual framework to decrypt the interplay between immunological, genetic, and brain systems. HERVs are normally silenced by cell machineries, but they can be activated following immune challenges and under pathological conditions (11). For instance, it has been observed that patients diagnosed with schizophrenia and bipolar disorder have altered HERV gene expression, with variable transcriptional levels and protein detection (1, 12, 13).
Applications WO2010/0033977A1 and US2019/263895A1, which clearly target the multiple sclerosis treatment, disclose in a very generic way that psychotic diseases such as schizophrenia or bipolar disorder, might be treated with antibodies directed against the envelope protein (ENV) of HERV-W (also noted anti-HERV-W ENV), since expression of HERV-W ENV has been observed in psychotic diseases as well as in multiple sclerosis.
However, whether the proteins expressed by HERV genes such as the envelope protein play an instrumental role in psychotic disorders and, most importantly, in which patients, still remains as unanswered questions with missing knowledge in the domain.
This precludes consistent indication for a targeted treatment in relevant patients when nosological definitions of psychiatric diseases are potentially resulting in heterogeneous populations without clearly identified etiology and with limited understanding of corresponding mechanisms of pathogenesis (14-16). To date, this absence of knowledge only allows targeting common downstream consequences that may modulate patients' symptomatology.
For example, alterations of the N-Methyl-D-aspartate receptors (NMDAR) signaling have been associated with behavioral deficits in cognitive and sensorimotor gating test (33), which are hallmarks of psychosis models in rodents. Thus, most psychomimetic drugs are antagonists of the N-Methyl-D-aspartate receptors (NMDAR) such as phencyclidine (1-phenylcyclohexylpiperidin), also called PCP.
Exploring the potential clinical and/or biological correlates of HERV-W ENV detection in patients diagnosed with Schizophrenia or with Bipolar disorder, the inventors evidenced different clusters of patients, across presently existing diagnoses. They identified a subgroup of patients with a significantly elevated level for a cytokine, in particular in the serum, and with detectable HERV-W ENV antigen and a subgroup without detectable HERV-W ENV antigen nor significant cytokine levels. These subgroups appear to represent biomarker-defined subgroups, only one of which is consistent with HERV-W ENV pathogenicity resulting in different clinical outcomes. Indeed, various clinical outcomes may occur with other pathogenic agents, e.g., Acquired Immune Deficiency Syndrome (AIDS) multifaceted diseases caused by HIV-1 retrovirus (17).
Thus, for the first time, the inventors discovered that unexpectedly among the patients diagnosed with a psychotic diseases, such as schizophrenia or bipolar disorder, the patients who really expressed HERV-W ENV did not include all the patients with such diagnoses but only a particular subgroup. They further discovered that this subgroup of patients had a significantly elevated cytokine level in the serum compared to the other patients with the same diagnosis.
This unexpected discovery is a step forward with respect to the prior art. Indeed, it now appears that not all the patients diagnosed with a psychotic disease, such as schizophrenia or bipolar disorder, can be treated with an anti-HERV-W ENV antibody. The inventors have highlighted that only 30 to 50% of the patients diagnosed with schizophrenia or bipolar disorder, express HERV-W ENV (see
Their discovery allows to select the eligible patients to an anti-HERV-W ENV antibody treatment, among all the patients diagnosed with a psychotic disease, such as schizophrenia or bipolar disorder. The knowledge of this new indication has rendered possible the research of a diagnostic-driven therapeutic treatment for this subgroup of patients. This represents a breakthrough in therapeutics for psychoses, for prior art made it impossible to perform regulatory clinical trials showing efficacy of products targeting HERV proteins, as of others, since about 50% of patients with a diagnosis of schizophrenia and about 70% for bipolar disorder, do not involve HERV-W ENV in the pathogenesis of their disease. This would render any statistical analysis of treatment efficacy not significant, simply because of this unseen and unknown majority of irrelevant patients included on the basis of existing diagnoses. Thus, no specific therapeutic product could be appropriately selected and developed, even less validated in clinics.
Unexpectedly, the inventors have discovered that the in vivo use of an antibody targeting HERV-W ENV could efficiently treat the neurobiological dysfunctions and psychotic symptoms in this subgroup of individuals. They have shown the efficacy of an anti-HERV-W ENV antibody to treat this subgroup of psychotic patients expressing HERV-W ENV and having a high level of a cytokine in a body fluid sample, in particular in the serum.
In addition, the Inventors have developed an anti-HERV-W ENV antibody having advantages compared to the already known anti-HERV-W ENV antibodies. The developed antibody recognizes a conformational epitope and is able to induce in neurons, the relocation of the NMDAR, in particular the relocation of the NMDA receptor containing the GluN2B subunit, into the synapses.
This diagnostic-driven therapy combined with the properties of this novel anti-HERV-W ENV antibody, make it an effective treatment of the subgroup of patients.
In this context, the inventors have highlighted that HERV-W ENV altered NMDAR-mediated synaptic transmission in hippocampal networks, which appeared to be mediated via microglia and the release of pro-inflammatory cytokines such as IL-1β or IL-6. This involves innate immunity and pro-inflammatory cytokines in an HERV-W ENV-triggered specific pathogenic pathway. The glutamatergic GluN2B-containing NMDA receptor is normally present at the synapses, but an exposure to HERV-W ENV was shown to cause a surprising dispersal of GluN2B away from the synapses. Because of its biochemical nature, i.e. a protein macromolecule, HERV-W ENV was not expected to display molecular interactions leading to similar effects than those mediated by the small chemical compounds used to interfere with receptors of neuronal neurotransmitters but, surprisingly, the inventors have demonstrated its direct involvement in the psychotic phenotype. The delocalization of GluN2B-containing NMDA receptor renders them non-functional at synapse. The effect of the HERV-W ENV protein in animals reproduced features of psychotic disorders such as schizophrenia or bipolar disorder, and was accompanied by secretions of pro-inflammatory cytokines.
The Inventors showed that HERV-W ENV, detected in a biologically-defined relevant group of psychotic patients, is capable of generating a dysfunction in the NMDAR organization in conjunction with an induction of cytokine production, which affects long-term plasticity within glutamatergic synapses and produces behavioral deficits associated with psychosis.
Surprisingly, the use of an anti-HERV-W ENV antibody prevents the displacement from synapses and/or induces the relocation into synapses of GluN2B-containing NMDA glutamatergic receptor, and thus provides a beneficial effect on the pathogenic dysfunction of NMDA receptor synaptic neurotransmission. The inventors have therefore demonstrated that the use of an anti-HERV-W ENV antibody (also noted here anti-ENV or anti-ENV antibody) could specifically and efficiently reverse the pathogenic effects at the level of the GluN2B-containing NMDA receptor, when impacted through its biodistribution over neuronal synapses by HERV-W ENV.
In prior art, no identified nosological sub-groups could provide a defined indication for treating a relevant group of patients, i.e., schizophrenic or bipolar patients in which HERV-W ENV drives pathogenesis and not those from other sub-group(s) without HERV-W ENV and pro-inflammatory cytokine involvement. Thus, the efficiency of a treatment of psychotic disease with an anti-HERV-W ENV antibody along with a newly defined indication was totally unexpected, as there was no indication that this protein needed to be targeted in a biologically-defined sub-population of patients with diagnosis of psychotic disease and that the use of such antibody could thus target the delocalization of NMDAR, as shown to be mediated by HERV-W ENV-induced cytokines.
Therefore, the invention relates to an anti-HERV-W ENV antibody or such a pharmaceutical composition thereof, combined with diagnostic biomarkers comprising HERV-W ENV and cytokines detection in body fluids, for use in the treatment of a subgroup of patients diagnosed with a psychotic disease and characterized with a high level of a cytokine in a body fluid sample, in particular in the serum, said cytokine being in particular a pro-inflammatory cytokine, more particularly IL-6, IL-1β and/or TNF-α.
In a particular aspect, the invention relates to a diagnostic method to identify if a patient diagnosed with a psychotic disease, belongs to a subgroup of patients suffering from psychotic disease as defined in the present disclosure, comprising:
In another particular aspect, the invention relates to a follow-up method of the efficacy of a treatment of a group of a patient suffering from a psychotic disease and having a high level of a cytokine, comprising the quantification of said cytokine and/or the detection of HERV-W ENV in a patient biological sample.
In another particular aspect, the invention relates to an anti-HERV-W ENV antibody which induces in neurons, the relocation of the GluN2B-containing NMDA receptor into the synapses, and in particular which binds to the conformational epitope of HERV-W ENV defined by the two distant linear sequences depicted in SEQ ID NO: 10 and in SEQ ID NO: 11, and more particularly which comprises each of the complementary-determining regions (CDRs) set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.
In a still another particular aspect, the invention relates to a pharmaceutical composition comprising such an anti-HERV-W ENV antibody and a pharmaceutically acceptable excipient.
As used herein, the terms “human endogenous retrovirus” and “HERV”, refer to the human endogenous retroviruses that comprise the virus belonging to the type-W endogenous retrovirus family, usually named “HERV-W”.
“HERV-W” is a family of human endogenous retroviruses that was unravelled in human genome from the initial discovery of “Multiple Sclerosis associated Retrovirus”, MSRV, a human retrovirus first isolated from patients with multiple sclerosis. Therefore, when studies mention “LM7” (first isolate described from MS), “MS-retrovirus”, “MSRV”, “Syncytin”, “HERV-W 7q”, “ERVW-E1”, “ERVW-E2”, “HERV-W copies from X chromosome” or “HERV-W”, they all designate HERV-W elements.
As used herein, the term “MSRV” refers to a specific endogenous retrovirus which is a member of the HERV-W family. In the context of the present invention, the expressions “HERV-W” and “MSRV” both designate HERV-W elements. Specifically, the expressions “HERV-W Envelope protein”, “HERV-W ENV”, “MSRV-ENV” and “ENV” altogether refer to the same envelope proteins. Typically, possible few variations in amino acid sequence do not prevent the binding of specific anti-ENV antibodies for therapeutic use in a HERV-W ENV associated psychotic disease.
As used herein, the term “treating” or “treatment”, as used herein, means reversing, alleviating, inhibiting the progress of one or more symptoms of the disorder or condition to which such term applies.
As used herein, the term “antibody” refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen. As such, the term “antibody” should be understood broadly and encompasses not only immunoglobulin molecules, but also antibody fragments, as well as derivatives of antibodies. In a particular embodiment, the antibody is an antibody directed against HERV-W ENV and inducing in neurons, the relocation of the GluN2B-containing NMDA receptor into the synapses. In a particular embodiment, the antibody comprises all the 6 CDRs as depicted in SEQ ID NO: 1 to 6.
As used herein, the expression “fragment of antibody” refers to a portion of such an antibody that mimics the hypervariable region, such as a CDR (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3). The fragments of antibody according to the present invention retain the binding affinity and specificity of said antibody. Such fragments are functional equivalents of said antibody and they bind substantially to the same epitope as said antibody. Examples of fragments of antibody include but are not limited to heavy chain, light chain, VL, VH, Fv, Fab, Fab′, F(ab)2, and F(ab′)2.
As used herein, the expression “derivative of antibody” refers to a fragment of the antibody of the invention, preferably including the six CDRs of said antibody, fused to at least one sequence different from the natural sequence (e.g. a linker sequence of another species . . . ), said derivative having binding affinity and specificity to HERV-W ENV comparable to that of the antibody of the invention. The derivatives according to the present invention retain the binding affinity and specificity of said antibody. Such derivatives are functional equivalents of said antibody and they bind at substantially the same epitope as said antibody. Examples of derivatives of antibody include, but are not limited to scFv, (scFv)2 and diabodies.
In natural antibodies, two heavy chains (HC) are linked to each other by disulfide bonds and each heavy chain is linked to a light chain (LC) by a disulfide bond. There are two types of light chain, lambda (l) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains.
Typically, the light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CH1, CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine the binding site specific to the antigenic epitope. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody binding site and the antigenic epitope. Antibody binding sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Complementarity Determining Regions or CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3, respectively. An antigen-binding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. Framework Regions (FRs) refer to amino acid sequences interposed between CDRs.
As used herein, the term “chimeric antibody” refers to an antibody which comprises a VH domain and a VL domain of an antibody from any species, preferably mouse, and a CH domain and a CL domain of a human antibody.
According to the invention, the term “humanized antibody” refers to an antibody having variable region framework and constant regions from a human antibody but retains the CDRs of an antibody from any species, preferably mouse.
The term “Fab” denotes an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, in which about a half of the N-terminal side of H chain and the entire L chain, among fragments obtained by treating IgG with a protease, papain, are bound together through a disulfide bond.
As used herein, the term “F(ab′)2” refers to an antibody fragment having a molecular weight of about 100,000 and antigen binding activity, which is slightly larger than the Fab bound via a disulfide bond of the hinge region, among fragments obtained by treating IgG with a protease, pepsin.
As used herein, the term “Fab′” refers to an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, which is obtained by cutting a disulfide bond of the hinge region of the F(ab′)2.
The expressions “A single chain Fv” or “scFv”” refer to a polypeptide which is a covalently linked VH::VL heterodimer, and usually expressed from a gene fusion including VH and VL encoding genes linked by a peptide-encoding linker. “dsFv” is a VH::VL heterodimer stabilised by a disulfide bond. Divalent and multivalent antibody fragments can form either spontaneously by association of monovalent scFvs, or can be generated by coupling monovalent scFvs by a peptide linker, such as divalent sc(Fv)2.
The term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
As used herein, the expression “antibody of the invention” refers to an antibody directed against, i.e. that specifically binds to HERV-W Envelope protein (HERV-W ENV), preferably against HERV-W ENV protein of the type-W human endogenous retrovirus family (HERV-W), more preferably against the envelope protein of MSRV, more preferably against the extracellular domain the envelope protein of MSRV. In one embodiment, the antibody according to the invention is an antibody directed against HERV-W ENV and inducing in neurons, the relocation of the GluN2B-containing NMDA receptor, into the synapses. The antibody of the invention typically binds to the conformational epitope.
In particular, the antibody of the invention binds to the conformational epitope defined by the two distant linear sequences depicted in SEQ ID NO: 10 and in SEQ ID NO: 11.
Preferably, the antibody of the invention comprises all the 6 CDRs as depicted in SEQ ID NO: 1 to 6.
Within the meaning of the invention the expressions “anti-ENV antibody”, “anti-HERV-W ENV antibody”, “antibody directed against HERV-W ENV” and “antibody targeting HERV-W ENV” are used indifferently and are equivalent.
As used herein, the term “biological sample” as used herein refers to any biological sample obtained for the purpose of evaluation in vitro. In the present invention, the sample or patient sample may comprise any body fluid or disease-specific tissue and lesions. Examples of body fluid include blood, nipple aspirate fluid, urine, saliva, synovial fluid and cerebrospinal fluid (CSF). In particular, a blood sample can be serum or plasma.
The present invention relates to a diagnostic-driven therapeutic treatment of patients diagnosed with a psychotic disease, said patients having been tested for biomarkers showing a high level of a cytokine in a body fluid sample and/or an expression of HERV-W ENV in a body fluid sample, and further treated with an antibody directed against HERV-W envelope protein (anti-HERV-W ENV antibody).
In particular, the present invention relates to an anti-HERV-W ENV antibody for use in the treatment of a group of patients diagnosed with a psychotic disease and having a high level of a cytokine in a body fluid sample, in particular in a blood sample, more particularly in the serum or the plasma.
In particular, the present invention relates to an anti-HERV-W ENV antibody for use in the treatment of a group of patients diagnosed with a psychotic disease and characterized with a high level of a cytokine in a body fluid sample, in particular in a blood sample, more particularly in the serum or the plasma.
In the present disclosure, the expressions “group of patients”, “subgroup of patients” and “cluster of patients” are used indifferently and are equivalent.
A patient suffering from a psychotic disease is a patient with psychotic symptoms or diagnosed with a psychotic disease.
In other words, a patient diagnosed with a psychotic disease is a patient suffering from a psychotic disease with psychotic symptoms and clinically diagnosed by a practitioner.
In particular, said psychotic disease is selected from the group consisting of schizophrenia, bipolar disorder, schizoaffective psychosis and schizophreniform disorder. These pathologies are defined according to the DSM IV or V classification or to an appropriate classification for psychotic diseases (25-28). The diagnosis of psychotic disease, and more particularly of this list of psychotic diseases, is well known from the practitioners. More particularly, said psychotic disease is schizophrenia or bipolar disorder.
In the present disclosure “having a high level of a cytokine” means “has been tested with a high level for a cytokine” or “characterized with a high level of a cytokine”.
A “high level for a cytokine” or “high level of a cytokine” or “elevated level of a cytokine” as used herein means that the level of said cytokine within the group of patients is significantly higher than those of healthy persons. As an example, a high level of a cytokine is a level of a cytokine higher than 0.5 pg/ml for IL-6 in the serum, higher than 0.05 pg/ml for IL-1β in the serum, higher than 1.25 pg/ml for TNF-α in the serum. Of note, the thresholds of cytokines quoted in the present disclosure vary with the kits, antibodies, techniques, protocols, devices and platforms used for their dosages and their sensitivity (proportion of true-positives) and their specificity (proportion of true-negatives). Presented values are only indicative and one should adapt them according to manufacturers' instructions or assess them with a given platform on heathy individuals. In other words, it means that the level of said cytokine in the group of patients is significantly above normal level of said cytokine. This normal value is given by kit suppliers and/or can be determined from a panel of healthy individuals confirmed without any possible starting or ongoing infection or inflammation. A person skilled in the art perfectly knows how to measure and how to determine such a threshold or normal level for a given cytokine according to the different techniques, protocols and devices available on the market.
This definition applies to the high levels of the specific cytokines (such as IL-6, IL-1β and TNF-α) quoted in the present disclosure.
Typically, the level of a cytokine is measured in a body fluid sample, in particular in a blood sample, more particularly in the serum or in the plasma.
In the present disclosure, said cytokine is in particular a pro-inflammatory cytokine. More particularly, said pro-inflammatory cytokine is selected in the group consisting of IL-6, IL-1β and TNF-α.
Thus, in a particular embodiment, the present invention relates to an anti-HERV-W ENV antibody for use in the treatment of a group of patients diagnosed with a psychotic disease and having a high level for a cytokine in a body fluid sample, in particular in a blood sample, more particularly in the serum, said cytokine being in particular a pro-inflammatory cytokine, more particularly IL-6, IL-1β and/or TNF-α.
In a particular embodiment, the present invention relates to an anti-HERV-W ENV antibody for use in the treatment of a group of patients diagnosed with a psychotic disease and characterized with a high level for a cytokine in a body fluid sample, in particular in a blood sample, more particularly in the serum, said cytokine being in particular a pro-inflammatory cytokine, more particularly IL-6, IL-1β and/or TNF-α.
In a particular embodiment, said psychotic disease is Schizophrenia and said cytokine with a high level in a body fluid sample is preferably IL-6.
In a particular embodiment, said psychotic disease is Bipolar disorder and said cytokine with a high level in a body fluid sample is preferably IL-1β. In a more particular embodiment, said group of patients diagnosed with Bipolar disorder and having a high level for IL-1β in a body fluid sample, further have a high level of TNF-α in a body fluid sample.
In a particular embodiment, HERV-W ENV has been detected in the patients of said group. Since the subgroup of patients presenting a high level of cytokines corresponds to the subgroup of patients expressing HERV-W ENV, the detection of the HERV-W ENV is not mandatory and may be optional. In particular, the HERV-W ENV is detected in a biological sample. More particularly, the HERV-W ENV is detected in a blood sample, more particularly in the serum or in the plasma. HERV-W ENV is detected with an anti-HERV-W ENV antibody such as an anti-HERV-W ENV antibody according to the present disclosure.
The expression of HERV-W ENV can be characterized by the detection of HERV-W DNA, RNA, antigen or protein, following the below-described conditions.
HERV-W family was discovered after MSRV, a human retrovirus first isolated from patients with multiple Sclerosis (18-20). Identification of the expression of HERV-W ENV is well-known by a person of ordinary skill in the art. Associated diseases or syndromes are defined by the presence in corresponding patients either (i) of specific HERV-W RNA or antigens, preferably detected in body fluids (blood, cerebrospinal fluid, urine . . . ), either (ii) of elevated DNA or RNA copy number in cells or tissues from organs with lesions or dysfunctions, either (iii) of specific MSRV/HERV-W proteins or antigens in cells or tissues involved in the process of the disease or of the clinical syndrome, or (iv) of HERV-W proteins or antigens in body fluids of individuals with the disease or expressing the clinical syndrome (see international application WO2019201908A1—Method for the detection of the soluble hydrophilic oligomeric form of HERV-W envelope protein). Examples of such detection assays with various technical conditions and targets have been described (21-23). All these conditions of detection exclude the detection of the physiological HERV-W copy, Syncytin-1 when expressed in normal and physiological conditions of its expression, i.e. during pregnancy (24).
In particular, in the present disclosure, the expression of HERV-W ENV is carried out by the detection of the protein HERV-W ENV in a sample. For this detection, an anti-HERV-W ENV antibody may be used. More particularly, said anti-HERV-W ENV antibody may be the one described in the present application.
Additionally, it may be a murine antibody comprising:
The above-mentioned light and heavy variable regions are disclosed in Table 1:
In such an embodiment, the psychotic disease may be also called “HERV-W ENV associated psychotic disease”. Among the patients suffering from a psychotic disease, said group of patients is then a group of patients suffering from a psychotic disease, having a high level for a cytokine in a body fluid sample, in particular IL-6, IL-1β and/or TNF-α, and wherein HERV-W ENV has been detected, for example by detecting HERV-W DNA, RNA, antigen or protein, following the above-described conditions. In a particular embodiment, the present invention applies to the group of patients suffering from a psychotic disease, in whom the detection of the HERV-W ENV is characterized by the detection of HERV-W DNA, RNA, antigen or protein and/or having a high level for a cytokine in a body fluid sample.
Thus, in a particular embodiment, the present invention relates to an anti-HERV-W ENV antibody for use in the treatment of a group of patients diagnosed with a psychotic disease and having a high level of a cytokine in a body fluid sample, in particular in a blood sample, more particularly in the serum or the plasma, and/or having an expression of HERV-W ENV detected in a body fluid sample, in particular in a blood sample, more particularly in the serum or the plasma.
In a particular embodiment, the present invention relates to an anti-HERV-W ENV antibody for use in the treatment of a group of patients diagnosed with a psychotic disease and characterized with a high level of a cytokine in a body fluid sample, in particular in a blood sample, more particularly in the serum or the plasma, and/or having an expression of HERV-W ENV detected in a body fluid sample, in particular in a blood sample, more particularly in the serum or the plasma.
In a particular embodiment, the present invention relates to an anti-HERV-W ENV antibody for use in the treatment of a group of patients as above mentioned, wherein said anti-HERV-W ENV antibody induces the relocation of the NMDA receptor in neurons, in particular of the GluN2B-containing NMDA receptor, into the synapses.
This means that said anti-HERV-W ENV antibody is able to reverse the dispersal of NMDAR from neuronal synapses paralleling cytokine production by microglia, in which these neuroreceptors can recover a normal functional activity. This relates particularly to the glutamatergic GluN2B-containing NMDA receptor. In fact, as demonstrated in Example 2, HERV-W ENV causes the loss of functional activity of the NMDAR by inducing its delocalization from the physiologically relevant synaptic area. The particular dispersal of the GluN2B-containing NMDA receptor away from the synapses to the postsynaptic compartment (i.e., in neuronal membrane regions out of the synapses), leads to characteristic symptoms of psychotic disorders.
Typically, the Example 2 teaches how to identify the therapeutic activity of an antibody anti-HERV-W on the relocation of the GluN2B-containing NMDA receptor from the postsynaptic compartment to the synapses, in hippocampal neurons. Animal hippocampi are widely used in neurology, as they constitute an animal cell model for the study of psychotic disorders and as they correspond to an easily accessible brain area.
As neurons in hippocampus are equivalent to neurons found in other brain areas for their global synaptic functions and for their need for synaptic localization of receptors to neurotransmitters, observations made on hippocampal neurons are extrapolated to neurons in other brain areas. Thus, although the brain areas involved in different psychotic pathologies may vary, the present observations made on neurons of the hippocampus are representative of neurons located in other brain areas.
In a particular embodiment, the present invention relates to an anti-HERV-W ENV antibody for use in the treatment of a group of patients as above mentioned, wherein said antibody binds to HERV-W ENV.
In a particular embodiment, the present invention relates to an anti-HERV-W ENV antibody for use in the treatment of a group of patients as above mentioned, wherein said antibody binds to a conformational epitope of HERV-W ENV. More particularly, said conformational epitope is defined by the two distant linear sequences depicted in SEQ ID NO: 10 and in SEQ ID NO: 11.
In a particular embodiment, the present invention relates to an anti-HERV-W ENV antibody for use in the treatment of a group of patients as above mentioned, wherein said antibody comprises each of the 6 CDRs as depicted in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.
In a particular embodiment, the present invention relates to an anti-HERV-W ENV antibody for use in the treatment of a group of patients as above mentioned, wherein said antibody induces in neurons, the relocation of the NMDA receptor, in particular of the NMDA receptor containing the GluN2B subunit, into the synapses, and wherein said antibody comprises each of the 6 CDRs as depicted in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.
In a particular embodiment, the present invention relates to an anti-HERV-W ENV antibody for use in the treatment of a group of patients as above mentioned, wherein said antibody induces in neurons, the relocation of the NMDA receptor, in particular of the NMDA receptor containing the GluN2B subunit, into the synapses, wherein said antibody specifically binds to the conformational epitope defined by the two distant linear sequences depicted in SEQ ID NO: 10 and in SEQ ID NO: 11 and comprises each of the 6 CDRs as depicted in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.
In a particular embodiment, said anti-HERV-W antibody for use in the treatment of a group of patients as above mentioned, is a monoclonal antibody or a humanized monoclonal antibody. More particularly, said antibody is an IgG such as an IgG1 or an IgG4. More particularly, it is a humanized IgG4 monoclonal antibody or an IgG1 monoclonal antibody.
In a particular embodiment said anti-HERV-W antibody for use in the treatment of a group of patients as above mentioned, comprises:
It may also be a fragment or a derivative of an anti-HERV-W antibody described above, selected from the group consisting of a Fv, Fab, F(ab′)2, Fab′, dsFv, scFv, sc(Fv)2, a diabody.
Diagnostic Method to Identify a Subgroup of Patients Diagnosed with a Psychotic Disease
The present application also relates to a diagnostic method to identify if a patient diagnosed with a psychotic disease, belongs to a subgroup of patients suffering from psychotic disease as defined in the present disclosure, comprising:
In particular said cytokine is a pro-inflammatory cytokine, more particularly IL-6, IL-1β and/or TNF-α.
In particular, the psychotic disease is selected from the group consisting of schizophrenia, bipolar disorder, schizoaffective psychosis and schizophreniform disorder.
For example, when the patient is diagnosed with schizophrenia, the level of IL-6 is preferably quantified. For example, when the patient is diagnosed with bipolar disorder, the level of IL-1β is preferably quantified. In case of bipolar disorder, in addition the level of TNF-α may also be quantified.
Typically, the level of cytokine is quantified in a body fluid sample, in particular in a blood sample, more particularly in the serum or in the plasma.
If the patient presents a high level of said cytokine in the body fluid sample, it means that said patient belongs to a subgroup of patients suffering from psychotic disease and characterized by a high level of a cytokine in a body fluid sample, and also characterized by the expression of the HERV-W ENV.
The definition of “high level of a cytokine” given above also applies here.
Examples of methods for detecting HERV-W ENV are given previously in the present disclosure. In particular, the expression of HERV-W ENV is carried out by the detection of the protein HERV-W ENV in a biological sample. For this detection, an anti-HERV-W ENV antibody may be used. More particularly, said anti-HERV-W ENV antibody may be the one described in the present application.
Additionally, it may be a murine antibody comprising:
In the present disclosure, HERV-W ENV is detected in a biological sample, in particular in a body fluid sample such as a blood sample, more particularly in the serum or in the plasma. Since the subgroup of patients presenting a high level of a cytokine corresponds to a subgroup of patients expressing HERV-W ENV, i. e. a subgroup of patients wherein HERV-W ENV is detected, identifying this subgroup of patients suffering from a psychotic disease therefore does not necessitate both quantifying the level of a cytokine and detecting HERV-W ENV. Thus, analysing only one of these parameters is sufficient.
Thus, in a particular embodiment, the present application relates to a diagnostic method to identify if a patient diagnosed with a psychotic disease belongs to a subgroup of patients suffering from psychotic disease and characterized with a high level of a cytokine in a body fluid sample, comprising:
The invention further relates to a murine antibody comprising:
Said murine antibody comprises the CDRs of SEQ ID NOs:1-6.
The invention further relates to a kit of detection of HERV-W ENV in a biological sample, in particular in a body fluid sample such as a blood sample, more particularly in the serum or in the plasma, comprising an anti-HERV-W ENV as described in the present disclosure. In particular, it comprises an anti-HERV-W ENV comprising each of the CDRs set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. More particularly, it comprises an anti-HERV-W ENV comprising:
The kit may also comprise a murine antibody comprising:
In another aspect, the invention also relates to a method for treating a group of patients diagnosed with a psychotic disease, such as schizophrenia or bipolar disorder, and having a high level of a cytokine in a body fluid sample, comprising administering to said patients an effective amount of an anti-HERV-W ENV antibody, in particular the one as described in the present disclosure.
In particular, the invention relates to a method for treating a group of patients diagnosed with a psychotic disease, such as schizophrenia or bipolar disorder, and characterized with:
In another aspect, the invention also relates to the use of an anti-HERV-W ENV antibody, such as the one described in the present disclosure, for the manufacture of a medicament for treating a group of patients diagnosed with a psychotic disease and having a high level of a cytokine in a body fluid sample.
In particular, the invention also relates to the use of an anti-HERV-W ENV antibody, such as the one described in the present disclosure, for the manufacture of a medicament for treating a group of patients diagnosed with a psychotic disease characterized with:
The invention also relates to a method for treating a group of patients diagnosed with a psychotic disease and characterized with:
All the embodiments described above apply to these other aspects of the invention.
In particular, the invention also relates to a method of treatment of a patient diagnosed with a psychotic disease, comprising the steps of:
In particular said cytokine is a pro-inflammatory cytokine, more particularly IL-6, IL-1β and/or TNF-α. In particular, the level of said cytokine is quantified in a blood sample, more particularly in the serum or in the plasma. To quantify the level of the cytokine, one or more specific biomarkers of said cytokine can be used. To detect the expression of HERV-W ENV an anti-HERV-W ENV antibody can be used such as one disclosed in the present application can be used. HERV-W ENV is detected in a biological sample, in particular in a body fluid sample such as a blood sample, more particularly in the serum or in the plasma.
If said step 1 shows a high level of a cytokine in a body fluid sample and/or if said step 2 shows the detection of HERV-W ENV, then the method of the invention comprises in addition a step of providing an antibody as described in the present disclosure to said patient. It should be noted that preferably step 1 is firstly carried out. In this embodiment, when step 1 shows a high level of a cytokine in a body fluid sample, step 2 may be optional.
To determine a “high level of a cytokine”, the definition of “high level of a cytokine” given above also applies here.
A follow-up may then be established using specific biomarkers of the cytokine(s) relevant for the patient or an anti-HERV-W ENV antibody such as one disclosed in the present application.
In a particular embodiment, the invention also relates to a method of treatment of a patient diagnosed with schizophrenia, comprising the steps of:
If said step 1 shows a high level of IL-6 and/or if said step 2 shows the detection of HERV-W ENV, then the method of the invention comprises in addition a step of providing the antibody of the invention to said patient.
In a particular embodiment, the invention also relates to a method of treatment of a patient diagnosed with bipolar disorder, comprising the steps of:
If said step 1 shows a high level of IL-1β and/or if said step 2 shows the detection of HERV-W ENV, and optionally step 3 shows a high level of TNF-α, then the method of the invention comprises in addition a step of providing the antibody of the invention to said patient.
The invention also relates to a method for monitoring the response to a treatment of a patient diagnosed with a psychotic disease and having a high level of a cytokine in a body fluid sample, said method comprising the following steps:
In particular said cytokine is a pro-inflammatory cytokine, more particularly IL-6, IL-1β and/or TNF-α. In particular, the level of cytokine is quantified in a blood sample, more particularly in the serum or in the plasma. To quantify the level of the cytokine, one or more specific biomarkers of said cytokine can be used. To detect the expression of HERV-W ENV, an anti-HERV-W ENV antibody can be used such as the one described in the present application. HERV-W ENV is detected in a biological sample, in particular in a body fluid sample such as a blood sample, more particularly in the serum or in the plasma. For example, if the psychotic disease is schizophrenia, said cytokine is preferably IL-6. For example, if said psychotic disease is bipolar disorder, said cytokine is preferably IL-1β, and optionally in addition preferably TNF-α.
The decrease of the cytokine level and/or the decrease of HERV-W ENV protein in the patient are indicators of the efficacy of the treatment. In addition, a behavioral study and/or clinical rating scales of the patients may be carried out to analyse an improvement of the general status of the patients and thus to confirm the efficacy of the treatment and/or to adapt its dosage to the patient.
Typically, in the present application, the step of detection and/or quantification may be performed according to the routine techniques, well known of the person skilled in the art. Typically, said step comprises contacting a biological sample such as a body fluid sample of the patient with selective reagents such as probes, primers, ligands or antibodies, and thereby detecting the presence of nucleic acids or proteins of interest originally in the sample.
The present invention also relates to an antibody directed against HERV-W ENV and inducing in neurons, the relocation of the NMDAR, in particular of the NMDA receptor containing the GluN2B subunit, into the synapses.
There are two main subtypes of the NMDA receptor (also noted NMDAR) in neurons. The NMDAR which contains the GluN2A subunit, called GluN2A-containing NMDA receptor (GluN2A-NMDAR), and the NMDAR which contains the GluN2B subunit, called GluN2B-containing NMDA receptor (GluN2B-NMDAR).
The present invention also relates to an anti-HERV-W ENV antibody binding to a conformational epitope of HERV-W ENV.
The term “conformational” opposes to the term “linear”. In the present invention, it means that the epitope is seen as two separated and distant linear amino acid sequences that are significantly detected by the same monoclonal antibody in classical epitope mapping protocols with overlapping peptides covering the primary amino acid sequence of the protein. The conformational epitope is formed when the protein is folded into a specific three-dimensional shape that presents these two distant amino acid sequences as contiguous ones, therefore targeted as a single unique epitope consisting in the joint sequences.
Preferably, the anti-HERV-W ENV antibody binds to the conformational epitope of HERV-W ENV defined by the two distant linear sequences depicted in SEQ ID NO:10 and in SEQ ID NO:11.
It is well-known by a person ordinary skilled in the art how to generate antibodies binding to a specific epitope defined by its amino acid sequence (29).
The present invention further relates to an antibody comprising each of the complementary-determining regions (CDRs) set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.
The present invention also relates to an anti-HERV-W ENV antibody which induces in neurons, the relocation of the GluN2B-containing NMDA receptor into the synapses, and which comprises each of the complementary-determining regions (CDRs) set forth in SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3 SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.
The present invention also relates to an anti-HERV-W ENV antibody which induces in neurons, the relocation of the GluN2B-containing NMDA receptor into the synapses, and which binds to the conformational epitope of HERV-W ENV defined by the two distant linear sequences depicted in SEQ ID NO: 10 and in SEQ ID NO: 11.
The present invention also relates to an anti-HERV-W ENV antibody binding to the conformational epitope of HERV-W ENV defined by the two distant linear sequences depicted in SEQ ID NO: 10 and in SEQ ID NO: 11, and comprising each of the complementary-determining regions (CDRs) set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.
The present invention also relates to an anti-HERV-W ENV antibody binding to the conformational epitope of HERV-W ENV defined by the two distant linear sequences depicted in SEQ ID NO: 10 and in SEQ ID NO: 11, and comprising each of the complementary-determining regions (CDRs) set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, and which induces in neurons, the relocation of the GluN2B-containing NMDA receptor into the synapses.
In one embodiment, the antibody of the invention comprises:
The above-mentioned complementarity determining regions (CDRs) are disclosed in Table 2:
In one embodiment, the present invention relates to a fragment or a derivative of the antibody described above, selected from the group consisting of a Fv, Fab, F(ab′)2, Fab′, dsFv, scFv, sc(Fv)2, a diabody.
In a preferred embodiment, the antibody of the invention is a monoclonal antibody. Monoclonal antibodies of the invention are monovalent, bivalent, multivalent, monospecific or bispecific. In another embodiment, the antibody directed against HERV-W ENV is a binding fragment or a conjugate. For examples antibodies of the invention may be conjugated to a growth inhibitory agent, cytotoxic agent, or a prodrug-activating enzyme.
Another type of amino acid modification of the antibody of the invention may be useful for altering the original glycosylation pattern of the antibody. By “altering” is meant deleting one or more carbohydrate moieties found in the antibody, and/or adding one or more glycosylation sites that are not present in the antibody. Glycosylation of antibodies is typically N-linked. “N-linked” refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites).
In a particular embodiment, the anti-HERV-W ENV antibody according to the invention is a monoclonal antibody, a chimeric monoclonal antibody, or a humanized monoclonal antibody.
Preferably, said antibody anti-HERV-W ENV is an IgG, in particular an IgG1 or an IgG4, preferably an IgG4.
More preferably, the antibody of the invention is a humanized monoclonal antibody, more preferably a humanized IgG4 monoclonal antibody or a humanized IgG1 monoclonal antibody.
Said humanized antibody may be produced by obtaining nucleic acid sequences encoding for CDRs domain by inserting them into an expression vector for animal cell having genes encoding a heavy chain constant region identical to that of a human antibody; and a light chain constant region identical to that of a human antibody, and expressing the expression vector by introducing it into an animal cell. The humanized antibody expression vector may be either of a type in which a gene encoding an antibody heavy chain and a gene encoding an antibody light chain exists on separate vectors or of a type in which both genes exist on the same vector (tandem type). In respect of easiness of construction of a humanized antibody expression vector, easiness of introduction into animal cells, and balance between the expression levels of antibody H and L chains in animal cells, a tandem type of the humanized antibody expression vector is more preferable. Examples of the tandem type humanized antibody expression vector include pKANTEX93, pEE18 and the like. Methods for producing humanized antibodies based on conventional recombinant DNA and gene transfection techniques are well known in the art. Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting, veneering or resurfacing, and chain shuffling. The general recombinant DNA technology for preparation of such antibodies is also known.
Thus, an embodiment of the invention relates to a monoclonal humanized antibody comprising:
The present invention also relates to an anti-HERV-W ENV antibody described above for a therapeutic use or for use as a medicament.
In one embodiment, the present invention relates to an antibody directed against HERV-W ENV and inducing in neurons, the relocation of the GluN2B-containing NMDA receptor, into the synapses, for a therapeutic use for use as a medicament.
In one embodiment, the present invention relates to an antibody directed against HERV-W ENV for a therapeutic use for use as a medicament, wherein said antibody binds to the conformational epitope of HERV-W ENV defined by the two distant linear sequences depicted in SEQ ID NO: 10 and in SEQ ID NO: 11.
In one embodiment, the present invention relates to an antibody directed against HERV-W ENV for a therapeutic use for use as a medicament, wherein said antibody comprises each of the complementary-determining regions (CDRs) set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.
These three embodiments may be combined with each other.
A further object of the invention relates to a pharmaceutical composition comprising an antibody directed against HERV-W ENV as described above, or a fragment or a derivative thereof, and a pharmaceutically acceptable excipient. In particular, the pharmaceutical composition comprises an effective amount of said antibody.
The present invention also relates to such a pharmaceutical composition for use in the treatment of a group of patients suffering from a psychotic disease and having a high level for a cytokine in a body fluid sample, as previously described.
Any therapeutic agent of the invention as above described may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
“Pharmaceutically” or “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.
The pharmaceutical compositions of the invention can be formulated for a topical, oral, intranasal, intraocular, intravenous, intrathecal (directly in the cerebrospinal fluid), intramuscular or subcutaneous administration and the like.
Preferably, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
The doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.
To prepare pharmaceutical compositions, an effective amount of the antibody directed against HERV-W ENV, or a fragment or a derivative thereof, may be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
Preferably, the antibody directed against HERV-W ENV of the invention can be formulated into a buffer in which it was solubilized, stored and injected to patients. Preferably, said buffer comprises 20 mM histidine, 5% sucrose, and 0.01% polysorbate 20.
For parenteral administration in an aqueous solution, for example, the solution may be suitably buffered and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the patient being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual patient.
In preferred embodiments, the antibody of the invention is formulated to be administered by intravenous or by intrathecal injection.
In all the legends and the figures, “Cont.” means Control.
We designed this study to determine whether HERV-W derived envelope-protein (ENV) could play a role in the known glutamatergic, namely NMDAR, disturbance of developmental neuropsychiatric disorders. We first showed that the ionotropic function of the receptor (NMDAR antagonists are related to psychotic like effects) was intact after ENV exposure and that instead, the surface diffusion of the NMDAR-GluN2B subtype was sensible to ENV. The recombinant ENV buffer was used as control and in addition, heat inactivation and neutralization with an specific ENV-antibody proved involvement of HERV-W ENV. At the commence of single particle tracking experiments the sample size was on the one hand, determined by referring to previous studies from the laboratory and on the other hand, calculated (Power & Sample Size Calculator, Statistical Solutions, LLC) with a power factor of 0.6-0.8 and α of 0.5 which in our condition was translated to 4-13 cells per condition depending upon the SD of the sample. For all type of experiments a minimum of 3 independent cultures were used per condition. Cultures were chosen at random and experimental conditions were alternated throughout the live imaging experiments, the experimenter was blind to the condition during removal of unspecific trajectories while all other analyses were done on a fully automated basis. Immunocytochemistry showed that the expression of the important synaptic GluN2A/2B NMDAR-subunit balance was affected. All analysis involving immunohistochemistry/immunocytochemistry were performed blindly. We controlled that no toxicity was induced by ENV in our neuronal networks by adding Propidium Iodide and visualizing healthy cell bodies post ENV exposure. We next measured the global NMDAR-mediated activity, as well as on NMDAR-dependent long-term synaptic potentiation (LTP) and observed an increase in calcium frequency and ablation of cLTP induction in the ENV treated cultures. In the chemical LTP experiments a variation above 10% of GluAlSEP intensity in single synapses during baseline were considered as unstable and hence this spine was excluded, for calcium imaging, spines with more than 2 calcium burst after D-APV application were considered as non NMDAR and excluded. We then transfected neuronal cultures with a plasmid containing the HERV-W ENV and observed similar results. An plasmid with an empty vector was used as control. We then asked if specific ENV expression could affect animal's behavior. For this, we expressed the ENV in the hippocampus of rat pups from postnatal day 0 and studied its impact on a series of behavioral studies. We controlled for cellular toxicity by evaluating TUNEL stained cells and weight gain of the animals and, the presence and extent of ENV expression was confirmed by immunohistochemistry and western blot analysis. Expression of GFP and additionally an empty vector was used in control animals. For behavioral studies the predetermined minimum sample size of 12 animals was based on previous studies. In our hands, a sample size of 13-18 animals in PPI were used, which corresponded to a power factor of 0.6-0.75 and a of 0.5 and in the x-link and MK-801 experiments a sample size of 11 animals corresponded to a power factor of 0.55 and <0.8 respectively. Rat pups from the same litter were assigned to different experimental groups in a randomized manner and groups were pseudo-randomized on each behavioral testing day. As predetermined, potentiation of PPI in response to prepulses and in the MK-801 challenge test, stereotypic behavior were used as exclusion criteria's. During animal studies the experimenter was not blind to the animal's condition since all analysis were done on a fully automated basis at the end of the experiment, after collecting the complete data set. As we observed deficits in several behavioral tests we then investigated the animal's hippocampal NMDAR expression in order to relate our in vitro data to a possible underlying cause for these observations. Protein expression studies were conducted with 9 animals per condition and replicated 2-3 times. By specifically neutralizing the ENV during development we observed a clear improvement of the behavioral response. The data showed that HERV-W ENV expression in the hippocampus induce behavioral deficits and modulate the synaptic NMDAR maturation during development.
Expression of the ENV in serum from 7 patients with neuropsychiatric disorders was detected by ELISA. Serum samples from 6 individuals with no known brain disease were used as controls and sample sizes were based on the availability of samples. In a pilot experiment, we stimulated neuronal networks transfected with plasmids containing the main neurotransmitter receptors (AMPA, NMDA, Dopamine and GABA) with our serum samples and observed that ENV containing serum specifically induced NMDAR alterations.
Cytokine levels were examined using a Milliplex Map Kit (RECYTMAG-65K, Millipore). Culture medium, from 3 different cultures, collected 5 min after vehicle (Control) or ENV (10 μg/ml) application were processed according to the manufacturer's recommendations and mean fluorescent intensities were obtained using a Luminex xPONENT software on a Bioplex™ MAGPIX reader (BioRad, Hercules, USA). Data was normalized to control within each experiment.
Recombinant ENV (full-length MSRV envelope protein of 548aa; ENV pV14; GenBank accession no. AF331500) was produced by PX'Therapeutics (Grenoble, France) according to quality control specifications of (GeNeuro Geneva, Switzerland). Endotoxin removal was done by polishing batches through Mustang Q Acrodisc followed by filtration on 0.22 μm filter Stericup (Merck, Darmstadt, Germany). Endotoxin levels for ENV batches used were between 13.6-92.3 EU/ml as measured by the limulus amebocyte lysate test. Influence of endotoxin on our results was excluded by observations after heat inactivation, 100° C. for 30 min.
Neurons were transfected with the postsynaptic marker Homer 1c-DsRed at 7 div. High resolution single molecular tracking of NMDAR's were achieved after 10 min incubation at 37° C. with antibodies against extracellular epitopes of either the GluN2A or the GluN2B subunits (Alomone Labs, Jerusalem, Israel, Table S3) at 11-14 div. After 10 min incubation with Quantum dots (QDs) 655 (Invitrogen, Thermo Fisher Scientific, Massachusetts, USA) in medium with 1% BSA (Sigma-Aldrich, Missouri, USA), selected regions of interest (ROI)s (with Homer-1c expressing neurons) were imaged for 500 consecutive frames with an acquisition time of 50 ms on a Nikon eclipse Ti epifluorescent microscope using an EMCCD camera (Evolve, Photometric, Tucson, USA). Acquisition was made with MetaMorph software (v.7.7.11.0, Molecular Devices, Sunnyvale, USA). The instantaneous diffusion coefficient (D) was calculated for each trajectory from linear fits of the first four points of the mean square displacement (MSD) versus time function using MSD(t)=<r2>(t)=4Dt. To determine the distribution of single QD complexes, frame stacks were obtained and after binarisation of the synaptic signal the complexes were automatically located into synaptic (Homer-1c positive area including surrounding 2 pixels) and extrasynaptic compartments. The percentages of synaptic locations per stack in relation to the total amount were calculated and the two highest deltas (pre-post) were excluded as outliers from each group. Data were projected on a single background image, providing high-resolution distribution of receptor/QD complexes and their trajectories. All single particle analysis was completed using the Palmtracer v1.0 plugin in MetaMorph software (Molecular Devices). The effect of ENV (GeNeuro) or LPS (Sigma-Aldrich) compared to respective vehicle (Controls) on receptor surface diffusion were addressed using 2 approaches: i) after 5 min bath application in the imaging chamber following an initial baseline acquisition or, ii) 24 h after protein application to the culture medium in the dish. Recombinant IL-1β (R&D Systems, Minnesota, USA) were only evaluated after 5 min application. TLR-4 involvement was studied by blocking the receptor through pre-incubation for 30 min with an antiTLR-4 neutralizing Ab (20 μg/ml, Affymetrix, Wien, Austria) at 37° C. before onset of QDexperiment. The specificity of the TLR-4 neutralizing antibody was confirmed by principal TLR4 staining on Iba-1 positive microglia cells (
Mixed cultures of hippocampal neurons and glia cells were prepared from E18 Sprague-Dawley rats. In brief, cells were plated at a density of 300-350×100 cells per dish on poly-lysine coated coverslips and were maintained in Gibco neurobasal medium (Thermo Fisher Scientific, Massachusetts, USA) containing 3% horse serum for approximately 4 days in vitro (div) at which the medium were changed to a serum-free neurobasal medium. Banker type “glia free” hippocampal cultures were prepared in two steps. Briefly, first glia feeder cultures were prepared in poly-lysine coated dishes from hippocampus then, after two weeks, hippocampal neurons (from the same type of preparation as for the glia cells) were cultured on poly-lysine coated coverslips which were suspended above the glia layer. Cells were kept at 37° C. in 5% C02 for 22 div at maximum. Human embryonic kidney cells (HEK) 293 were plated on glass coverslips in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific) with 10% fetal calf serum and used one day later.
Cells were transfected using either Effectene (Qiagen, Hilden, Germany) according to the manufacturer's recommendations or by phosphate calcium transfection. The plasmid encoding HERV-W ENV protein consisted in the reference MSRV-env gene inserted into a phCMV vector allowing expression in transfected human cells: phCMV-MSRV env from GeNeuro, Switzerland. The inserted env synthetic nucleic acid sequence is encoding HERV-W envelope protein as described in databases (Genbank Ref. AF 331500).
Recombinant ENV (full-length MSRV envelope protein of 548 aa; ENV pV14; GenBank accession no. AF331500) was produced by PX'Therapeutics (Grenoble, France) according to quality control specifications of GeNeuro (Geneva, Switzerland). Endotoxin removal was done by polishing batches through Mustang Q Acrodisc followed by filtration on 0.22 μm filter Stericup (Merck, Darmstadt, Germany). Endotoxin levels for ENV batches used were between 13.6-92.3 EU/ml as measured by the limulus amebocyte lysate test. Influence of endotoxin in our results was excluded by observations after heat inactivation, 100° C. for 30 min as previously described (18).
Before human serum incubation, neurons (10 div) were transfected with either the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA)-A1 containing; the N-Methyl-D-Aspartate receptors (NMDA)-N1 containing; the gamma-Aminobutyric acid (GABA)-γ2 containing subunits fused to the Super Ecliptic pHluorin (SEP) or the dopamine (D)-1 containing subunit fused to Cyan Fluorescent Protein (CFP). Serum samples were slowly thawed and following incubation, with ENV-positive or ENV-negative serum samples at 20% for 15 min at 37° C., live immunostaining of surface receptors were conducted with an anti-GFP antibody for 10 min at 4° C. (heat inactivation of complement factors in serum samples was considered but not applied due to the known heat sensitivity of the ENV-protein). Live staining was followed by 15 min fixation in 4% paraformaldehyde (PFA). In general, fixation was followed by quenching in 50 mM NH4Cl, blocking and incubation with secondary Ab's coupled to Alexa fluorophores for 1 h in 1% bovine serum albumin (BSA) (Sigma-Aldrich, Missouri, USA) at room temperature (R.T.). For staining of fixed cells and tissues incubation overnight with primary antibodies at 4° C. was done after an initial block and permeabilization step in 1% BSA (Sigma-Aldrich) and 0.1% Triton X-100 (Sigma-Aldrich) for 30 min. Cells were mounted in Mowiol (Calbiochem (Merck)) or Vectashield®+DAPI (Vector Laboratories, Burlingame, CA). Surface or intracellular staining of ENV in HEK-cells transfected with cytosolic enhanced green fluorescent protein (EGFP) and MSRV-ENV was obtained after 48 h with an anti-ENV antibody ((GN-mAb 01; GeNeuro Switzerland)). For the microglia activation study, cells were fixed and stained for ionized calcium-binding adapter molecule 1 (Iba1) 5 min after LPS (serotype 026:B6, Sigma-Aldrich) (1 μg/ml), ENV (PX'Therapeutics) (1 μg/ml) or Vehicle (Control) application. Images were either collected on a video confocal spinning-disk system (Leica DMI6000B, Wetzlar, Germany) with a CoolSNAP HQ2 camera (Photometrics, Tucson, USA) or, on a Nanozoomer (Hamamatsu, Japan). Cluster area was obtained using a fixed threshold approach based on integrated fluorescence levels in ImageJ. Microglia perimeter (μm) and area (μm2) was obtained from all microglia present on entire coverslips and a transformation index (TI) was calculated (perimeter of cell) 2/4pi (cell area) based on previous publications (27).
Neurons transfected with GCaMP3 or GCaMP6 at 10 div were transferred into Tyrode's solution containing (in mM): 110 NaCl, 5 KCl, 25 HEPES, 15 D-glucose, 2 CaCl2) and 2 MgCl2. For isolation of NMDAR dependent transients the neurons were then moved to Mg2+free Tyrode's with 5 μM Nifedipine (Tocris) and 5 μM Bicuculline (Tocris) 15 min before imaging. Time-lapse images were acquired with MetaMorph software (Molecular Devices) at 20 Hz on a Nikon eclipse Ti epifluorescent microscope with an EMCCD camera (Evolve, Photometric). To asses immediate effects tree time lapse movies (3000 frames) were successively recorded: Pre (baseline), Post (5 min after bath application of PBS (Blank), Vehicle (Control) or ENV and, APV (5 min after 50 μM D-(−)-2-Amino-5-phosphonopentanoic acid (APV, Tocris) bath application). For prolonged effects two time lapse movies were recorded. The first, 24 h after Vehicle (Control), ENV (1 μg/ml), Vehicle+IL-ra (250 ng/ml) or ENV+IL-ra addition or, 4 days after transfection with the ENV-gene or an empty-vector, and the second, 5 min after APV bath application. Time-lapse movies were concatenated and realigned in ImageJ (NIH) with the PoorMan3DReg plugin (Michael Liebling). Fluorescence from calcium transients vs. time was measured within individual ROIs (spines) manually defined by the experimenter (ImageJ, NIH). All pixels within each ROI were averaged to give a single time course associated to the ROI. Mean normalized fluorescence (ΔF/F) was calculated by subtracting each value with the mean of the previous 5 s values lower than P50 (p) and dividing the result by μ. Positive calcium transients were identified following a two-step procedure: initially, ΔF/F traces were smoothened by convoluting the raw signal with a 10 s squared kernel. Using custom-written Matlab routines, true positive NMDA transients (with minimum 1 see between transients) were defined on an automated basis where the threshold was set at 5*SD of APV average trace. Pairwise cross-correlation of transients between spines on the same neuron was computed using a time window of 0.5 seconds. We corrected the correlation values by subtracting the mean correlation obtained by shuffling the inter-transient time for individual spines (repeated 100 times).
Chemically Induced Potentiation (cLTP).
Live hippocampal neurons, co-transfected at 10 div with GluA1-SEP and Homer 1c-DsRed, were incubated (12 div) overnight with ENV (1 μl/ml) or nothing (Control) at 37° C. Chemically induced long-term potentiation (cLTP) was provoked by bath co-application of 200 μM Glycine (Tocris) and 5 μM Picrotoxin (Tocris) for 4 min as previously described (24). cLTP was always applied after a period (2×5 min) of baseline acquisition and the medium was carefully replaced by fresh equilibrated and heated medium after induction. GluA1-SEP fluorescence signal was then recorded every 5 min during the following 30 min. Synapses were outlined using the synaptic Homer 1c signal and GluA1-SEP intensity (ImageJ, NIH) was followed over time within these synaptic areas and then exclusively normalized to baseline if synapses showed a stable baseline (variation below 10% between −10 and −5 min). All images were collected on a video confocal spinning-disk system (Leica, DMI6000B, Wetzlar, Germany) with a CoolSNAP HQ2 camera (Photometrics, Tucson, USA).
Pregnant rats (Sprague Dawley) were purchased from Janvier (France) and on P 0-1 male pups from the same litter were assigned to different groups in a randomized manner: Control, Control+ or ENV. Rats were kept at constant ambient temperature (21±1° C.) with ad libitum access to food and water. Every effort was made to minimize the number of animals used and their suffering. Animal procedures were conducted in accordance with the European Community guidelines (Directive 2010/63/EU) regulating animal research, and were approved by the local Bordeaux Ethics Committee (APAFIS #3420-2015112610591204). Postnatal electroporation was done in newborn pups between P0-1, as previously described. Briefly, pups were anesthetized by hypothermia and injected with deoxyribose nucleic acid (DNA) constructs coding for cytosolic EGFP to identify transfected cells (Control-group) in combination with an empty vector (Control+-group) or, with phCMV-MSRV ENV (clone pV14, AF 331500) (ENV-group). Approximately 2 μg of DNA in 8 μl of PBS and 0.1 μl of Fast Green were injected into lateral ventricles, immediately followed by electroporation with five electrical pulses (150 V, 50-ms duration, 1-s interval between pulses) delivered by a pulse generator (BTX, Harvard apparatus, ECM830, San Diego, CA). Pups were reanimated on a thermal blanket at 37° C. and quickly returned to their mother.
Experiments were conducted during the light cycle (14.00-20.00) by the same experimenter as the one who handled the animals during their lifespan. Their weights were monitored every week and at P 21 the pups were weaned and housed 2-5 rats from the same litter and with the same treatment/cage. Five different study-groups were used: i) the animals in the first group, Control (n=13), ENV (n=13), were subjected to a series of behavioral tests in the following order: (1) at P35-37, locomotor activity and anxiety, (2) habituation/adaptation to the open field and (3) at P56-58 the prepulse inhibition test (PPI) and 2 days after (4) social recognition and interaction. ii) The animals in the second study-group, Control (n=30), ENV (n=30), was either exposed to Clozapine/Vehicle treatment before PPI or/and to a MK-801 challenge on the day after PPI. iii) The third study group, Control (n=11), Control+(n=14), was tested in the PPI and, iv) the fourth study-group, ENV (n=22), was subjected to a crosslink protocol (see below for stereotaxic injection) on the day before PPI. v) The animals in the last group Control (n=13), ENV (n=24), were treated with the ENV neutralizing-Ab during an early postnatal period. All animals were naïve to the apparatuses when first presented with the tests and acclimatized to the room for at least 1 h before onset. Groups were pseudo-randomized on each behavioral testing day. During the open field and PPI tests the experimenter was not blind to the animal's condition although behavioral data collection was done using a fully computer controlled setup and in an unbiased way. In contrast, the investigator was blinded to the treatment during scoring analysis in the social recognition and interaction test. See
Open field. Locomotor activity was measured in an open field arena (54 cm long×54 cm wide×40 cm high) with light settings at approximately 5 lux. Novelty-induced locomotion was assessed by video tracking the rat which was allowed to freely explore the empty arena during 2×10 min on day 1 out of 3 consecutive test days. From the recordings on day 1, anxiety was evaluated as the time spent within a center zone comprising 50% of the arena during the first 10 min. Then, habituation/adaptation to context was assessed as a decrease of locomotor activity on day 2-3. For MK-801 studies, Control (n=13) and ENV (n=12) rats were given an intraperitoneal (i.p.) injection with saline and left to explore the arena during one hour. Then, a MK-801 (0.5 mg/kg, Tocris) injection was administered (i.p.) and animals were monitored for two additional hours. One animal was excluded due to stereotypic behavior after injection. Total distance travelled was extracted with the IDtracker software (43) and analyzed with automatized custom-written MATLAB routines.
PPI was performed using a Panlab startle chamber (Harvard, San Diego Instruments). Each PPI session lasted for approximately 31 min and began with a 5 min acclimatization period with a constant background noise. The session consisted of 8 different trial types: a no pulse, a startle pulse (120 decibel (dB) at 8 kHz, 40 ms) that was or, was not preceded by tree prepulses at +4, +8, and +12 dB above a 74 dB background noise (20 ms, interval 100 ms) and, the prepulses alone. Each session started with 10 startle pulses (ITIs 70 sec) followed by a counterbalanced pseudorandom order of the 8 trials×6 and ended by a final block of 10 startle pulses. Baseline data from different groups were initially pooled seeing that the effect of Envgene insertion on the overall PPI measured at the three prepulses was similar: 1) two-way ANOVA, group factor; F(1,72)=11.23, **P=0.0013, n=13 and, 2) two-way ANOVA, group factor; F(1,102)=9.597, **P=0.0025, n=18. Potentiation in response to the prepulses was observed in both animal groups and these animals were excluded from the final data set. Prepulse inhibition is expressed as % PPI and was calculated by (100*((S-PP)/S), where S=average response on startle-only trials and PP=average response on prepulse+startle trials. We confirmed that the DNA load was trivial to the behavioral outcome (study-group iii) by comparing GFP animals with GFP+empty vector electroporated animals (
This protocol was adjusted from (43). The day before testing, 3 rats (1 study rat (Control or ENV), and 2 target rats (naïve)) with similar weight (±5%) from different litters were assigned to be tested together. Target rats were then habituated to the experimental cage (a housing cage) in the experimental room during 1 h. At the day of experiment, the study rat was habituated to the experimental cage for 1 h. Thereafter, without any training the test started and the first target rat was placed into the same experimental cage (3×1 min, ITI 3 min) and social recognition was recorded. Subsequently during a last interaction session the first target rat was removed and the second target rat was placed into the experimental cage together with the study rat and social interaction was recorder for 10 min. In the last session, control study rats showed increased interaction during the first minute meaning that the rats were once again interested in the unfamiliar rat (second target rat). The full last session (10 min) was then scored in order to measure social interaction. Study and target rats were not used in this paradigm more than one time. Videos were scored in a blinded fashion for the time the study rat actively engaged in social interacting behaviors (sniffing, grooming, close following, and crawling over/under). No aggressive behaviors were noted during the sessions.
Brains were removed between 2-14 days after PPI and either the whole brain; was rapidly frozen in isopentane (Sigma-Aldrich) placed in liquid nitrogen or, transferred to ice cold artificial cerebral fluid for dissection of the hippocampal areas and then frozen in liquid nitrogen. The frozen hippocampal tissue was later processed for Western blot as described below. For immunohistochemistry, 20 μm thick coronal tissue sections were cut on a microtome-cryostat, thaw-mounted onto superfrost ultra plus (Thermo Scientific) slides, and stored at −20° C. until further processing. Animals at the age P7, Control (n=5) ENV (n=5) and, P59-70 Control (n=5) ENV. (n=5) were anesthetized with pentobarbital (50 mg/kg) and transcardially perfused with 4% paraformaldehyde in 0.1M phosphate buffer. Brains were removed and postfixed overnight at 4° C. and 50-μm-thick slices were prepared with a VT1200S Leica vibratome. Slices were washed three times with PBS and left in 0.03% acid-PBS at 4° C. for later use.
Perfused tissue were blocked and permeabilized in BSA (Sigma-Aldrich) and 0.1% Triton X-100 (Sigma-Aldrich) for 2 h at R.T. After rinsing samples were incubated with primary Ab's in 2% BSA (Sigma-Aldrich) and 0.2% Triton X-100 (Sigma-Aldrich) overnight at 4° C. Secondary Ab's coupled to Alexa fluorophores were incubated for 2 h at R.T. in the same solution as the primary Ab's. Sections were mounted using Mowiol (Calbiochem) or Vectashield®+DAPI (Vector Laboratories, Burlingame, CA). All images were collected on a video confocal spinning-disk system (Leica DMI6000B, 63X) with a CoolSNAP HQ2 camera (Photometrics) or on a Nanozoomer (Hamamatsu).
DNA fragmentation was histologically examined using the in situ Apoptosis Detection System Fluorescein (TUNEL, Promega, Madison, WI). Frozen tissue sections from P7 electroporated animals were stained according to the manufacturer's recommendations and mounted with Mowiol (Calbiochem).
Dissected hippocampus from control and ENV electroporated rats (˜P70) were processed by subcellular fractionation (
Figures were assembled in ImageJ (NIH) and Adobe Photoshop (Adobe Systems, San Jose, CA), only contrast and brightness were adjusted to optimize the image quality. All statistical analysis was performed in GraphPad Prism 6 (GraphPad Software, San Diego, USA). In brief, statistical analysis using alpha=0.5 were conducted. For behavioral studies a predetermined sample size was based on the literature. Here, a sample size of 13-18 animals were used in the PPI, which correspond to a power factor of 0.6-0.75 and a of 0.5 and, in the cross-link and MK-801 experiments a sample size of 11 animals correspond to a power factor of 0.55 and <0.8 respectively. Parametric statistical tests were applied when data passed the D'Agostino & Pearson omnibus normality test hence, two-tailed Student's t-test with or without Welch's correction, one- or two-tailed paired Student's t-test or two-way ANOVA followed by Bonferroni's multiple comparisons test were conducted. Comparisons between groups with data from non-gaussian distributions were performed using nonparametric test: either the two-tailed unpaired Mann-Whitney test or, for multiple groups the Kruskal-Wallis test followed by Dunn's multiple comparison test. For direct comparison of distributions, Kolmogorov-Smirnov test was used. For behavioral testing, the PPI results were analyzed using the factors group and prepulse by two-way ANOVA followed by Bonferroni's multiple comparisons test for group and prepulse intensity. A one-way ANOVA analysis, followed by Tukey's multiple comparisons test for treatment was used in the crosslink and Neutralizing Ab protocols. RM two-way ANOVA considering the factors treatment and time followed by Fisher's LSD post-hoc analysis were used for analysis of the MK-801 response and a two-way ANOVA for TUNEL data with factors treatment and area. Significance levels were defined as *P<0.05, **P<0.01, ***P<0.001.
In a first experiment, we collected sera from patients suffering from psychotic disorders (schizophrenia and bipolar disorder) or from control healthy subjects in order to test their impact on the content of key neurotransmitter receptors in hippocampal neuronal networks containing neurons, astrocytes and microglia (
Surprisingly, when glia-free neuronal cultures (without astrocyte or microglia) were exposed to recombinant ENV, GluN2B-NMDAR synaptic dynamics remained stable (
The functional alteration of neuronal functions by HERV-W ENV therefore reveals to be highly specific, at both levels of glutamatatergic neuroreceptor subclass and of microglia-mediated inflammatory cytokine production. Thus, pro-inflammatory cytokines and the most relevant one, IL-1β, involved in this specific neuronal dysfunction, are not induced by environmental factors such as infectious agents but by the HERV-W ENV protein itself when expressed and/or released in a neuron-glia tissue environment. Since no such specific effect of IL-1β, alone or in association with IL-6 and/or TNF-α, has ever been described nor could be expected, it becomes obvious that the contextual induction and synergistic effects of HERV-W ENV are key to this ENV-specific pathogenic mechanism.
This also unravels the existence of a specific biological continuum, based on these ENV-glia/cytokine-mediated neuronal dysfunctions, in psychiatric diseases classified in different diagnostic categories until now, such as Schizophrenia and Bipolar Disorder.
Present nosological entities only rely upon psychological evaluations with dedicated tests and scales, as well as on the clinical, mostly behavioral, symptomatology.
The present elucidation of this HERV-W ENV-induced neuronal dysfunction is therefore providing a causal, mechanistic and biological definition that underlies symptoms and disease evolution across different diagnostic categories associated with HERV-W ENV and, IL-1β and/or Il-6 and/or TNF-α, cytokine-driven synergistic effects on brain cells.
A new biologically-defined nosological entity can therefore comprise sub-groups of patients with Schizophrenia, with Bipolar Disorder or with other present psychiatric diagnoses, when resulting from the presently discovered synergistic effect between HERV-W ENV and such cytokines. Depending on upstream, probably environmental and temporal factors (11), this HERV-W expression may be activated in various brain regions, in which neuronal synapses with GluN2B-NMDAR are involved in various behavioral, cognitive and other nervous system functions. This also implies neurodevelopmental impairment when HERV-W ENV and cytokine-driven synergistic effects occur during embryogenesis and early life development of brain areas and functions. This unexpectedly unravels a previously unknown but common pathogenic process between different nosocomial entities of present psychiatric diagnoses. One major aspect of HERV-W ENV-induced pro-inflammatory cytokines is the presence of Il-1 and/or of related pro-inflammatory cytokines, IL-6 and/or TNF-alpha, as observed to be co-expressed in the presently described experiments, but also detectable in body fluids of these patients (cf. Example 5).
Most psychomimetic drugs (e.g. PCP) are NMDAR antagonists. Despite its biochemical nature, i.e. a protein macromolecule that cannot have the molecular interactions of the small chemical compounds used to interfere with receptors of neuronal neurotransmitters, HERV-W ENV blocked NMDAR-mediated synaptic transmission in hippocampal networks.
An effect of HERV-W ENV protein on neuronal receptors was never described nor could be expected, since only known to interact with TLR4 (30) receptor and V-beta chains of T-cell receptor (31), which are not present nor expressed in neuronal cells. Moreover, these results also provided the evidence that HERV-W ENV did not interfere with neuronal receptors through presently known mechanisms since neither NMDAR-mediated calcium transients, nor NMDAR-mediated evoked EPSCs, were altered by various concentration of HERV-W ENV (
A specific antibody directed against HERV-W ENV was found to inhibit this pathogenic effect (
To test whether TLR4 activation could unexpectedly trigger this effect, a well-known TLR4 agonist, lipopolysaccharide (LPS), was applied onto hippocampal networks. Consistently, LPS was capable to rapidly disperse synaptic GluN2B-NMDAR (
As schizophrenic patients may be chronically exposed to HERV-W ENV (11), we investigated how long-term exposure to ENV alters hippocampal network properties and animal behavior. After 24 h of ENV exposure, synaptic GluN2B-NMDAR were still laterally dispersed, an effect that was fully blocked by a selected anti-ENV antibody (
Because a bulk exposure of HERV-W ENV may not reflect the bioavailability of the protein in the patients' brains, HERV-W ENV was genetically-expressed for several days in only 3% of hippocampal cells (
In psychotic diseases, alterations of the NMDAR signaling and synaptic plasticity have been associated with behavioral deficits in cognitive and sensorimotor gating (i.e. prepulse inhibition, PPI) tests (40), which are hallmarks of psychosis models in rodents. Following postnatal electroporation, HERV-ENV electroporated cells were observed in hippocampal astrocyte/radial glial (GS+) or non-glial (GS−) cells (
Altogether, this study demonstrates that the HERV-W ENV, detected in a large fraction of psychotic patients (12, 42), is capable of generating a dysfunction in the NMDAR organization and long-term plasticity within glutamatergic synapses. This dispersal of the synaptic receptor NMDAR away from the synapses produces behavioral deficits associated to psychosis. This makes HERV-W ENV a therapeutic target of interest for new therapeutic strategies, with novel mechanism of action, in psychotic patients.
It also demonstrates that an anti-ENV antibody could specifically and efficiently reverse and/or prevent these pathogenic effects at the level of a unique neuronal receptor of neurotransmitters, GluN2B when impacted through its biodistribution over neuronal synapses by HERV-W ENV. The use of an anti-ENV antibody induces in neurons, the relocation of the NMDAR, in particular of the GluN2B-containing NMDA receptor, into the synapses. This therapeutic effect of anti-ENV antibody translates, in vivo, in the reversal or prevention of psychosis-related behavioral abnormalities.
This interplay between HERV-W ENV and glutamate synapse thus adds a novel and unexpected layer in our understanding of how endogenous retroviruses could play an instrumental role in psychiatric diseases. Indeed, it now emerges that HERV-W activation can alter glutamatergic synapse development and functions through convergent, yet different (including synergistic induction of cytokines), pathways from what was shown from previous knowledge.
Further analyses were then conducted which evidenced that neonatal ENV expression tunes glutamatergic synapse maturation and induces behavioral deficits associated to psychosis, effects which are reversed by the neutralizing anti-HERV-W ENV antibody.
The following in vivo study was conceived when considering that the proper maturation of glutamatergic synapses requires a switch from immature GluN2B-rich to mature GluN2A-rich synapses (44). This fundamental process occurs during the postnatal period (first two postnatal weeks) in which neuronal networks and cognitive functions are established. Synaptic alterations occurring during this time window often lead to adult behavioral dysfunctions associated with psychiatric disorders, such as psychosis (44, 45, 46). Considering that the GluN2A/2B synaptic ratio was altered in vitro by ENV, we measured the GluN2A- and GluN2B-NMDAR synaptic content (
Because the maturation of glutamatergic synapses occurs during the first postnatal weeks, we postulated that the deleterious effect of ENV on the NMDAR signaling and synapse maturation is prominent during this period. To test this hypothesis, ENV rats received injections of ENV-neutralizing antibody from P4 to P12 (3 injection;
These data indicate that ENV expression in the early postnatal period alters the synaptic GluN2A/B-NMDAR maturation, and drives the emergence of behavioral abnormalities in adults, which can be prevented and/or restored using successive in vivo injections of the neutralizing anti-HERV-W ENV antibody.
The VL Amino-Acid Sequence Along with Identified CDR Sequences is Presented in
The VH Amino-Acid Sequence Along with Identified CDR Sequences is Presented in
VL region corresponds to a rearranged and productive IGK sequence of Kappa isotype that has no identical sequence found in databases (Uniprot databank-UniprotKB).
VH region corresponds to a rearranged and productive IGH sequence of Ig2a isotype that has no identical sequence found in databases (Uniprot databank-UniprotKB).
The experiments were performed by Pepscan, The Netherlands, as recommended by the manufacturers using ELISA plates coated with synthetic peptides overlapping over the primary HERV-W/MSRV-ENV sequence. The test antibody was diluted from 250 ng/ml to 1 μg/ml. The read-out values indicate the mean luminometric OD measured with each successive peptide.
The epitope mapping using overlapping peptides of 15 amino-acids all over HERV-W sequences (MSRV-ENV; Genbank ref. AAK18189.1; Locus AF331500_1; 542 aa) clearly identified two distant sequence stretches that were significantly detected by ENV-W2.3 antibody (
This clearly indicates that these sequences must be jointly detected by this antibody in the native protein, with 3D folded structure presenting these two peptide regions, LFNTTLTRLHE (SEQ ID NO: 10) and LYNHVVP (SEQ ID NO: 11), as contiguously detected aminoacids, i.e., as a conformational epitope (
This epitope detection characterizes this antibody, along with sequences of its CDR and of its variable light and heavy chains (VL and VH).
In conclusion, for a therapeutic application in humans, a humanized antibody or any immune-tolerated molecule is normally required to treat the patients. Therefore, the unique recognition characteristics of such humanized antibody or of equivalent therapeutic molecule, is determined by (i) CDR sequences well known to be involved in the epitope recognition and/or by (ii) the original structure of the conformational epitope joining two distant linear regions of HERV-W ENV pathogenic protein (cf. Examples 3 and 4) and/or by (iii) its ability to induce in neurons, the relocation of the NMDAR, in particular of the GluN2B-containing NMDA receptor, into the synapses.
The present invention therefore provides a therapeutic product for a novel indication in patients previously diagnosed with symptomatology- and psychologically-defined criteria, representing sub-groups of such diagnostic categories and now representing biologically defined novel entity(ies), including the one characterized by the detection of HERV-W ENV antigen in parallel with elevated levels of Il-1beta (>0.05 μg/ml) and/or TNF-alpha (>1.25 μg/ml) and/or IL-6 (>0.5 μg/ml) in patients previously diagnosed as “Schizophrenia” or “Bipolar Disorder”. Of note, these thresholds of cytokines in serum vary with the kits, antibodies, techniques and platforms used for their dosages. Presented values are only indicative and one should adapt them according to manufacturers' instructions or assess them with a given platform. In such cases, when not having an expression confined to the deep brain tissues, HERV-W ENV antigen detection in circulating fluids may be a complementary and highly indicative biomarker.
Pilot studies including patients diagnosed with schizophrenia or bipolar disorder investigated current diagnostic parameters, routine laboratory biological markers and HERV-W antigen detection in the serum. Routine laboratory biological tests were performed according to manufacturer's instruction for the use of diagnostic kits, e.g., for cytokines, and, in general, according to present recommendations and regulations for medical biology analyses in France, where the analyses were performed. Concerning the detection of HERV-W ENV antigen in sera and the quantification of its circulating soluble form, all analyses were performed according to the conditions provided in the patent published under ref. WO2019201908 (A1) and entitled “Method for the detection of the soluble hydrophilic oligomeric form of HERV-W envelope protein”. Results for HERV-W ENV soluble antigen in sera are expressed as “Inter-Experiment Standardized Result”, corresponding to the area under the curve (AUC) of the specific HERV-W ENV soluble antigen peak calculated from the immunocapillary WES platform, normalized for inter-experiments variations using the mean+2×standard deviation of series of healthy controls (lower limit of specificity/positivity cut-off value: CO) in each experiment to adjust all data to those of the first experiment used as a reference. Thus the measured AUC of each sample in each different experiment were multiplied by the ratio of corresponding CO to that of the first experiment, which adjusted all values to the same mean of non-specific background signal also taking into account the impact of inter-individual variation with twice the standard deviation as used for the determination of the minimal value of specific signal (CO). Though variations were not important, this optimized further correlation analyses with data from all subjects thereby avoiding inter-experiment slight variations to impact statistical results. Thus, all measured values were standardized with a CO of 15, above or equal to which all results indicated the specific detection and quantification of HERV-W ENV antigen. Below this CO, values correspond to non-specific background signal generated by the components of the sample and by the technical protocol with non-significant variations among negative samples (“technical noise”).
To identify possible subgroups, we used unbiased two-step cluster analyses (48), in which we concomitantly integrated measures of HERV-W positivity (cut-off for positivity: IESR>15; imputed as categorical variable) and serum cytokines (IL-lb, IL-4, IL-6, IL-8, TNF-a, IFN-y; all LN-transformed; imputed as categorical variables) from subjects with schizophrenia (SZ, n=29), bipolar disorder (BP, n=43) and matched healthy controls (HC, n=32). Missing variables for some subjects led to a reduction in sample size. The final number of subjects included in the cluster analysis was: n(HC)=29, n(SZ)=18 and n(BP)=30. The cluster analysis was run without predetermining the number of clusters, thereby avoiding bias in terms of identifying the number of possible clusters. Bayesian Criterion (BIC) was used to estimate of the maximum number of clusters, whereas the log—likelihood method was used as distance measure [1]. Following stratification of HC, SZ and BP subjects into subgroups, serum cytokines and clinical variables (age of disease onset, Montgomery-Asberg Depression Rating Scale [MADRS] score, Young Mania Rating Scale [YMRS] score, Positive and Negative Syndrome Scale [PANSS] scores, and daily chlorpromazine [CPZ] equivalents) were analyzed by one-way or two-way analysis of variance (ANOVA), followed by Tukey's post-hoc tests for multiple comparisons whenever appropriate. All statistical analyses were performed using SPSS Statistics (version 25.0, IBM, Armonk, NY, USA) and Prism (version 8.0; GraphPad Software, La Jolla, California), with statistical significance set at p<0.05.
Patients diagnosed with Schizophrenia (SZ), bipolar disorder (BP) and healthy controls without known psychiatric, inflammatory, infectious or metabolic disease (HC), had given informed consent to participate to a multiparametric study validated by relevant ethical committee, including clinical and treatment data in parallel with the detection and/or dosage of serum biomarkers including HERV-W ENV antigen and cytokines in their serum.
Data collected from patients and controls are presented at
In
These results were further analyzed for potential correlation or co-stratification (cluster analysis) with other parameters. Significant clustering was essentially found with elevated concentration of certain cytokines in sera. In
Further detailed analyses of different cytokines are presented in
Correlation and/or clustering with clinical or treatment parameters were also identified and are presented in
In
These data obtained with clinical rating scores used for present psychiatric diagnoses, illustrate the discrepancy between patients classification as “SZ or BP” based on clinical parameters and a classification as “CL1 or CL2 with psychotic symptoms” (to exclude HC from CL1) based on objective biological parameters causing pathogenic effects.
Finally and of major importance, in
Therefore, though an equivalent scale for daily treatment dose evaluation in BP is not available, the more severe disease course and/or a resistance to current anti-psychotic drugs in patients with SZ with CL2 biomarkers calls for the use of other treatment strategies, implicitly suggesting to neutralize the pathogenic effects of CL2 biomarkers and preferentially those of the major predictor of CL2 (
Thus, a multiparametric analysis of clinical, therapeutic and serum biomarkers data from a population comprising healthy individuals and patients diagnosed with schizophrenia or bipolar disorder, has independently led to the same evidences as those obtained from previous preclinical studies of HERV-W ENV pathogenic effects, calling for a targeted treatment neutralizing this human endogenous retroviral protein. The in vitro and in vivo efficiency of the antibody from the present invention therefore provide a unique therapeutic tool for the treatment of patients with either Schizophrenia or bipolar disorder and clustering with similar biological parameters: HERV-W ENV antigen positivity and elevated pro-inflammatory cytokine, which can be detected in blood samples, e.g., in serum (here designated as CL2).
Treating patients from CL1 with this antibody is not relevant, in the absence of the targeted pathogenic protein and its elevated cytokine correlates. This implies that clinical efficacy of this antibody in patients should only be assessed in patients matching with CL2 criteria, as presently defined and not in groups of any patients diagnosed either with schizophrenia and/or with Bipolar Disorder. It may correspond to new nosological definitions or, at least, to newly defined subgroups of patients across the two types of clinical diagnoses (SZ and BP), but the most probable etiological and pathogenic heterogeneity between patients with such diagnoses is consistent with the need to define the relevant target population before applying a treatment specific for a pathogenic protein that is not involved in all cases. For routine practice and practical conditions, results from CL2 analyses also show that IL-1β, 11-6 and TNF-α dosages in serum may serve as surrogates for routine CL2 classification.
In conclusion, the present invention provides unexpected and meaningful evidences of (i) a previously unknown mode of action of HERV-W ENV protein on neurons relevant for psychiatric diseases, (ii) a new monoclonal antibody with novel CDR sequences targeting a conformational epitope of HERV-W ENV protein that inhibits its newly discovered pathogenic effects on neurons in an animal model of psychotic behavior/symptomatology induced by HERV-W ENV and (iii) new indications for appropriate therapeutic use of this new antibody to treat relevant sub-groups of patients, presently diagnosed with schizophrenia or bipolar disorder, with new biomarker-based criteria defining new nosological sub-groups or entities.
These biomarkers, combined or alone, may therefore be used in face of relevant clinical symptoms for validating the accuracy of a treatment with this antibody in a given patient.
| Number | Date | Country | Kind |
|---|---|---|---|
| 20305561.1 | May 2020 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/064364 | 12/20/2021 | WO |
