Ifn-signature as a biomarker for herpes simplex encephalitis related symptoms

Abstract

A method for diagnosing a probability of a human patient with encephalopathy symptoms has or may develop a herpes simplex encephalitis (HSE) related symptom

Description

[0001] IFN-signature as a biomarker for herpes simplex encephalitis related symptoms

[0002] Field of the invention

[0003] The present invention relates to a method for diagnosing a probability of a human patient with acute encephalopathy symptoms has or may develop a herpes simplex encephalitis (HSE) related symptom.

[0004] Background of the invention

[0005] Herpes simplex virus (HSV) is a virus that produces different viral infection symptoms in humans.

[0006] Infection with HSV is manifested with different related symptoms - such as e.g. watery blisters in the skin or mucous membranes, lesions heal with a scab characteristic, herpes viral meningitis (HSV meningitis), and herpes simplex encephalitis (HSE - alternatively termed herpes viral encephalitis).

[0007] Accordingly, a patient with HSV may develop different symptoms / diseases - i.e., the art describes different methods for diagnosing a probability in a patient for developing different HSV related symptoms - such as e.g. HSV related diseases and / or complications.

[0008] Encephalopathy refers to abnormal mental status secondary to any disorder. In modern usage, encephalopathy does not refer to a single disease, but rather to a syndrome of overall brain dysfunction; this syndrome has many possible organic and inorganic causes - such as e.g. mitochondrial encephalopathy, glycine encephalopathy, herpes simplex encephalitis (HSE), etc.

[0009] As known in the art - acute encephalopathy symptoms refer to symptoms of encephalopathy in a patient that have been developed in a relatively short time frame (e.g. hours or days).

[0010] Encephalitis refers when encephalopathy is secondary to an inflammatory disorder of the brain (e.g. Autoimmune causes, virus, etc.). Most causes of encephalitis have an acute presentation (symptoms develop in e.g. hours or days).

[0011] Herpes simplex encephalitis (HSE) (alternatively termed Herpesviral encephalitis) is the most frequent sporadic infectious encephalitis in high-income (or non-tropical) countries, with an incidence of 2-4 cases per million persons each year and causes a high morbidity and mortality.

[0012] Distinction between HSE and other causes of acute encephalopathy is difficult and may hold up important management decisions as early treatment is essential to decrease the burden of neurological residual symptoms.

[0013] Currently the distinction between HSE and other causes of acute encephalopathy is only possible with invasive medical tests (lumbar puncture evaluation of cerebrospinal fluid [CSF] to look for inflammatory signs and specific microbiological tests for HSV in CSF). In addition, microbiological tests for HSV diagnosis in CSF have low sensitivity (first 3 days of symptoms) and non-optimal specificity; and do not allow distinction of HSE and HSV meningitis.

[0014] Furthermore, in a confirmed case of HSE, in addition to sequelae caused by the infection, more than 25% of patients develop new neurological symptoms within 1-2 months after HSE, many of them in association with auto-antibodies against neuronal surface proteins (termed autoimmune encephalitis (AE) post-HSE).

[0015] Distinction between AE post-HSE and recrudescence of residual deficits or new manifestations related to persistent or reactivation of the viral infection also is difficult and may hold up treatment decisions.

[0016] The article by Armangue T et al (Lancet Neurol. 2018;17(9):760-772) describes the presence of CSF neuronal antibodies at day 21 after HSE onset as a diagnostic predictor of AE post-HSE.

[0017] Chronic or persistent activation of the type I interferon (IFN) pathway has been described in autoimmune disorders such as systemic lupus erythematous or autoinflammatory diseases such as monogenic type I interferonopathies including Aicardi-Goutieres Syndrome (AGS) Rice et al. (Lancet Neurol. 2013;12(12):1159-1169). In these diseases, the chronic activation of the type I IFN pathway has been measured with an “IFN-signature” which quantifies RNA of multiple genes in the blood that are induced by high levels of type I IFN - see e.g. the article of Kim et al. (J. Interferon & Cytokine Rech. 2018;38(4):171-185) and (Rice et. al 2013).

[0018] As discussed in e.g., Kim et al. 2018 - the term “IFN-signature” is a well know term in the art, and it relates to measurement of elevation of interferon (IFN)-response genes (IRGs) expression (e.g. via measurement of elevated / higher RNA expression levels). According to the art - the term “type I IFN- signature” relates to measurement of elevation of type I IFN response genes (IRGs) expression.

[0019] The article by Liu Benke et al (J Neurol Sciences 424 (2021) 117394) discloses a study showing that of serum CXCL 13, IL-6, BAFF, CXCL 10 and MMP was significantly increased on day 21 following Japanese encephalitis (JE) caused by Japanese encephalitis virus (JEV) in subjects that developed autoimmune encephalitis over those that did not.

[0020] Without being limited to theory - at the filing date of the present patent application - the present inventors were not aware of a single prior art document that directly and unambiguously describes the use of a blood type I IFN-signature as a diagnostic biomarker for herpes simplex encephalitis (HSE) related symptoms / diseases / complications.

[0021] Summary of the Invention. A problem to be solved by the present invention relates to the provision of a novel improved method for diagnosing a probability of a human patient with acute encephalopathy symptoms has or may develop a herpes simplex encephalitis (HSE) related symptom comprising the steps.

[0022] The solution is based on that the present inventors identified that a blood type I IFN-signature can be used as a diagnostic biomarker for predicting if a patient with acute encephalopathy symptoms has or may develop a herpes simplex encephalitis (HSE).

[0023] For instance, and as discussed in working Examples herein - the IFN-signature may be used as a diagnostic biomarker for diagnosing a probability for that a human patient is developing HSE-related symptoms such as e.g. autoimmune encephalitis (AE) post-HSE or for distinguishing a probability of a patient for developing HSV meningitis or more severe HSE as such, and also to distinguish HSE instead of other causes of encephalopathy.

[0024] As discussed above, the article of Armangue T et. al. describes the use of neuronal antibodies as a diagnostic biomarker for HSE-related symptoms / diseases.

[0025] However, and as discussed above - at the filing date of the present patent application - the present inventors were not aware of a single prior art document that directly and unambiguously describes the use of blood type I IFN-signature as a diagnostic biomarker for herpes simplex encephalitis (HSE) related symptoms / diseases / complications.

[0026] In patients with acute encephalopathy, an advantage of using a blood IFN-signature as a supporting method for HSE diagnosis compared to e.g., only use conventional or current methods that only available in CSF, is that this IFN-signature can be measured in blood which is a much less invasive procedure. So in patients with low risk of having HSE or at onset of the disease (e.g. patient with fever and only mild and / or transient encephalopathy) the use of blood IFN-signature (negative result) can be used to rule out in high probability the change of the patient of having HSE. In patients with high risk of having HSE (e.g. very suggestive symptoms as fever, encephalopathy, seizures, MRI temporal lesions and / or aphasia or speech disorders) in which microbiological CSF studies are negative, the use of blood IFN-signature (positive result) can be used to support the clinical diagnosis and treat accordingly.

[0027] In patients with confirmed HSE, as demonstrated / discussed herein - an advantage of using an IFN- signature as described herein compared to e.g., use of antibodies in the prior art (see e.g., Armangue T et al), relates to that the use of CSF antibodies as a predictor for this complication has important clinical limitations. First, up to 50% of patients with HSE may develop neuronal antibodies but only half of them will develop symptoms of AE post-HSE. Second, at the time point of day 21 after HSE onset, these antibodies are not detectable by commercial panels in most of these patients. Third, the assessment of CSF antibodies at day 21 implies the necessity of repeating the lumbar puncture at day 21 after HSE onset, and this is currently not performed in most medical centers as per clinical practice in the setting of HSE. Thus, there is a need for developing a novel blood biomarker that may help identifying patients at risk of developing AE post-HSE, and that may be able to distinguish between patients with this autoimmune complication and patients that suffer from viral complications.

[0028] Accordingly, a first aspect of the present invention relates to a method for diagnosing a probability of a human patient with acute encephalopathy symptoms has or may develop a herpes simplex encephalitis (HSE) related symptom comprising the steps of:

[0029] (i): measuring, in a blood sample obtained from the patient, the expression level of at least one type I interferon (IFN)-response gene (IRG) to obtain a patient type I IFN-signature;

[0030] (ii): comparing the measured patient type I IFN-signature of step (i) to a control group of human persons with a reference control type I IFN-signature with the same IRG as in step (i);

[0031] (iii): analyzing the compared data of step (ii) to identify if the patient type I IFN-signature of step (i) is deviating from the control IFN-signature of step (ii); and

[0032] (iv): deciding if the possible deviation in step (iii) relates to an elevated patient IFN-signature of step (i) as compared to the control IFN-signature of step (ii) and if the patient IFN-signature of step (i) is elevated, then is the patient IFN-signature defined as a positive IFN-signature, indicating a relatively high probability for that the human patient has or may develop a HSE related symptom.

[0033] As understood - the term “relatively high probability” of step (iv) should be understood as a relatively higher probability as compared to if the patient IFN-signature is not an elevated patient IFN-signature - i.e. it is not a positive patient IFN-signature, but to the contrary what herein may be termed a negative patient IFN-signature.

[0034] The term “(i): measuring, in a blood sample obtained from the patient” of step (i) of the first aspect reads “obtained” in past tense - i.e. as understood by the skilled person the blood sample has earlier been obtained from the patient and the earlier taking of the sample is therefore not part of the method of the first aspect as such.

[0035] Said in other word, the blood sample is earlier obtained in a suitable way and then provided to the site (e.g. laboratory), where the in vitro method of the first aspect as such is performed.

[0036] Embodiments of the present invention are described below, by way of examples only.

[0037] Drawing description

[0038] Figure 1 : Discussed in working Example herein - illustrate IFN-signature is more elevated for HSE as compared to HSV meningitis.

[0039] Figure 2: Discussed in working Example herein - illustrate IFN-signature is more elevated for HSE as compared to HSV meningitis - data for individual genes are shown.

[0040] Figure 3: Discussed in working Example herein - illustrate IFN-signature at day 21 is more elevated for AE post-HSE patients than patients who do not develop this complication.

[0041] Figure 4: Shows a correlation between median of 6 genes included in the IRG signature according to Rice et al and the median of the 6 genes most freguently elevated in the HSE patients.

[0042] Figure 5: The 2 GM-IRG score using the 2 genes IFI27 and IFI44L showed good correlation with the 6 GM-IRG score, kappa index 0.8202, p<0.0001.

[0043] Definitions

[0044] In general - technical terms used herein should be understood as the skilled person would understand them in the present context in view of the prior art.

[0045] In line with this - below are provided comments to some of the herein relevant used technical terms.

[0046] The term “autoimmune encephalitis (AE) post-HSE" relates to the known HSE related symptom AE post-HSE. As discussed above, distinction between AE post-HSE and recrudescence of residual deficits or new manifestations related to persistent viral infection is difficult and may hold up treatment decisions, and the article by Armangue T et al. describes the presence of CSF neuronal antibodies at day 21 after HSE onset as a diagnostic predictor of AE post-HSE. The skilled person (e.g. medical personal) knows how to determine if a patient has AE post-HSE or not.

[0047] The term “control group of human persons" relates to human persons used to obtain the “control type I IFN-signature” of step (ii) of the first aspect. As discussed herein, depending on e.g. the specific overall diagnostic purpose of the method as described herein - the control group may be a healthy control (HC) group, a group of persons with herpesviral meningitis (HSV meningitis), etc.

[0048] The term “control type I IFN-signature" of step (ii) of the first aspect relates to the control / reference level of the control type I IFN-signature used in the method of the first aspect.

[0049] As understood by the skilled person, the control / reference level of the control type I IFN-signature (i.e. the “control type I IFN-signature” as such) may be a known control type I IFN-signature (e.g. described in the prior art - see e.g. herein relevant cited articles) or may have been obtained “de novo" at e.g. a relevant site (e.g. a hospital, clinic, etc.) of interest (see e.g. working Examples herein).

[0050] The skilled person knows how to make a herein relevant control type I IFN-signature - i.e. knows how to e.g. optimize the control type I IFN-signature (e.g. including more human person in the control group, choose preferred IFN-signature measurement time, etc) in order for that the control IFN-signature has an expression level that is representative of the control group of interest (e.g. a healthy control group, a HSV meningitis related control group, etc).

[0051] The term "deviating” relates to a difference, or variation from a standard or control group.

[0052] The term "deciding” of step (iv) of the first aspect relates to the deductive medical decision phase - i.e. decide if the patient IFN-signature of step (i) is an elevated patient IFN-signature as compared to the control IFN-signature of step (ii) or not (i.e. if it is a “positive IFN-signature” or not as defined herein). As said in step (iv) - if it is a positive (i.e. elevated) IFN-signature, then is this indicating a relatively high probability for that the human patient has or may develop a HSE related symptom - i.e. provide a probability diagnosis of the patient of interest, which can be used to e.g. make optimal specific / preferred treatment of the patient of interest.

[0053] The term “encephalopathy” refers to abnormal mental status secondary to any disorder. In modern usage, encephalopathy does not refer to a single disease, but rather to a syndrome of overall brain dysfunction; this syndrome has many possible organic and inorganic causes - such as e.g. mitochondrial encephalopathy, glycine encephalopathy, herpes simplex encephalitis (HSE), etc.

[0054] The term “encephalitis” refers when encephalopathy is secondary to an inflammatory disorder of the brain (e.g. Autoimmune causes, virus, etc.). Most causes of encephalitis have an acute presentation (symptoms develop in hours or days).

[0055] The term “acute encephalopathy symptoms” refers to symptoms of encephalopathy in a patient that have been developed in a relatively short time frame (e.g. hours or days) - such as when a patient has abnormal level of consciousness or abnormal mental status for the last hours or days. The skilled person (e.g. medical personal) knows how to determine if a patient has acute encephalopathy symptoms or not. The acute encephalopathy symptoms may relate to a patient with suspected encephalitis (i.e. not know for sure if patient has encephalitis). As understood, a patient with confirmed encephalitis (e.g. HSE) has acute encephalopathy symptoms (e.g. HSE related symptoms).

[0056] In the present context, a human patient with acute encephalopathy symptoms may e.g. also be a patient that e.g. has had confirmed encephalitis (e.g. HSE) and at day of performing the method of the first aspect maybe does not anymore have significant symptoms (as discussed herein could e.g. be of relevance for diagnosing a probability for maybe developing (AE) post-HSE).

[0057] In relation to a human patient with acute encephalopathy symptoms as such - the skilled person (e.g. doctor) does not as such have to know if the patient is a patient with herpes simplex virus (HSV) or not.

[0058] The term "healthy control (HC) group” in relation to an example of a control group of human persons of step (ii) first aspect - relates to a healthy group of persons who had no known signs of illnesses or chronic inflammatory illnesses at the time when the blood sample was obtained. In the present context, the skilled person (e.g. medical personal) knows how to determine if a person is considered healthy or not.

[0059] The term “herpesviral meningitis (HSV meningitis)" is well known to the skilled person. The skilled person (e.g. medical personal) knows how to determine if a patient has HSV meningitis or not.

[0060] The term “herpes simplex encephalitis or (HSE)” relates to a viral infection that affects the brain. It is caused by the herpes simplex virus (HSV). HSE commonly presents with acute symptoms such as fever, headache, altered mental status, confusion, seizures, focal neurological deficits, and behavioral changes. These symptoms may progress rapidly and become more severe if left untreated. The skilled person (e.g. medical personal) knows how to determine if a patient has HSE symptoms or not.

[0061] The term “HSE related symptom" relates to HSE related symptoms. The skilled person (e.g. medical personal) knows how to determine if a patient has a HSE relates symptom or not.

[0062] As discussed herein - a HSE relates symptom may e.g. be autoimmune encephalitis (AE) post-HSE, HSE as such, etc. In accordance with the art, and as understood by the skilled person in the present context - term “HSE related symptom” is understood to include e.g. HSE related diseases and / or complications - e.g. a patient with a HSE related disease also has a HSE related symptom.

[0063] The term “herpes simplex virus” or “HSV" relates to members of the herpesviridae family. There are two types of HSV: herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2). The skilled person knows or can routinely determine by for example PCR analysis if a human patient is a patient with herpes simplex virus (HSV) or not.

[0064] The term “HSE onset’ relates to the initial appearance or occurrence of symptoms or signs of HSE - i.e., it signifies the time when the symptoms of the disease appear in the body or when a patient first experiences the manifestations of HSE. The skilled person (e.g. medical personal) knows how to determine HSE symptoms and thereby the “HSE onset’ day.

[0065] The term “HSV disease onset’ relates to the initial appearance or occurrence of symptoms or signs of HSV presence - e.g. symptoms such as e.g. fever. The skilled person (e.g. medical personal) knows how to determine HSV presence related symptoms and thereby the “HSV disease onset’ day.

[0066] The term “IFN-signature" relates to a well know term in the art (see e.g. Kim et al. 2018), and it relates to the measurement of the expressions levels of one or more interferon (IFN)-response genes (IRG) - e.g. by measurement of elevated / higher RNA expression. The IFN-signature may be based on one or more IRG - i.e. at least one (IFN)-response gene (IRG).

[0067] The first aspect herein reads: “if the patient IFN-signature of step (i) is elevated, then is the patient IFN- signature defined as a positive IFN-signature” - in line with this, if the patient IFN-signature of step (i) is not elevated, then may it herein be termed a negative IFN-signature.

[0068] The term “patient" relates in relation to e.g. “a human patient with acute encephalopathy symptoms” essentially to any person with acute encephalopathy symptoms. Similar - “a human patient with herpes simplex virus (HSV)” relates essentially to any person with HSV. In the present context, a human patient with acute encephalopathy symptoms may e.g. also be a patient that e.g. has had confirmed encephalitis (e.g. HSE) and at day of performing the method of the first aspect maybe does not anymore have significant symptoms (as discussed herein could e.g. be of relevance for diagnosing a probability for maybe developing (AE) post-HSE).

[0069] The term “positive IFN-signature" relates to an IFN-signature that exhibit an elevated level of expression as compared to an IFN-signature from a human control group. Thus, a positive IFN signature indicates the activation of the innate immune response, particularly the production of type I interferons, in response to viral infection or other stimuli.

[0070] The term “probability” relates to an increased likelihood or susceptibility of an individual to have or to develop a particular condition or disease. It implies a higher probability or inclination towards a particular outcome or characteristic, although it does not guarantee that the outcome will necessarily occur.

[0071] The term “type I IFN-signature" relates to measurement of elevation of type I IFN response genes (IRGs) expression. The skilled person knows or may routinely determine if a IFN of interest is a type I IFN or not - i.e. knows or may routinely determine if a IFN-signature is a type I IFN-signature or not (see e.g. Kim et al. 2018).

[0072] The term “viral complications" in relation to the HSE symptoms as discuses herein, relates to viral complications of HSE or when symptoms of the patient are directly related to a direct virus effect. The skilled person (e.g. medical personal) knows how to determine if a patient has viral complications of HSE or not.

[0073] Detailed description of the invention

[0074] Measuring expression level - step (i) of first aspect

[0075] As discussed above - step (i) of the first aspect reads:

[0076] “(i): measuring, in a blood sample obtained from the patient, the expression level of at least one type I interferon (IFN)-response gene (IRG) to obtain a patient type I IFN-signature”. Without being limited to theory - it is believed that the technical disclosure herein (e.g. experimental data of Examples herein) makes it plausible that one may obtain herein relevant useful diagnosis by using only one IRG gene (see e.g. Figure 2).

[0077] As discussed in working Examples herein - very good / reliable results were obtained by use of 6 different IRGs - but based on the technical disclosure herein and the common general knowledge of the skilled person - it is believed to be plausible that use of less IRGs may also provide herein useful results.

[0078] Preferably, the patient IFN-signature of step (i) is made based on measurement of at least 2 different IRGs, more preferably at least 3 different IRGs, even more preferably at least 4 different IRGs (such as at least 5 different IRGs), and most preferably at least 6 different IRGs.

[0079] The measuring of the expression level in step (i) may be done according to the art - e.g. by measuring the mRNA levels of the respective IRGs.

[0080] According to the art, known methods for measuring the mRNA levels of the respective IRGs include e.g. Reverse Transcription Polymerase Chain Reaction (RT-PCR), Quantitative Real-Time PCR (qPCR) that allows for real-time monitoring of the amplification process, or by Microarray Analysis where the mRNA from the sample is reverse transcribed into cDNA and then labelled with fluorescent tags. The labelled cDNA is hybridized with e.g. an microarray, and the fluorescent signals emitted from the bound cDNA molecules are detected and quantified.

[0081] In working Examples herein - the mRNA levels of the respective IRGs were measured by NanoString Technologies (Seattle, WA), which uses molecular “barcodes” and an optical scanner to detect and simultaneously count up to several hundred unique target gene sequences in a single hybridization reaction without requiring amplification. The skilled person knows how such methods are performed.

[0082] The prior art describes several IRGs - i.e. the skilled person knows several different IRGs and may, based on the technical disclosure herein and common general knowledge - identify herein relevant suitable IRGs.

[0083] In working Examples herein useful results were obtained using the 28 genes (Entrez gene symbol): CXCL10, DDX60, EPSTI1, GBP1, HERC5, HERC6, IFI27, IFI44, IFI44L, IFI6, IFIT1, IFIT2, IFIT3, IFIT5, ISG15, LAMP3, LY6E, MX1, OAS1, OAS2, OAS3, OASL, RSAD2, RTP4, SIGLEC1, SOCS1, SPATS2L, USP18.

[0084] In Yao et al. (Hum Genomics Proteomics 2009; DOI: 10.4061 / 2009 / 374312), a combination of 20 of these genes (EPSTI1, HERC5, IFI27, IFI44, IFI44L, IFI6, IFIT1, IFIT3, ISG15, LAMP3, LY6E, MX1, OAS1, OAS2, OAS3, RSAD2, RTP4, SIGLEC1, SPATS2L, USP18), and also and additional gene PLSCR1 not included in the 28 genes were used - so it is believed that this gene PLSCR1 may be considered equivalently useful.

[0085] Accordingly, in a preferred embodiment - the at least one IRG of step (i) is at least one IRG selected from the group of genes consisting of (Entrez gene symbol): CXCL10, DDX60, EPSTI1, GBP1, HERC5, HERC6, IFI27, IFI44, IFI44L, IFI6, IFIT1, IFIT2, IFIT3, IFIT5, ISG15, LAMP3, LY6E, MX1, OAS1, OAS2, OAS3, OASL, RSAD2, RTP4, SIGLEC1, SOCS1, SPATS2L, USP18, and PLSCRI.

[0086] As known in the art - the Entrez gene symbol or gene name is a standardized and unique identifier used to represent a specific gene in the National Center for Biotechnology Information (NCBI) Entrez Gene database. Each gene symbol is a short, alphanumeric abbreviation or name assigned to a specific gene based on its biological function, gene family, or other relevant characteristics - see e.g. table 2 of Kim et al and the NCBI.

[0087] Accordingly, the Entrez gene symbol is a unique identifier of herein discussed IRG genes - i.e. based on the Entrez gene symbol (e.g. CXCL10, DDX60, etc.) the skilled person knows the relevant sequence of the IRG gene.

[0088] As known - an Entrez gene symbol may relate to a human and / or not human gene - for instance, the Entrez gene symbol CXCL10 relates to a human gene (Gene ID: 3627) and a mouse gene (Gene ID: 15945).

[0089] Since the method of the first aspect relates to a method for diagnosing a human patient - the relevant gene is of course a human gene.

[0090] In table below is shown examples of herein preferred IRGs

[0091]

[0092] 1Maglott D, et al. Entrez Gene: gene-centered information at NCBI. Nucleic Acids Res. 2007; 35: D26-

[0093] D31

[0094] In working Examples herein useful results were obtained using the 6 genes most expressed in HSE patients or AGS patients in the tested cohort IFI27, IFI44, IFI44L, ISG15, RSAD2, and SIGLEC1. However, in Rice et al - IFIT1 was used instead of IFI44 and based on the technical disclosure herein and the skilled person common general knowledge - it is believed that these two genes may be considered equivalently useful.

[0095] Accordingly, more preferably the at least one IRG of step (i) is at least one IRG selected from the group of genes consisting of (Entrez gene symbol): IFI27, IFI44, IFI44L, ISG15, RSAD2, IFIT1, IFI6, USP18, and SIGLEC1 - even more preferably, the at least one IRG of step (i) is at least one IRG selected from the group of genes consisting of (Entrez gene symbol): IFI27, IFI44, IFI44L, ISG15, RSAD2, IFIT1 and SIGLEC1 - and most preferably, the at least one IRG of step (i) is at least one IRG selected from the group of genes consisting of (Entrez gene symbol): IFI27, IFI44, IFI44L, ISG15, RSAD2, and SIGLEC1.

[0096] Comparing the measured type I IFN-siqnature - step (ii) of the first aspect

[0097] As discussed above - step (ii) of the first aspect reads:

[0098] “(ii): comparing the measured patient type I IFN-signature of step (i) to a control group of human persons with a reference control type I IFN-signature with the same IRG as in step (i)”

[0099] Based on the technical disclosure herein and the common general knowledge - the skilled person knows how make such a comparison - i.e. comparing the patient type I IFN-signature with the control type I IFN-signature by comparing the respective IRG expression levels (e.g. measured via mRNA). In working Examples were e.g. used the known so-called “Geomean-score-based IFN score calculation” as described in e.g. Kim et al., and / or the median of the Z scores of all genes included in each signature (e.g. 6 or 28 gene typel IFN-signature). - i.e. this may be the preferred methods for comparing the data of the relevant IFN-signatures.

[0100] As understood - the term “median” is only of technical relevance if more than one IRG gene is used.

[0101] If more than one IRG is used (e.g. 6 different IRGs) then is the Z-score herein the median Z-score.

[0102] As discussed in working Examples herein and in Kim et al. - a Z-score for each gene is calculated according to the below equation.

[0103] > — > Normalized gene count patient— mean Normalized gene counts of the HCs

[0104] -score ( s ) e n e—Standard deviation (SD) of the normalized gene counts in the HCs

[0105] In relation to the equation above - may be noted that the abbreviation “HC” is herein (e.g. in working Examples) used in relation to healthy control.

[0106] However, and as discussed herein - the “control group of human persons” of step (ii) of the first aspect is not always healthy control persons (may e.g. alternatively be a group of persons with HSV meningitis).

[0107] Accordingly, and as understood by the skilled person in the present context - the abbreviation “HC” in the above Z-score equation refers to “control group of human persons” as such of a step (ii) of the first aspect.

[0108] If not said otherwise herein - it is understood that the “control group of human persons” of step (ii) is preferably a “healthy control (HC) group”.

[0109] As understood - if the expression level of patient type I IRG (i.e. Normalized gene count patient) is higher than control type I IRG (i.e. mean Normalized gene counts of the HCs) - then will the Z-score be above 0. If the expression level of patient type I IRG is equal than control type I IRG - then will the Z-score be 0. Usually a Z-score is considered elevated (positive) if the Z score is >1 or 2 (equivalent to >1 or 2 standard deviations above the HC levels of the mRNA levels of those genes).

[0110] - i.e. there is a positive or elevated IFN-signature as defined herein - see e.g. step (iv) of first aspect.

[0111] As discussed in working Examples herein and in Kim et al. - a way to compare the results is by using the so-called type I IFN standardized score (alternatively termed Z score), which is the summatory of the Z scores for each of the genes included in the type I IFN signature as described by Kim et al.

[0112] Also, other methods such levels of mRNA quantifications of the genes superior to HC group (e.g. 1 or 2 standard deviations above the HC levels of the mRNA levels of those genes) as described by Rice et al. The skilled knows how to correlate median Z-score values to other known measuring / calculating methods such as e.g. Z score, 1 or 2 fold change of normalized gene counts, Geomean score, median Z scores, standardized Z base score, 1 or 2 fold change in mRNA levels of each gene or of the summatory of the genes - i.e. the skilled person knows how to e.g. correlate a specific Z-score to a corresponding relevant e.g. Geomean score.

[0113] Accordingly, and as understood by the skilled person - it is herein not essential how the comparison / analysis of the relevant IFN-signature data is actually done (i.e. by, median Z-score, standardized based Z score, Geomean score, two fold change in mRNA levels, etc.).

[0114] Analyzing the compared data of step (ii) - step (Hi) of the first aspect.

[0115] As discussed above - step (iii) of the first aspect reads:

[0116] “(iii): analyzing the compared data of step (ii) to identify if the patient type I IFN-signature of step (i) is deviating from the control IFN-signature of step (ii)”

[0117] The skilled person knows how make such an analysis - e.g. based on Z-score, median Z-scores and / or Geomean score values as discussed above.

[0118] Deciding if the patient IFN-signature is a positive IFN-signature - step (iv) of the first aspect

[0119] As discussed above - step (iv) of the first aspect reads:

[0120] (iv): deciding if the possible deviation in step (iii) relates to an elevated patient IFN-signature of step (i) as compared to the control IFN-signature of step (ii) and if the patient IFN-signature of step (i) is elevated, then is the patient IFN-signature defined as a positive IFN-signature, indicating a relatively high probability for that the human patient has or may develop a HSE related symptom”

[0121] Based on the technical disclosure herein and the common general knowledge - the skilled person knows how to decide / determine if patient IFN-signature is an elevated patient IFN-signature (i.e. a positive IFN-signature) - e.g. based on Z-score, median Z-score, Geomean score or other method calculations values as discussed above.

[0122] As understood - a positive patient IFN-signature (i.e. with higher IRG expression level than control type I IFN-signature) has a positive Z-score - i.e. a Z score >0.

[0123] The skilled person knows how to properly determine e.g. relevant measurement and uncertainty and standard deviations - i.e. it is understood that a positive patient IFN-signature is understood to be elevated or positive as determined by the skilled person in relation to relevant uncertainty parameters - such as e.g. measurement uncertainty and standard deviations. Preferably, - a positive patient IFN-signature (i.e. with higher IRG expression level than control type I IFN-signature) has a positive Geomean score if the geomean score of the IRG included in the IFN- signature are > 95 percentile of the Geomean score in HC.

[0124] Preferably, for each gene, positive Z-score - e.g. a Z score of each gene is >1 .25, such as e.g. >1 .96. Preferably, a positive medium elevated Z-score is a medium Z-score >1.5 (between 1 and 2 standard deviations).

[0125] HSE symptoms of step (iv) is AE post-HSE - an embodiment of first aspect.

[0126] As discussed in e.g. working Examples herein - the present inventors identified that the method as described herein may be particular useful for diagnosing a probability for possible development of autoimmune encephalitis (AE) post-HSE or viral complications.

[0127] As discussed herein (see e.g. working Examples and Figure 3) - compared to a healthy control (HC) group and measured around 21 days after HSE onset - relatively lower or medium elevated IFN signature values (e.g. median Z score of the 6 genes included in the example signature >1 .5 but Z <4) relate in particular to a relatively high probability for (AE) post-HSE and higher values (median Z scores of the 6 IRG included in the signature > 4) relate to viral complications.

[0128] Without being limited to theory - even though the working Examples herein refers to median Z score >1 .5 - it is believed that herein useful results for e.g. (AE) post-HSE may be obtained if the median Z score is positive - i.e. with an elevated patient IFN-signature around 10 to 40 days after HSE onset.

[0129] Accordingly, in a preferred embodiment - the method for diagnosing of the first aspect and / or embodiments thereof is a method:

[0130] - wherein the control group of human persons of step (ii) of the first aspect is a healthy control (HC) group; and

[0131] - wherein the patient has HSE and the measuring in step (i) of the first aspect is done in a period from 10 to 40 days after HSE onset, preferably from 15 to 30 days after HSE onset, and more preferably at day 21 after HSE onset; and

[0132] - wherein the HSE related symptoms of step (iv) of the first aspect is autoimmune encephalitis (AE) post-HSE or viral complications.

[0133] The term “has HSE” of the embodiment immediately above includes a patient that have had HSE, but a time measuring in step (i) maybe does not have any remaining significant HSE symptoms.

[0134] In relation to the preferred embodiment immediately above - preferably, the positive patient IFN signature of step (iv) of the first aspect has a Z score (preferably a median Z score) between 1 .5 to 4, determined according to the method identified in the specification; and the HSE related symptoms of step (iv) is autoimmune encephalitis (AE) post-HSE.

[0135] In relation to the preferred embodiment immediately above - it may be that a very high positive patient IFN signature of step (iv) of the first aspect has Z score (preferably a median Z score) >4, determined according to the method identified in the specification - which is indicating a probability for that the human patient is developing viral complications HSE related symptoms.

[0136] As discussed above - the skilled knows how to correlate e.g. median Z-score values to other known measuring / calculating methods such as e.g. standard deviations of normalized gene counts, Geomean score, Standardized Z score (summatory of Z scores), fold changes of mRNA levels, etc.

[0137] Accordingly, and as understood by the skilled person - it is herein not essential how the comparison / analysis of the relevant IFN-signature data is actually done (i.e. by, Z-score, Geomean score, etc.).

[0138] Consequently, in relation to e.g. the embodiment above referring to “Z score between 1.5 to 4” - it is not required that the IFN-signatures have been comparted / analyzed with Z score as such - i.e. other measuring / calculating methods may be used (e.g. Geomean score) and then correlated to “Z score between 1 .5 to 4”.

[0139] HSE related symptoms of step (iv) is HSE (distinguish between probability for meninqitis / HSE) - an embodiment of first aspect.

[0140] As discussed in e.g. working Examples herein - the present inventors identified that the method as described herein may be particular useful for distinguish between probability for development of HSV meningitis or the more severe HSE as such.

[0141] As illustrated in e.g. Figures 1 and 2 - the present inventors identified - that if the patient IFN-signature is positive relatively close to HSV disease onset as compared to a HSV meningitis control IFN- signature - then is this indicating a relatively high probability for that the patient develops HSE instead of HSV meningitis.

[0142] As known in the art - the clinical outcome of HSE may be relatively bad / poor, and usually relatively good for HSV meningitis - i.e., it is important to early get knowledge / diagnosis of the possible patient probability.

[0143] Accordingly, in a preferred embodiment - the method for diagnosing of the first aspect and / or embodiments thereof is a method:

[0144] - wherein the control group of human persons of step (ii) of first aspect is a group of persons with herpesviral meningitis (HSV meningitis); and

[0145] - wherein the human patient with encephalopathy symptoms is a human patient with herpes simplex virus (HSV); and - wherein the measuring in step (i) of first aspect is less than 7 days after HSV disease onset; and

[0146] - wherein the HSE related symptoms of step (iv) of first aspect is HSE.

[0147] As understood in relation to the embodiment immediately above - if the patient IFN-signature of step (iv) is negative, then is this an indication for that the patient does not have a relatively high probability for getting HSE as such - i.e. the patient may be understood to more likely get the less severe HSV meningitis as compared to the more severe HSE.

[0148] As understood by the skilled person and in line with working Examples herein - the HSV meningitis control type I IFN-signature of step (ii) relates to a control IFN-signature - i.e. a control IFN-signature where the IRG expression levels also were measured relatively close to disease onset and where it is known that the patients of this group later develops HSV meningitis.

[0149] The skilled person knows how to make a herein relevant control type I IFN-signature - such as a HSV meningitis control type I IFN-signature - i.e. knowns how to e.g. optimize the control type I IFN- signature (e.g. including more human person in the control group, choose preferred IFN-signature measurement time, etc.) in order for that the control IFN-signature has an expression level that is representative of the control group of interest (e.g. a healthy control group, a HSV meningitis related control group, etc.).

[0150] Preferably, the positive patient IFN-signature of step (iv) first aspect has a Z score of at least 2 times higher than the HSV meningitis control type I IFN-signature of step (ii) of first aspect, determined according to the method identified in the specification.

[0151] Remarks generally relevant to all aspects / embodiments herein.

[0152] As discussed above, in relation to a human patient with encephalopathy symptoms as such - the skilled person (e.g. doctor) does not as such have to know if the patient is a patient with herpes simplex virus (HSV) or not.

[0153] This may e.g. be a situation of relevance, if the overall purpose of the method is to identify / diagnose if the patient with encephalopathy symptoms has or may develop a HSE related symptom or the patient has the encephalopathy symptoms for other reason (as e.g. mitochondrial encephalopathy, glycine encephalopathy, etc).

[0154] It is evident - that a patient that has HSE is a patient with herpes simplex virus (HSV).

[0155] In any case - it may be relevant / preferred to test if the patient is a patient with herpes simplex virus (HSV) - e.g. via routine PCR technique. As discussed herein - in step (iv) of the first aspect may be identified / diagnosed the probability of different types of HSE related symptoms - e.g. HSE as such, (AE) post-HSE, etc.

[0156] As understood - based on this highly valuable diagnosis information may be patient be treated in optimal / preferred way.

[0157] For instance, if the identified / diagnosed HSE related symptom is (AE) post-HSE - then may the patient be treated in optimal / preferred way according to the art.

[0158] Similarly, if the identified / diagnosed HSE related symptom is e.g. viral complication or e.g. HSE as such - then may the patient be treated in optimal / preferred way according to the art.

[0159] EXAMPLES

[0160] Material and methods Study design and participants

[0161] Patients of all ages with HSE diagnosed between January 1st, 2014, and December 31 st, 2021 , were prospectively included in one of two cohorts depending on whether the recruitment was at the onset of the viral infection (Cohort A) or by the time they presented with relapsing neurological symptoms (Cohort B). In Cohort A, patients from 28 participating centers in Spain were recruited at symptom onset of HSE; and in Cohort B, patients from Spain or other countries were recruited as soon as their physicians suspected AE post-HSE. For all patients, the diagnosis of HSE was confirmed with CSF PCR for HSV type 1 or 2 (tested at the local institutions) and brain MRI showing lesions compatible with HSE (studies performed at local institutions, with all images centrally reviewed at IDIBAPS- Hospital Clinic of Barcelona). Patients identified at Hospital Clinic or Sant Joan de Deu Childrens' Hospital of Barcelona who during the same period developed meningeal symptoms with CSF PCR positive for HSV1 or 2, and brain MRI without parenchymal lesions, were included as “HSV meningitis”. Blood-extracted RNA samples from 22 healthy controls (HCs) and from 11 patients with monogenic type I interferonopathy Aicardi Goutieres Syndrome (AGS) were used as controls for the IFN signature studies (described below). DNA samples from 36 Spanish patients with anti-NMDAR encephalitis not related to HSE were used as controls for HLA studies (described below).

[0162] For Cohort A, clinical information was provided by the treating physicians using standardized questionnaires at several time points: at HSE diagnosis, after discontinuation of acyclovir (~21 days after symptom onset) and at 2, 6, and 12 months after HSE onset. IgG antibodies against NMDAR and other neural surface proteins were systematically examined in paired CSF / serum samples obtained at diagnosis and on day 21 , and in serum at 2, 6, and 12 months. All neural antibody studies were performed at IDIBAPS-Hospital Clinic of Barcelona using established techniques.24, 25 Investigators performing antibody testing were blind to clinical information, and treating physicians were blind to antibody results unless patients developed new or unexplained progression of neurological deficits. If this occurred, new paired CSF / serum samples were obtained and the results, along with those of previous samples, were provided to the corresponding physician.

[0163] For Cohort B, clinical information was provided by the treating physicians using a standardized questionnaire, and serum and CSF samples were obtained by the time of symptom presentation suggesting AE post-HSE. In this setting, the results of antibody studies were immediately provided to the treating physicians.

[0164] Patients were considered to have AE post-HSE, if within the first 12 months after completing treatment with acyclovir they developed new onset CNS symptoms or worsening of pre-existing deficits that lasted for more than 24 hours, accompanied by negative CSF PCR for HSV, positive CSF neuronal surface antibodies, and absence of alternative etiologies (e.g., metabolic derangement, drug toxicity, stroke).

[0165] Regarding treatment, all patients received intravenous acyclovir (60mg / kg / day in children <12 years; 30mg / kg / day in children >12 years and adults; in all cases divided into three daily doses) for 14-21 days. Concurrent use of steroids, and use of immunotherapy for patients who developed AE, were at physician’s discretion. Clinical outcome was assessed with the modified Rankin scale (mRS)26 or the mRS adapted for children.27

[0166] In addition to the mentioned CSF / serum antibody studies, we obtained biological samples that included whole blood for DNA studies, and from Cohort A since January 2017, serial blood samples collected in PAXgene® tubes for RNA investigations at HSE onset, 21 days, and at 2, 6 and 12 months after HSE onset.

[0167] Fifty-one patients from cohort A and 48 patients from cohort B were included in a previous study focused on symptoms and antibody studies;2 no data about HLA, WES or IFN studies have been previously reported.

[0168] Essentially - measurement of Expression levels of Type I IFN signatures genes was done as described in Kim et al - i.e. based on similar primers (e.g. PCT primers), etc.

[0169] Accordingly, based on common general knowledge, herein cited prior art (e.g. Kim et al), and the technical information below - the skilled person may routinely repeat the examples below to e.g. verify the below discussed results.

[0170] Just as a note and as known in the art - the specific choice of primers (e.g. PCT primers) are as such not essentially - i.e. one way vary primer sequence and still herein relevant similar results.

[0171] Total RNA was extracted using PAXgene® Blood RNA Kit (Qiagen). RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies) and RNA integrity was determined with Agilent 2100 Bioanalyzer (Agilent Technologies). Expression levels of 28 type I IFN inducible or IFN response genes (IRG): CXCL10, DDX60, EPSTI1 , GBP1 , HERC5, HERC6, IFI27, IFI44, IFI44L, IFI6, IFIT1 , IFIT2, IFIT3, IFIT5, ISG15, LAMP3, LY6E, MX1 , OAS1 , OAS2, OAS3, OASL, RSAD2, RTP4, SIGLEC1 , SOCS1 , SPATS2L, USP18; and 4 housekeeping genes (ALAS1 , HPRT1 , TBP and TUBB) were determined using the nCounter® Analysis System (NanoString Technologies) at the Functional Genomics Core facility of IDIBAPS.20 Counts of target genes were normalized (normalized gene counts) to internal synthetic positive controls and housekeeping genes. Then, a Z-score (Zs) for each IFN inducible gene or IRG was calculated after normalization with the mean and standard deviation (SD) of the control group (healthy controls, HCs):

[0172] > — > Normalized gene count patient— mean Normalized gene counts of the HCs

[0173] -score ( s ) e n e—Standard deviation (SD) of the normalized gene counts in the HCs

[0174] Each type I interferon-stimulated genes were considered overexpressed or activated if the Zs of the gene was above 1.96 (>percentile 95 or 2 SD). To assess if the main components of the type I IFN pathway were activated, a previously validated score in monogenic interferonopathies was used, that calculates the geomean of the 28 type I IFN response genes (divided by 10) (Kim et al. 2018).

[0175] Geomean score

[0176] Where g1 to g28 corresponds to the normalized gene counts for each of the 28 IFN response genes, and for the score reporting, the geomean was divided by 10, which scales the ranges of the IRG-GM score to be similar to the ranges of Zscore-based (standardized) score, an alternative method for calculation of the IFN score described in Kim et al.

[0177] Given that in monogenic type I interferonopathies such as the Aicardi-Goutieres syndrome (AGS) and in autoimmune disorders such as systemic lupus erythematous, simplier type I IFN signature scores that include a combination of 5 or 6 of the indicated IRG are commonly used to assess the type I IFN pathway activation (Kim et al, Table 3), we first assessed whether this paradigm was also applicable to HSE. To this end, we used the geometric mean of 6 out of the 28 above indicated type I IFN response genes that had the higher Z scores in our patients with HSE and AGS, divided by 10, to generate the 6 IRG-GM score; the 6 genes included: IFI27, IFI44, IFI44L, ISG15, RSAD2 and SIGLEC1 :

[0178] 6 IFN response genes geomean (IRG-GM) Score =(6^(g1*g2*..g6)) / 10. g1 ...g6 = Normalized gene count for each of the 6 IFN response genes (IRG)

[0179] The 95th percentile of the 22 HCs for the 28-IRG-GM and 6-IRG-GM scores were calculated as reported by Kim et al. 2018 and served to establish the cutoff between normal and elevated scores: 109.1 IC95% (783-114.3) for the 28-IRG-GM and 130.6 IC95% (85.8-136.2) for the 6-IRG-GM.

[0180] Results

[0181] The results show that the 6-IRG-GM and the 28-IRG-GM scores were highly correlated (Pearson’s r=0.967, kappa index for positive / negative scores: 0.86 for all samples), and with other combinations of IRG genes (data not shown). Due to this correlation along with the mildly increased sensitivity and simplicity of the 6-IRG-GM score compared with the 28-IRG-GM score, we used the 6-IRG-GM score for the subsequent analyses.

[0182] Overall, blood RNA samples obtained at the indicated time points were available for the IFN signature from 54 patients of Cohort A. Samples obtained at HSE onset (<7 days from symptom onset) were available from 21 patients; of these, 19 (91%) showed a positive IFN signature (positive 6-IRG-GM score). In the acute phase of HSE, the IFN signature scores were similar to those of patients with AGS interferonopathy (median Zs of the 6 IRG: 12.6 [IQR 7.1 ; 22.4] in HSE vs 25 [20.0; 38.1] AGS, p=0.0727). These scores were significantly higher than those of HC (median Zs of the 6 IRG: -0.4 [- 0.6; 0.44] (p<0.001) and patients with HSV1-2 meningitis (median Zs of the 6 IRG: 1 .5 IQR [-0.3; 1 ,8] p<0.0037, Figure 1 and 2). Only one of 7 (14%) patients with HSV1-2 meningitis had a positive IFN signature at disease onset. In this case, the IFN signature was permanently elevated during follow-up and was found to be related to a chronically active systemic inflammatory disease (Crohn’s disease and epidermolysis bullosa). The other 6 patients with meningitis had none (1 patient) or 1 IFN response gene activation (5 patients, all IFI27), in comparison with a median of 6 / 6 (IQR 6-6) activated genes in HSE or AGS patients (p=0,0003). Of the 6 IFN response genes included in the IFN signature, IFI27 had the highest Zs in patients with HSE (median Zs of the 6 IRG: 50 IQR [17; 187]) or AGS (median Zs of the 6 IRG: 69 IQR [50; 164]).

[0183] Among HSE patients with RNA samples available at subsequent time points, the percentage of cases with positive IFN signatures progressively decreased with time: 8 / 40 (20%) at ~21 days, 4 / 32 (13%) at 2 months, 4 / 28 (14%) at 6 months, and 2 / 22 (9%) at 12 months. Similarly, the median Zs of the 6 genes and the number of activated genes decreased with time. The median Zs of the 6 genes in patients with HSE at 21 days after disease, onset was mildly increased compared with those of healthy controls (median Zs of the 6 IRG: 0.5 [-0,4; 0.9] vs median Zs or the 6 IRG: -0.4 [-0,7; 0,4], p=0.0454); no differences were identified in subsequent time points.

[0184] Considering that the activation of the IFN pathway should predominantly occur in the acute phase of the viral infection (e.g., <7 days from onset of HSE), 14 of 54 (26%) patients had an unexpected IFN signature (>7 days after HSE onset and initiation of acyclovir). Of these 14 patients, 7 had a positive signature at day 21 and became negative afterwards, another 5 patients had a transient positive signature during follow-up, in which an activation of the IFN pathway by a non-HSE intercurrent viral infection cannot be excluded, and the remaining 2 patients had a sustained positive IFN signature for several months (>3 time points during follow-up); both cases had an atypical HSE course that suggested a persistent viral infection.

[0185] We next assessed whether the IFN signature was associated with development of neuronal antibodies or AE after HSE. Studies on day 21 (which is when neuronal antibodies start becoming positive or detectable), showed that patients who developed AE had higher median IFN signature compared with those who did not develop AE (median Zs of the 6 IRG: 1.4 [0.6; 2.0] vs 0.2 [-0.4; 0.8], p=0.03). No differences were found between the median INF signature of patients with HSE who developed neuronal antibodies without symptoms of AE and those with HSE who did not develop neuronal antibodies (median Zs of the 6 IRG: 0.7 [-0.2; 5.2] vs -0.2 [-0.6; 0.8], p=0.11) - see e.g. Figure 3.

[0186] Logistic regression analysis showed that having a moderate increase of the 6 gene INF signature at day 21 (median Zs of the 6 IRG >1.5 but <4) was a risk factor for AE (OR 33.0, IQR [2.6-424.0] p= 0.007). However, a very high increase of the IFN signature at day 21 (median Zs of the 6 IRG: 9.5 IQR [7.4; 29.5]) associated with viral complications. When assessing other time points (HSE onset, and 2, 6 or 12 months follow-up) we did not find differences in the IFN median Zs between patients who developed AE and those who did not.

[0187] These findings suggest that in patients who develop AE post-HSE the IFN pathway activation decreases more slowly than in patients that do not develop this complication, such that by day 21 after HSE onset only a moderate increase in the IFN signature (median Zs of the 6 IRG <4) may still be present. However, a very high increase of the IFN signature (median Zs of the 6 IRG >4), or a persistently increased IFN signature, is associated with a persistent viral infection.

[0188] Combination of risk factors for AE

[0189] Multivariate logistic analysis in patients of Cohort A showed that the presence of CSF neuronal antibodies at the 3 week follow-up (OR 5.2; 95%CI 1 .6-16.8, p=0.005) and the absence of HLA-A*02 (OR 6.2; 95%CI 1 .8-22.0, p=0.004) were both independent risk factors for developing AE post-HSE. If the IFN signature at day 21 was included in the multivariate analysis (n=40), having a moderate increase of blood 6 IRG-IFN signature at day 21 (median Zs of the 6 IRG >1.5 but <4) was the only risk factor for developing AE post-HSE that remained statistically significant (OR 34.8 IQR [1 .7-691 .9]).

[0190] Conclusions

[0191] The results show that the 6-IRG-GM and the 28-IRG-GM scores were highly correlated and due to this correlation along with the mildly increased sensitivity and simplicity of the 6-IRG-GM score, this score for the subsequent analyses.

[0192] Samples obtained at HSE onset (<7 days from symptom onset) were available from 21 patients; of these, 19 (91 %) showed a positive IFN signature (positive 6-IRG-GM score).

[0193] The median Zs of the 6 genes in patients with HSE at 21 days after disease onset (available from 40 patients), was mildly increased compared with those of healthy controls.

[0194] Studies on day 21 (which is when neuronal antibodies start becoming positive or detectable), showed that patients who developed AE had higher median IFN signature compared with those who did not develop AE, but lower median IFN signature compared with those who developed viral complications.

[0195] EXAMPLE 2: IFN signature scores using a lower number of genes (2, 3, 4 or 5 genes) Methods

[0196] We (i.e. the inventors) also explored if changes in some of the genes including in the IRG-GM scores also would be equivalent in terms of the IFN signature. For example, we compared the 6 IRG-GM using the 6 genes most elevated in HSE patients in our cohort (IFI27, IFI44, IFI44L, ISG15, RSAD2 and SIGLEC1) with the 6 IRG-GM score using the 6 genes most elevated in AGS patients according Rice et al. (IFI27 IFIT1 IFI44L ISG15 RSAD2 SIGLEC1).

[0197] Additionally, given that the 6 IRG-GM score correlated well with the 28 IRG-GM score, and that the 6 IRG-GM score showed mildly increased sensitivity and specificity that the 28 IRG-GM score, we then explored if simpler scores including a smaller number of genes also correlated well with the 6 IRG-GM. To this end, we used the geometric mean of 2, 3, 4 or 5 out of the 28 above indicated type I IFN response genes that had the higher Z scores in our patients with HSE and AGS, divided by 10, to generate the 2, 3, 4 or 5 IRG-GM scores; using a combination of the 6 genes included in the 6 IRG- GM: IFI27, IFI44, IFI44L, ISG15, RSAD2 and SIGLEC1 : n IFN response genes geomean (IRG-GM) Score =(n^(g1*g2*..gn)) / 10. g1 ...gn = Normalized gene count for each of the n IFN response genes (IRG) n=number of genes included in the signature (2 to 5)

[0198] The 95th percentile of the 22 HCs for the different IRG-GM scores were calculated as reported by Kim et al. 2018 and served to establish the cutoff between normal and elevated scores.

[0199] Results:

[0200] Comparison between of different 6 IRG-GM scores including the 6 genes most frequently elevated in our HSE patients (IFI27, IFI44, IFI44L, ISG15, RSAD2 and SIGLEC1) or the 6 genes most frequently elevated in AGS patients according Rice et al. (IFI27, IFIT1 , IFI44L, ISG15, RSAD2 and SIGLEC1) showed that both 6 IRG-GM correlated perfectly and were equivalent (kappa index 1.0, p<0.0001) - see Figure 4, which show a correlation between median of 6 genes included in the IRG signature according to Rice et al and the median of the 6 genes most frequently elevated in the HSE patients.

[0201] Similar to Figure 4 good correlation were identified between the GM-IRG score calculated using the 6 genes included in the IRG signature according to Rice et al or using the 6 genes most frequently elevated in the HSE patients.

[0202] Comparison of the GM IRG scores using a lower number of genes and the 6 GM-IRG score showed also a good correlation.

[0203] For example, the comparison of the GM IRG score using the 5 genes (IFI27, IFI44, IFI44L, IFI6 and RSAD2) or the 4 genes (IFI27, IFI44, IFI44L and RSAD2) showed a very good correlation with the 6 GM-IRG (kappa index 0.9584, p<0.0001 and kappa index 0.9183, p<0.0001 respectively). Although the IFI27 gene was one of the most relevant genes, comparison of signatures excluding this gene also showed good correlation (index kappa >0.8) with the 6 GM-IRG score. As an example, the signature of 4 genes (IFI44, IFI44L, IFI6, RSAD2) or (IFI44L ISG15 RSAD2 SIGLEC1) but excluding the IFI27 gene showed index kappa of 0.8015 p<0.0001 and index kappa 0.8179, p<0.0001 respectively in comparison with the 6 GM-IRG score.

[0204] Signatures using a lower number of genes as 2 or 3 also showed good correlation with the 6 GM-IRG signature. For example, the 3 GM IRG signature using the IFI27, IFI44L and the RSAD2 genes showed an index kappa of 0.8669, p<0.0001 compared with the 6 GM-IRG signature.

[0205] The 2 GM-IRG score using the 2 genes IFI27 and IFI44L also showed good correlation with the 6 GM- IRG score, kappa index 0.8202, p<0.0001 is shown in Figure 5.

[0206] The single use of one IRG also was useful and correlated well with the 6 GM-IRG.

[0207] For example the counts of the gene IFI44 correlated well with the 6 GM-IRG score, kappa index 0.8246, p<0.0001.

[0208] Conclusions

[0209] The results show that the 6-IRG-GM also correlates with different IRG-GM scores that uses a lower number of genes (2, 3, 4 or 5 genes) in different combinations, and that also correlates well with the determination of a single gene of the IRG.

[0210] REFERENCE LIST

[0211] 1 : Armangue T, Spatola M, Vlagea A, et al. Frequency, symptoms, risk factors, and outcomes of autoimmune encephalitis after herpes simplex encephalitis: a prospective observational study and retrospective analysis. Lancet Neurol. 2018;17(9):760-772.

[0212] 2. Rice Gl, Forte GMA, Szynkiewicz M, et al. Assessment of interferon-related biomarkers in Aicardi- Goutieres syndrome associated with mutations in TREX1 , RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1 , and ADAR: A case-control study. Lancet Neurol. 2013;12(12): 1159-1169.

[0213] 3. Kim H, De Jesus AA, Brooks SR, et al. Development of a Validated Interferon Score Using NanoString Technology. J Interf Cytokine Res. 2018;38(4):171-185.

[0214] 4. Yao et al. (Hum Genomics Proteomics 2009; DOI: 10.4061 / 2009 / 374312).

[0215] 5. Liu Benke et al (J Neurol Sciences 424 (2021) 117394).

Claims

CLAIMS1. A method for diagnosing a probability of a human patient with acute encephalopathy symptoms has or may develop a herpes simplex encephalitis (HSE) related symptom comprising the steps of:(i): measuring, in a blood sample obtained from the patient, the expression level of at least one type I interferon (IFN)-response gene (IRG) to obtain a patient type I IFN-signature;(ii): comparing the measured patient type I IFN-signature of step (i) to a control group of human persons with a reference control type I IFN-signature with the same IRG as in step (i);(iii): analyzing the compared data of step (ii) to identify if the patient type I IFN-signature of step (i) is deviating from the control IFN-signature of step (ii); and(iv): deciding if the possible deviation in step (iii) relates to an elevated patient IFN-signature of step (i) as compared to the control IFN-signature of step (ii) and if the patient IFN-signature of step (i) is elevated, then is the patient IFN-signature defined as a positive IFN-signature, indicating a relatively high probability for that the human patient has or may develop a HSE related symptom.

2. The method for diagnosing of claim 1 , wherein the at least one IRG of step (i) is at least one IRG selected from the group of genes consisting of the genes of the table below:

3. The method for diagnosing of claim 2, wherein the at least one IRG of step (i) is at least one IRG selected from the group of genes consisting of (Entrez gene symbol): IFI27, IFI44, IFI44L, ISG15, RSAD2, and SIGLEC1.

4. The method for diagnosing of any of the preceding claims, wherein the patient IFN-signature of step (i) is made based on measurement of at least 2 different IRGs, more preferably at least 3 different IRGs, even more preferably at least 4 different IRGs (such as at least 5 different IRGs), and most preferably at least 6 different IRGs.

5. The method for diagnosing of claim 4, wherein the at least 6 different IRGs are at least 6 different IRGs selected from the group of claim 2.

6. The method for diagnosing of claim 5, wherein the at least 6 different IRGs are at least 6 different IRGs selected from the group of claim 3.

7. The method for diagnosing of any of the preceding claims:- wherein the control group of human persons of step (ii) of claim 1 is a healthy control (HC) group; and- wherein the patient has HSE and the measuring in step (i) of claim 1 is done in a period from 10 to 40 days after HSE onset, preferably from 15 to 30 days after HSE onset, and more preferably at day 21 after HSE onset; and- wherein the HSE related symptoms of step (iv) of claim 1 is autoimmune encephalitis (AE) post-HSE or viral complications.

8. The method for diagnosing of claim 7, wherein the positive patient IFN-signature of step (iv) of claim 1 has a Z score between 1 .5 to 4, determined according to the method identified in the specification; andwherein the HSE related symptoms of step (iv) is autoimmune encephalitis (AE) post-HSE.

9. The method for diagnosing of any of the claims 7-8, wherein the method is of claim 4 and the Z score is median Z score.

10. The method for diagnosing of any of the claims 7-9, wherein the method is of claim 5 or is of claim 6.11 . The method for diagnosing of any of the claims 1 -6:- wherein the control group of human persons of step (ii) of claim 1 is a group of persons with herpesviral meningitis (HSV meningitis); and- wherein the human patient with acute encephalopathy symptoms is a human patient with herpes simplex virus (HSV); and- wherein the measuring in step (i) of claim 1 is less than 7 days after HSV disease onset; and- wherein the HSE related symptoms of step (iv) of claim 1 is HSE.

12. The method for diagnosing of claim 11 , wherein the positive patient IFN-signature of step (iv) of claim 1 has a Z score of at least 2 times higher than the HSV meningitis control type I IFN-signature of step (ii) of claim 1 , determined according to the method identified in the specification.

13. The method for diagnosing of any of the claims 11-12, wherein the method is of claim 4 and the Z score is median Z score.14 The method for diagnosing of claim 13, wherein the method is of claim 6.

15. The method for diagnosing of any of the preceding claims, wherein the measuring of the expression level in step (i) of claim 1 is done by measuring the mRNA levels of the respective IRGs.

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