METHODS OF TREATING FULMINANT VIRAL HEPATITIS
20220323579 · 2022-10-13
Inventors
Cpc classification
A61K31/522
HUMAN NECESSITIES
A61K31/513
HUMAN NECESSITIES
A61K31/675
HUMAN NECESSITIES
A61K31/498
HUMAN NECESSITIES
A61K48/005
HUMAN NECESSITIES
A61K2039/507
HUMAN NECESSITIES
A61K39/3955
HUMAN NECESSITIES
A61P1/16
HUMAN NECESSITIES
A61K31/454
HUMAN NECESSITIES
International classification
A61K39/395
HUMAN NECESSITIES
A61K31/454
HUMAN NECESSITIES
A61K31/498
HUMAN NECESSITIES
A61K31/513
HUMAN NECESSITIES
A61K31/522
HUMAN NECESSITIES
A61K31/675
HUMAN NECESSITIES
A61K31/7072
HUMAN NECESSITIES
A61K48/00
HUMAN NECESSITIES
A61P1/16
HUMAN NECESSITIES
Abstract
The invention relates to mutations and alterations in the inflammatory pathway, including IL-18BP and IL-10RB mutations, that are associated with the development of fulminant viral hepatitis following viral infection, such as following hepatitis virus infection. The invention relates to methods for treating or ameliorating viral hepatitis comprising administering IL-18BP, IL-18 antagonist, IFNγ antagonist or inhibitor, and/or IL-10RB or an IL-10 antagonist.
Claims
1. A method of treating or preventing fulminant viral hepatitis in a patient, said method comprising: (a) administering IL-18BP or IL-18 antagonist to a patient in need thereof, or upregulating the expression of IL-18BP in a patient in need thereof; (b) administering IL-10RB or IL-10 response mediator to a patient in need thereof, or upregulating the expression of IL-10RB in a patient in need thereof; or (c) administering an IFNγ antagonist or blocking agent to a patient in need thereof; wherein the patient comprises cells having a IL-18BP gene or protein variant wherein IL-18 binding of IL-18BP is absent or altered or an IL-10RB gene or protein variant wherein IL-10RB mediated IL-10 binding or IL-10 mediated signaling is absent or altered.
2. The method according to claim 1, wherein the IL-18BP gene or protein variant results in loss of function of IL-18BP.
3. The method according to claim 1, wherein the IL-18BP gene variant comprises at least one of: deletion of 10-25 nucleotides from the fourth and last intron, and deletion of 10-30 contiguous nucleotides from the fifth and last exon.
4. The method according to claim 3, wherein the IL-18BP gene variant comprises deletion of 19 nucleotides from the 4th and last intron, and deletion of 21 contiguous nucleotides from the 5th and last exon.
5. The method according to claim 1, wherein the patient comprises the following IL-18BP gene variant: NG_029021.1:g.7854_7893del; NM_173042.2:c.508-19_528del.
6. The method according to claim 1, wherein the IL-10RB gene or protein variant results in one or more amino acid substitution in the extracellular domain of IL-10RB.
7. (canceled)
8. The method according to claim 6, wherein the IL-10RB protein variant is substitution of the tryptophan at amino acid 100 of the IL-10RB protein, optionally wherein the substitution is with glycine or is W100G.
9. The method according to claim 1, wherein the patient is homozygous for the gene or protein variant.
10. (canceled)
11. The method according to claim 1, wherein the patient cells comprising the variant have at least 25%, 50%, or 75% more IL-18 as compared to cells from a person who is not suffering from fulminant viral hepatitis.
12. The method according to claim 1, wherein the patient cells comprising the variant have 10%-25%, 40%-60%, or 50-100% less IL-18BP or lower IL18BP mRNA levels as compared to cells from a person who is not suffering from fulminant viral hepatitis.
13. (canceled)
14. The method according to claim 1, wherein the cells are in the liver.
15. The method according to claim 1, wherein the patient is under the age of 30, 25, 20, 15, 10, or 5 years or is between the age of 5 and 25, 1 and 10, 10 and 20, or 10 and 15.
16. (canceled)
17. The method according to claim 1, wherein IL-18 antagonist comprises a drug that interferes with IL-18 mediated NK cell activation.
18. The method according to claim 1, wherein the IL-18 antagonist is an IL-18 antibody.
19. The method according to claim 1, wherein upregulating IL-18BP expression comprises genetic engineering to increase expression of IL-18BP in the cells comprising the gene variant, wherein the increase is more than 10%, more than 25%, or more than 50% as compared to the level of expression of IL-18BP before the genetic engineering.
20. The method according to claim 1, wherein the IFNγ antagonist is an anti-IFNγ antibody.
21. The method according to claim 1, wherein the patient has a primary infection comprising a liver-tropic virus.
22. The method according to claim 1, wherein the patient has a primary infection selected from the group consisting of: hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), and hepatitis E virus (HEV).
23. (canceled)
24. (canceled)
25. The method according to claim 1, further comprising administration of one or more antiviral therapies to the patient, wherein the antiviral therapies are selected from entecavir (Baraclude), tenofovir (Viread), lamivudine (Epivir), adefovir (Hepsera), telbivudine (Tyzeka), sofosbuvir (Daklinza), voxilapresvir (Vosevi), and pibrentasvir (Mavyret).
26. (canceled)
27. A method for evaluating and treating a patient with a liver tropic virus infection so as to alleviate, treat or prevent fulminant viral hepatitis comprising assessing the gene encoding IL-18BP and/or IL-10RB in the patient, or assessing the IL-18BP and/or IL-10RB in the patient to determine whether IL-18BP is mutated to prevent IL-18 binding or result in loss of function of IL-18BP and/or to determine whether IL-10RB encoded protein is altered by substitution of one or more amino acids in the extracellular domain of IL-10RB such that response to IL-10 signaling is altered, the method further comprising administering to the patient one or more of IL-18BP or an IL-18 antagonist, an IFNγ antagonist or blocking agent, or IL-10RB or an IL-10 signaling mediator, whereby fulminant viral hepatitis is alleviated, treated or prevented in the patient.
Description
DESCRIPTION OF THE FIGURES
[0038] The patent or patent application contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee.
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DETAILED DESCRIPTION
[0054] The present invention relates generally to methods of treating, alleviating or preventing fulminant viral hepatitis (FVH). The invention provides methods of treating, alleviating or preventing fulminant viral hepatitis (FVH), particularly in an individual or patient altered in the immune response or inflammatory response pathway. In embodiments of the method, an individual or patient may be altered in the expression, function, activity of IL-18BP, IL-10RB and/or or the IFNγ pathway. The invention provides mutations and alterations in the inflammatory pathway that are associated with the development of fulminant viral hepatitis following viral infection, such as following hepatitis virus infection. The invention provides mutations in the IL-18BP gene or protein and in the IL-10RB gene or protein which are associated with the occurrence of FVH in an individual or patient infected with a liver tropic virus, such as hepatitis virus, including hepatitis A virus. The invention provides approaches to assess an individual's or patient's susceptibility to or prevalence for FVH in the event of or upon infection with a liver tropic virus, such as hepatitis.
[0055] Fulminant viral hepatitis (FVH) is a rapid and severe impairment of liver functions (acute liver failure) with hepatic encephalopathy developing less than 8 weeks after the onset of jaundice, secondary to viral hepatitis. Clinically, FVH is observed as mainly due to hepatitis B virus (HBV), but also to hepatitis A virus (HAV). The present invention relates to methods of treating or preventing fulminant viral hepatitis in patients having a primary infection with a liver-tropic virus. Hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), and hepatitis E virus (HEV) are the most common liver-tropic viruses in humans. There is also the liver tropic hepatitis virus hepatitis D virus (HDV). A majority of the adeno-associated virus (AAV) serotype vectors exhibit liver tropism. Other viruses may cause fulminant hepatitis, for example Epstein-Barr virus (EBV) or herpes simplex virus. In addition, it is notable that coinfection of HBV/HCV with human immunodeficiency virus (HIV) infection and in AIDS patients is common. Estimated numbers of HBV and HCV infected subjects worldwide are significant (370 and 130 million subjects respectively) and of the 40 million known HIV positive subjects, 3 million are co-infected with HBV and 4.5 million with HCV (Williams R (2006) Hepatology 44:521-526).
[0056] Individuals or patients developing FVH upon or after or following or with viral infection, such as hepatitis virus infection, may be altered in the liver's anti-viral response pathway such that the virus and inflammatory response to the virus is not effective and significant liver pathology results, leading to FVH.
[0057] In embodiments of the invention, mutations in the IL-18BP gene or protein and mutations in the IL-10RB gene or protein are associated with FVH following hepatitis infection, such as hepatitis A virus infection. The IL-18BP mutations alter or block IL-18BP activity or IL-10RB activity. In some embodiments, IL-18BP is altered so that the mutant IL-18BP protein does not bind IL-18. In an embodiment, a deletion mutation in IL-18BP results in loss-of-function of IL-18BP. The IL-18BP mutations can result in uncontrolled IFNγ production and IFNγ-mediated pro-inflammatory activity. In some embodiments, IL-10RB mutations alter the ability of IL-10RB to bind as a receptor component to IL-10. In some embodiments, IL-10RB mutations alter the extracellular domain of IL-10RB. In some embodiments, IL-10 mediated response(s) are altered with IL-10RB mutation(s). In an embodiment, response to IL-10 is altered and response to IL-22 and IL-29 is not altered. In an embodiment, IL-10 mediated response(s) are altered such that FVH results with virus infection. IN an embodiment, IL-10 responses are altered such that IFNγ production and/or IFNγ-mediated activity and inflammation are not properly controlled or mediated.
[0058] Interleukin 18 (IL-18) is a strong inducer of Th-1 responses, interferon γ (IFN-γ) production, and stimulation of macrophages and natural killer (NK) cells. In fact, IL-18 was originally termed IFN-γ inducing factor. IL-18 binding protein (IL-18BP) is a natural secreted inhibitor of IL-18, has high-affinity binding for IL-18 and neutralizes the biologic activity of mature IL-18. This protein binds to and neutralizes IL-18, prevents the binding of IL-18 to its receptor, and thus inhibits IL-18-induced IFN-gamma production, resulting in reduced T-helper type 1 immune responses. IL-18BP is constitutively expressed and secreted in mononuclear cells and expression of IL-18BP can be enhanced by IFN-gamma.
[0059] Interleukin 10 receptor, beta subunit (IL-10 RB) is a subunit for the interleukin-10 receptor, also designated IL-10 Receptor subunit 2 (IL10R2) and cluster differentiation antigen CD Ag CDw210b. IL-10RB has the GeneID 3588 and UnitprotKB/SwissProt identifier Q08334. IL-10RB protein amino acid sequence (SEQ ID NO:2) is provided herein. The IL-10RB protein belongs to the cytokine receptor family and is an accessory chain essential for the active interleukin 10 receptor complex. Coexpression of IL-10RB and IL-10 receptor subunit 1 protein (also denoted IL-10RA) is required for IL10-induced signal transduction.
[0060] Interleukin-10 (IL-10) is a potent immunoregulatory cytokine that inhibits undesirable innate and acquired immune responses (Couper K N et al (2008) J Immunol 180: 5771-7; Moore K W et al (2001) Annu Rev Immunol 19: 683-765). Interleukin-10 is responsible for the limitation and eventual termination of inflammatory responses, which reduces the damage of self tissues in the process of pathogen eradication. In addition, IL-10 plays an important role in immune tolerance to protect self organs from autoimmunity. IL-10 antagonizes Th1 responses by inhibiting the production of IFN-γ. Interleukin-10 inhibits IFN-γ-induced monocyte/macrophage activation, subsequent cytokine production and the upregulation of costimulatory molecules.
[0061] IL-10 has emerged as a key immunoregulator during infection with viruses, bacteria, fungi, protozoa, ameliorating the excessive Th1 and CD8+ T cell responses (typified by overproduction of IFN-γ and TNF-α) that are responsible for much of the immunopathology associated with infections including Toxoplasma gondii, Trypanosoma spp., Plasmodium spp., Mycobacterium spp., and HSV. Thus, ablation of IL-10 signaling results in the onset of severe, often fatal immunopathology in a number of infections (reviewed in Couper K N et al (2008) J Immunol 180: 5771-7). IL-10 is the founding member of the so-called IL-10 family (including IL-10, IL-19, IL-20, IL-22, IL-24 and IL-26). Interaction of IL-10 with its receptor leads to the activation of STAT transcription factors, including STAT3, the transcription factor downstream of IL-10.
[0062] In the context of the acute inflammatory response (AIR), IL-10 binding to IL-10R activates the IL-10/JAK1/STAT3 cascade, where phosphorylated STAT3 homodimers translocate to the nucleus within seconds to activate the expression of target genes In fact, upon IL-10 treatment multiple Stat proteins become simultaneously activated, including STAT1 and STAT3.
[0063] IFN-gamma (IFNγ), or type II interferon, is a cytokine that is critical for innate and adaptive immunity against viral, some bacterial and protozoal infections. IFNγ has long been recognized as a signature proinflammatory cytokine that plays a central role in inflammation and autoimmune disease. IFNγ is an important activator of macrophages and inducer of Class II major histocompatibility complex (MHC) molecule expression. Aberrant IFNγ expression is associated with a number of autoinflammatory and autoimmune diseases. The importance of IFNγ in the immune system stems in part from its ability to inhibit viral replication directly, and also from its immunostimulatory and immunomodulatory effects.
[0064] The present invention relates to methods of treating or preventing fulminant viral hepatitis in patients having a primary infection with a liver-tropic virus and an IL-18BP gene variant. In an embodiment, the invention relates to methods of treating or preventing fulminant viral hepatitis in patients having a primary infection with a liver-tropic virus and an IL-18BP gene or protein variant which results in IL-18BP loss of function.
[0065] Methods to treat, alleviate or prevent FVH may include administering IL-18BP or otherwise expressing functional IL-18BP. Methods may include administering an IL-18 inhibitor or antagonist, or agent that blocks IL-18 expression or activity. In some embodiments, the invention includes a method of treating fulminant viral hepatitis in a patient by administering IL-18BP or IL-18 antagonist to a patient in need thereof, or upregulating the expression of IL-18BP in a patient in need thereof; wherein the patient cells have a IL-18BP gene variant.
[0066] As used herein, fulminant viral hepatitis is characterized as a rapid and severe impairment of liver functions (acute liver failure) in a patient having a liver-tropic viral primary infection. This may develop less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20 weeks after the onset of jaundice. In some instances, the rapid and severe impairment of liver functions (acute liver failure) develops between 1 and 20 weeks, 1 and 10 weeks, or 5 and 15 weeks after the onset of jaundice.
[0067] Without wishing to be bound by theory, it is believed that the activity of IL-18 is balanced by the presence of a high affinity, naturally occurring IL-18 binding protein (IL-18BP). In humans, increased disease severity can be associated with an imbalance of IL-18 to IL-18BP such that the levels of free IL-18 are elevated in the circulation. The loss of control or modulation of IL-18 via IL-18BP can thus result in uncontrolled IL-18 mediated activity and response, and can thus result in increased IFNγ productions, expression, activity or unregulated IFNγ.
[0068] The IL18BP gene encodes for the IL-18BP protein. The human wild type IL-18BP gene includes NC_000011.9 (71709955 . . . 71713965), on chromosome 11. (11913.4), based on human genome assembly GRCh37.p13.
[0069] Human IL-18BP is a 194 amino acid protein and has a Gene ID of 10068. Human IL-18BP sequence is known such as provided in NP_001034748.1, as indicated below (SEQ ID NO: 1):
TABLE-US-00001 1 mtmrhnwtpd lsplwv111c ahvvtllvra tpvsqtttaa tasvrstkdp cpsqppvfpa 61 akqcpalevt wpevevping tlslscvacs rfpnfsilyw lgngsfiehl pgrlwegsts 121 rergstgtql ckalvleqlt palhstnfsc vlvdpeqvvq rhvvlaqlwa glratlpptq 181 ealpsshssp qqqg
[0070] The IL18BP gene variant includes deletions in at least one region of the IL18BP gene. In some embodiments, this variant includes deletions in at least two regions of the IL18BP gene. In some embodiments, this variant includes deletions in three regions of the IL18BP gene. These regions include fourth intron, fifth exon, last exon, and last intron. As used herein, “IL18BP gene variant”, “IL18BP variant”, “IL-18BP gene variant”, “IL-18BP variant”, “gene variant”, and “variant” are used interchangeable, unless otherwise specified.
[0071] The IL-18BP variant includes nucleotide deletions or amino acid deletions in the protein such that encoded IL-18BP or IL-18BP protein is not functional and is thus a loss of function variant or mutation. In an embodiment, the IL-18BP variant is incapable of binding, successfully interacting with, or modulating IL-18. In an embodiment, the IL-18BP variant is a nucleotide deletion such that expression of IL-18BP is lost at the mRNA level, or such that an altered form of IL-18BP mRNA is generated and is unstable or is rapidly or effectively degraded. The IL-18BP variant includes less than 100 nt deletions or less than 50 nt deletions. In some embodiments, the deletions include 2-100, 30-50, 35-45, or 25-55 nucleotides. In some embodiments the deletion includes nucleotides of the fourth and last intron and also nucleotides of the fifth and last exon such that normal splicing does not occur.
[0072] In some embodiments, the variant includes at least one of: deletion of 10-25 nucleotides from the fourth and last intron, and deletion of 10-30 contiguous nucleotides from the fifth and last exon. In some embodiments, the variant includes deletion of 10-25 nucleotides from the fourth intron and last intron, and deletion of 10-30 contiguous nucleotides from the fifth exon and last exon.
[0073] In some embodiments, the variant includes at least one of deletion of 19 nucleotides from the fourth intron and last intron, and deletion of 21 contiguous nucleotides from the 5 exon and last exon.
[0074] In some embodiments, the variant includes 40-nucleotide deletion including deletion of 19 nucleotides from the fourth intron and last intron, and deletion of 21 contiguous nucleotides from the 5 exon and last exon.
[0075] In some embodiments, the variant includes a deletion of 20-40, 30-40, or 40 nucleotides from 71712811-71712850, of NC_000011.9.
[0076] In some embodiments, the variant includes NG_029021.1:g.7854_7893del and/or NM_173042.2:c.508-19_528del.
[0077] In a preferred embodiment, the patient is homozygous for the IL18BP gene variant. In some embodiments the patient is heterozygous for the IL18BP gene variant.
[0078] In some embodiments, the patient has at least one of the following mutations: a missense mutation in the ADAMTS1 gene (NM_006988:p.Lys648Arg), a missense mutation in the SLC6A19 gene (NM_001003841.2:p.Val551Met), a missense mutation in the TM7SF2 gene (NM_003273.3:p.Ala36Gly), a missense mutation in the ZNF324 gene (NM_014347.2:p.Ile385Met), and a missense mutation in the ZNF814 gene (NM_001144989.1:p.His407Asp).
[0079] In some embodiments, the cells of the patient suffering from FVH have at least 25%, 50%, or 75% more IL-18 as compared to cells from a person who is not suffering from fulminant viral hepatitis.
[0080] In some embodiments, the cells of the patient suffering from FVH have 10%-25%, 40%-60%, or 50-100% less IL-18BP as compared to cells from a person who is not suffering from fulminant viral hepatitis.
[0081] In some embodiments, the cells of the patient suffering from FVH have 50-100% less IL-18BP as compared to cells from a person who is not suffering from fulminant viral hepatitis.
[0082] In some embodiments, the cells of the patient suffering from FVH have IL18BP mRNA levels that are 10%-25%, 40%-60%, or 50-100% lower as compared to cells from a person who is not suffering from fulminant viral hepatitis.
[0083] Without wishing to be bound by theory, it is believed that the IL18BP gene and protein variants described herein result in loss of expression and/or loss of function in IL-18BP, and therefore, result in increased IL-18 in patients having this variant.
[0084] Treatment of patients having a FVH as described herein include administering a therapeutically effective amount of IL-18BP or IL-18 antagonist to a patient in need thereof. Recombinant human IL-18BP may be used. An example of recombinant human IL-18BP includes Tadekinig Alfa, AB2 Bio Ltd, Switzerland. As used herein, IL-18 antagonists include any protein or small molecule or agent or compound, including a chemical compound or peptide mimetic, that inhibits IL-18 mediated activation of NK cells. IL-18 antagonists include any protein or small molecule or agent or compound, including a chemical compound or peptide mimetic, that inhibits IL-18 mediated induction of IFNγ production, expression or activity.
[0085] In an embodiment, anti-IFNγ agent such as an agent, compound, molecule, peptide, peptide mimetic capable of blocking IFNγ or interfering with IFNγ-mediated signaling is administered. Anti-IFNγ agents or compounds, such as IFNγ antibodies, including blocking or neutralizing antibodies are known and available in the art. Anti-IFNγ antibodies have been shown to potentiate or enhance the immunogenicity of recombinant adenovirus vector vaccine immunization (Jackson S S et al (2011) Clin Vaccine Immunology 18(11):1969-1978). Blocking anti-IFNγ antibodies have been shown to affect disease pathogenesis in some experimental model systems, including in experimental allergic encephalomyelitis (EAE), diabetes, malaria, lung tumors and Trypanosome infections (Billiau, A H et al (1988) J Immunol 140:1506-1510; Billiau, A H et al (1987) Eur J Immunol 17:1851-1854; Debray-Sachs, M et al (1991) J Autoimmun 4:237-248; Grau, G E et al (1989) Proc Natl Acad Sci U.S.A 86:5572-5574; Matthys, P et al (1991) Eur J Cancer 27:182-187; Torrico, F et al (1991) J Immunol 146:3626-3632; Uzonna, J E et al (1998) J Immunol 161:5507-5515).
[0086] IFNγ antibodies have been tested in several autoimmune diseases, including RA, MS, uveitis, Type I diabetes and various autoimmune skin diseases (alopecia areata, psoriasis vulgaris, vitiligo) (reviewed in Skurkovich B (2003) Curr Opin Mol Ther 5(1):52-57; Miller C H T et al (2009) Ann NY Acad Sci 1182:69-79). XMG1.2, a rat anti-mouse IFN-IgG1, was described by Cherwinski et al. (Cherwinski, H M et al (1987) J. Exp. Med. 166:1229-1244) and has been used extensively in vivo in mice with various dosing regimens (Uzonna, J E et al (1998) J Immunol 161:5507-5515). Other IFNγ antibodies that have been evaluated and are known include Fontolizumab (HuZAF) a humanized anti IFNγ antibody, and AMG811 (Amgen), a fully human IFNγ antibody.
[0087] In some embodiments, the treatment described herein is initiated to a patient having a primary liver-tropic viral infection within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20 weeks of the onset of jaundice. In some embodiments, the treatment described herein is initiated in a patient having a primary liver-tropic viral infection and between 1 and 20 weeks, 1 and 10 weeks, or 5 and 15 weeks after the onset of jaundice.
[0088] In some embodiments, a patient having a primary infection including a liver-tropic virus and an IL18BP gene variant is administered IL-18BP or IL-18 antagonist in the absence of the onset of jaundice. Liver function or liver enzymes may be monitored so as to determine the appropriate timing or amount of administration. Monitoring of liver function is well known and within the capability of one skilled in the art or in clinical practice.
[0089] In some embodiments, a treatment for a patient having a primary infection including a liver-tropic virus and an IL18BP gene variant includes upregulating the expression of IL-18BP. Upregulating the expression of IL-18BP in the cells include increasing the expression of IL-18BP more than 10%, more than 25%, or more than 50% as compared to the level of expression of IL-18BP before increasing the expression.
[0090] Upregulating the expression of IL-18BP may be accomplished by genetic engineering. As used herein, “genetic engineering” includes transcription, or transcription and translation, of exogenously supplied nucleic acid to prevent, palliate and/or cure a disease or diseases.
[0091] In some embodiments, the invention includes inhibiting or preventing the development of FVH in a patient having the IL18BP gene variant described herein by administering IL-18BP or IL-18 antagonist to a patient in need thereof, or upregulating the expression of IL-18BP in a patient in need thereof.
[0092] An IL-18 antagonist may include an IL-18 antibody such as a neutralizing antibody, or an agent, compound, peptide, small molecule, peptide mimetic that inhibits IL-18 activity, expression or IL-18 binding to IL-18BP. Some exemplary small molecule inhibitors of IL-18 have been identifies and are known. For example, three compounds (NSC201631, NSC80734, and NSC61610) have been identified that disrupt hIL-18 binding to the ectromelia virus IL-18BP (Krumm B et al (2017) Scientific Reports 7:483). Through cell-based bioassay, it was shown that NSC80734 inhibits IL-18-induced production of IFN-γ in a dose-dependent manner. GSK 1070803 (GlaxoSmithKline) is a humanized monoclonal antibody that blocks IL-18 (McKie E A et al (2016) PLoSOne 11(3):e0150018, doi:10.1371/journal.pone.0150018).
[0093] The present invention relates to methods of treating or preventing fulminant viral hepatitis in patients having a primary infection with a liver-tropic virus and an IL10-RB gene or protein variant. In an embodiment, the invention relates to methods of treating or preventing fulminant viral hepatitis in patients having a primary infection with a liver-tropic virus and an IL-10RB gene or protein variant which results in an amino acid substitution in the extracellular domain of IL-10RB such that IL-10 mediated response is altered. In an embodiment, with the IL-10RB gene or protein variant, binding to IL-10 is altered. In an embodiment, response to IL-10 is altered and is hypomorphic, such that a partial loss of gene function, such as through reduced protein or RNA expression or reduced functional performance, but not a complete loss results. In one embodiment, some IL-10 mediated response but reduced response is demonstrated. In one embodiment, response to IL-10 is altered, but response to IL-22 and to IL-29 interleukins is normal or about the same as for a wild type IL-10RB protein. In one embodiment an IL-10RB mutation, such as a single amino acid substitution or a missense mutation is present whereby the IL-10RB mutant or variant does not respond to IL-10 but does respond to IL-22 and IL-29.
[0094] In an embodiment, the IL-10RB mutation is a substitution mutation of amino acid 100 of IL-10RB. In an embodiment, the IL-10RB mutation is a substitution mutation of the tryptophan amino acid 100 of IL-10RB. In an embodiment, the tryptophan is substituted with glycine, alanine, leucine, isoleucine or methionine. In an embodiment, the tryptophan is substituted with glycine or alanine. In an embodiment, the tryptophan is substituted with glycine. In an embodiment, the IL-10RB mutation corresponds to that of designated IL-10RB W100G or W100 and is a missense mutation resulting in a Tryptophan to Glycine single amino acid substitution mutation at amino acid 100 in the IL-10RB protein. The IL-10RB protein sequence is provided herein and as SEQ ID NO:2.
[0095] In an embodiment, the IL-10RB mutation is an extracellular domain missense mutation such that response to IL-10 is altered or absent. In an embodiment, the IL-10RB mutation is an extracellular domain missense mutation selected from S58R and Y59C. In an embodiment, the IL-10RB mutation is an extracellular domain missense mutation selected from S58R and Y59C, wherein the IL-10RB alleles do not respond to IL-10 but do respond to IL-22 and IL-29.
[0096] Methods to treat, alleviate or prevent FVH may include administering IL-10RB or otherwise expressing functional IL-10RB. Methods may include administering an IL-10 binding agent or agent that mediates IL-10 activity. Methods may include administering, expressing or increasing expression of the other IL-10 receptor subunit IL-10RA. Methods may include administering an IL-10 binding agent or agent that mediates IL-10 phosphorylation of STAT1. Methods may include administering an agent that mediates IL-10 phosphorylation of STAT1. Methods may include administering an agent that mediates IL-10 phosphorylation of STAT3. Methods may include administering an agent that mediates IL-10 phosphorylation of STAT1 and STAT3. Methods may include administering an agent that mediates activation of STAT1. Methods may include administering an agent that mediates activation of STAT3. Methods may include administering an agent that mediates activation of STAT1 and STAT3. Methods may include administering an agent that mediates IL-10 stimulation or IL-10 mediated stimulation of CXCL9. Methods may include administering an agent that stimulates CXCL9 expression or activity. In some embodiments, the invention includes a method of treating fulminant viral hepatitis in a patient by administering IL-10RB or IL-10 binding agent or IL-10 mediator to a patient in need thereof, or upregulating the expression of IL-10RB wild type or expressing IL-10RB wild type protein in a patient in need thereof; wherein the patient cells have a IL-10RB gene variant.
[0097] Without wishing to be bound by theory, it is believed that the activity of IL-10 is balanced by the presence of the IL-10 receptor subunits IL-10RB and IL-10RA which bind IL-10 and can mediate IL-10 activity and response. In humans, increased disease severity can be associated with an imbalance of IL-10 to IL-10RB such that the levels of free IL-10 are elevated in the circulation or such that IL-10 mediated signaling is altered or reduced. This can result in uncontrolled or alteration of control and mediation of other signaling pathways such as the IFNγ-mediated pathway and viral response and inflammatory response. The loss of control or modulation of IL-10 via IL-10RB can thus result in lack of IL-10 mediated controls or responses or altered IL-10 mediated response(s) and/or in uncontrolled IFNγ mediated activity and response, and can thus result in increased IFNγ productions, expression, activity or unregulated IFNγ.
[0098] The IL10RB gene and protein variant includes one or more substitution(s) or missense mutation(s) in at least one region of the IL-10RB, such as in the extracellular domain of IL-10RB. In some embodiments, this variant includes at least one substitution or missense mutation in at least one region of the IL-10RB, such as in the extracellular domain of IL-10RB. In some embodiments, this variant includes at least one substitution or missense mutation in the extracellular domain of IL-10RB. In some embodiments, the variant includes one or more amino acid substitution in the extracellular domain of IL-10RB such that IL-10 binding and/or IL-10 mediated response is altered. In some embodiments, the variant includes one or more amino acid substitution in the extracellular domain of IL-10RB such that IL-10 binding and/or IL-10 mediated response is altered or blocked, including wherein IL-22 and IL-29 response is maintained or close to that mediated by wild type IL-10RB. As used herein, “IL10RB gene variant”, “IL10RB variant”, “IL-10RB gene variant”, “IL-10RB variant”, “gene variant”, and “variant” are used interchangeable, unless otherwise specified.
[0099] In a preferred embodiment, the patient is homozygous for the IL10RB gene variant and expresses only altered IL-10RB protein, such as having an amino acid substitution in IL-10RB. In some embodiments the patient is heterozygous for the IL10RB gene variant.
[0100] In some embodiments, the patient has at least one of the following mutations: S31F, S58R, Y59C, C66Y, W100G, G193R, W204C. In some embodiments, the patient has at least one of the following mutations: S58R, Y59C, W100G. In an embodiment, the patient has the mutation W100G.
[0101] Treatment of patients having a FVH as described herein include administering a therapeutically effective amount of IL-10RB or an IL-10 binding agent or an IL-10 mediator to a patient in need thereof. Recombinant human IL-10RB may be used. Treatment of patients having a FVH as described herein include administering a therapeutically effective amount of IL-10RB and IL-10RA or an IL-10 binding agent to a patient in need thereof. Recombinant human IL-10RB may be used. A gene therapy vector, such as a liver tropic vector such as AAV may be utilized expressing IL-10RB. In an embodiment, a gene therapy vector, such as a liver tropic vector such as AAV may also be utilized expressing IL-10RA.
[0102] In some embodiments, the treatment described herein is initiated to a patient having a primary liver-tropic viral infection within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20 weeks of the onset of jaundice. In some embodiments, the treatment described herein is initiated in a patient having a primary liver-tropic viral infection and between 1 and 20 weeks, 1 and 10 weeks, or 5 and 15 weeks after the onset of jaundice.
[0103] In some embodiments, a patient having a primary infection including a liver-tropic virus and an IL10RB gene variant is administered IL-10RB or an IL-10 binding agent or an IL-10 mediator in the absence of the onset of jaundice. Liver function or liver enzymes may be monitored so as to determine the appropriate timing or amount of administration.
[0104] In some embodiments, a treatment for a patient having a primary infection including a liver-tropic virus and an IL10RB gene variant includes upregulating the expression or activity of IL-10RB. Upregulating the expression of IL-10RB in the cells include increasing the expression of IL-10RB more than 10%, more than 25%, or more than 50% as compared to the level of expression of IL-10RB before increasing the expression. In an embodiment, expression or activity of IL-10RA is also or concomitantly upregulated so as to mediate IL-10 binding and IL-10 mediated signaling.
[0105] Upregulating the expression of IL-10RB or expression of a wild type IL-10RB may be accomplished by genetic engineering. As used herein, “genetic engineering” includes transcription, or transcription and translation, of exogenously supplied nucleic acid to prevent, palliate and/or cure a disease or diseases.
[0106] In an embodiment, anti-IFNγ agent such as an agent, compound, molecule, peptide, peptide mimetic capable of blocking IFNγ or interfering with IFNγ-mediated signaling is administered. Anti-IFNγ agents or compounds, such as IFNγ antibodies, including blocking or neutralizing antibodies are known and available in the art, including as described herein.
[0107] As used herein, “inhibiting the development of”, “reducing the risk of”, “prevent”, “preventing”, and the like refer to reducing the probability of developing a FVH in a patient who may not have a primary liver-tropic viral infection, but may have a genetic predisposition to developing FVH. As used herein, “at risk”, “susceptible to”, or “having a genetic predisposition to”, refers to having a propensity to develop FVH. For example, a patient having the IL18BP gene variant described herein has increased risk (e.g., “higher predisposition”) of developing the FVH relative to a control patient having a “lower predisposition” (e.g., a patient without the IL18BP gene variant described herein).
[0108] In certain embodiments, the patient is a human 0 to 6 months old, 6 to 12 months old, 1 to 5 years old, 5 to 10 years old, 5 to 12 years old, 10 to 15 years old, 15 to 20 years old, 13 to 19 years old, 20 to 25 years old, 25 to 30 years old, 20 to 65 years old, 30 to 35 years old, 35 to 40 years old, 40 to 45 years old, 45 to 50 years old, 50 to 55 years old, 55 to 60 years old, 60 to 65 years old, 65 to 70 years old, 70 to 75 years old, 75 to 80 years old, 80 to 85 years old, 85 to 90 years old, 90 to 95 years old or 95 to 100 years old.
[0109] In certain embodiments, the patient is under the age of 30, 25, 20, 15, 10, or 5 years.
[0110] In certain embodiments, the patient is between the age of 5 and 25, 1 and 10, 10 and 20, or 10 and 15.
[0111] In some embodiments, the invention is directed to a method of treating FVH in a patient having an HAV, HBV, HCV, or HEV primary infection. In some embodiments, the invention includes a method of treating FVH in a patient having an HAV, HBV, or HEV primary infection. In some embodiments, the invention includes a method of treating FVH in a patient having an HAV, HBV, or HCV primary infection. In some embodiments, the invention includes a method of treating FVH in a patient having a HBV or HCV primary infection. In some embodiments, the invention includes a method of treating FVH in a patient having a HBV and HCV primary infection. In some embodiments, the invention includes a method of treating FVH in a patient having an HAV primary infection.
[0112] In some embodiments, the method described herein include co-administration of one or more antiviral therapies to the patient. Antiviral therapies include entecavir (Baraclude), tenofovir (Viread), lamivudine (Epivir), adefovir (Hepsera), telbivudine (Tyzeka), sofosbuvir (Daklinza), voxilapresvir (Vosevi), and pibrentasvir (Mavyret).
[0113] In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook et al, “Molecular Cloning: A Laboratory Manual” (1989); “Current Protocols in Molecular Biology” Volumes I-III [Ausubel, R. M., ed. (1994)]; “Cell Biology: A Laboratory Handbook” Volumes I-III [J. E. Celis, ed. (1994))]; “Current Protocols in Immunology” Volumes I-III [Coligan, J. E., ed. (1994)]; “Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic Acid Hybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “Transcription And Translation” [B. D. Hames & S. J. Higgins, eds. (1984)]; “Animal Cell Culture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells And Enzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide To Molecular Cloning” (1984).
[0114] As used herein, administering includes administering a therapeutically effective dose. As used herein, the phrase “therapeutically effective dose” or “therapeutically effective amount” refers to a dose or amount of an agent that provides for an improvement in either the onset of symptoms or the progression of symptoms associated FVH in comparison to a patient that has not received the agent.
[0115] Undesirable effects, e.g. side effects, are sometimes manifested along with the desired therapeutic effect; hence, a practitioner balances the potential benefits against the potential risks in determining what an appropriate “effective amount” is. The exact amount required will vary from patient to patient, depending on the species, age and general condition of the patient, mode of administration and the like. Thus, it may not be possible to specify an exact “effective amount”. However, an appropriate “effective amount” in any individual case may be determined by one of ordinary skill in the art using only routine experimentation.
[0116] As used herein the terms treatment, treat, or treating refer to a method of reducing the effects of FVH or symptom of FVH. Thus in the disclosed method, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an FVH or symptom of FVH. For example, a method for treating FVH is considered to be a treatment if there is a 10% reduction in one or more symptoms of FVH in a patient as compared to a control. Thus the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the infection, condition, or symptoms of the infection or condition.
[0117] As used herein, the terms prevent, preventing, and prevention of a FVH refer to an action, for example, administration of a compound described herein, that occurs before or at about the same time a patient begins to show one or more symptoms of FVH, which inhibits or delays onset or exacerbation of one or more symptoms of the disease or disorder. As used herein, references to decreasing, reducing, or inhibiting include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater as compared to a control level. Such terms can include but do not necessarily include complete elimination.
[0118] The term “antibody” describes an immunoglobulin whether natural or partly or wholly synthetically produced. The term also covers any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain. CDR grafted antibodies are also contemplated by this term. An “antibody” is any immunoglobulin, including antibodies and fragments thereof, that binds a specific epitope. The term encompasses polyclonal, monoclonal, and chimeric antibodies. The term “antibody(ies)” includes a wild type immunoglobulin (Ig) molecule, generally comprising four full length polypeptide chains, two heavy (H) chains and two light (L) chains, or an equivalent Ig homologue thereof (e.g., a camelid nanobody, which comprises only a heavy chain); including full length functional mutants, variants, or derivatives thereof, which retain the essential epitope binding features of an Ig molecule, and including dual specific, bispecific, multispecific, and dual variable domain antibodies; Immunoglobulin molecules can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Also included within the meaning of the term “antibody” are any “antibody fragment”. As antibodies can be modified in a number of ways, the term “antibody” should be construed as covering any specific binding member or substance having a binding domain with the required specificity. Thus, this term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic.
[0119] An “antibody fragment” refers to and includes a molecule comprising at least one polypeptide chain that is not full length, including (i) a Fab fragment, which is a monovalent fragment consisting of the variable light (VL), variable heavy (VH), constant light (CL) and constant heavy 1 (CH1) domains; (ii) a F(ab′)2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a heavy chain portion of an Fab (Fd) fragment, which consists of the VH and CH1 domains; (iv) a variable fragment (Fv), which consists of the VL and VH domains of a single arm of an antibody, (v) a domain antibody (dAb) fragment, which comprises a single variable domain (Ward, E. S. et al., Nature 341, 544-546 (1989)); (vi) a camelid antibody; (vii) an isolated complementarity determining region (CDR); (viii) a Single Chain Fv Fragment wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al, Science, 242,423-426, 1988; Huston et al, PNAS USA, 85, 5879-5883, 1988); (ix) a diabody, which is a bivalent, bispecific antibody in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementarity domains of another chain and creating two antigen binding sites (WO94/13804; P. Holliger et al Proc. Natl. Acad. Sci. USA 90 6444-6448, (1993)); and (x) a linear antibody, which comprises a pair of tandem Fv segments (VH-CH1-VH-CH1) which, together with complementarity light chain polypeptides, form a pair of antigen binding regions; (xi) multivalent antibody fragments (scFv dimers, trimers and/or tetramers (Power and Hudson, J Immunol. Methods 242: 193-204 9 (2000)); (xii) a minibody, which is a bivalent molecule comprised of scFv fused to constant immunoglobulin domains, CH3 or CH4, wherein the constant CH3 or CH4 domains serve as dimerization domains (Olafsen T et al (2004) Prot Eng Des Sel 17(4):315-323; Hollinger P and Hudson P J (2005) Nature Biotech 23(9):1126-1136); and (xiii) other non-full length portions of heavy and/or light chains, or mutants, variants, or derivatives thereof, alone or in any combination.
[0120] Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contains the paratope, including those portions known in the art as Fab, Fab′, F(ab′).sub.2 and F(v), which portions are preferred for use in the therapeutic methods described herein.
[0121] Antibodies may also be bispecific, wherein one binding domain of the antibody recognizes a target of reference in the invention, and the other binding domain has a different specificity, e.g. to recruit an effector function or the like. Bispecific antibodies of use in the present invention include wherein one binding domain of the antibody recognizes a target of reference in the invention, including a fragment thereof, and the other binding domain is a distinct antibody or fragment thereof, including that of a distinct pathway antibody, immunomodulatory antibody, or viral antibody. The other binding domain may be an antibody that recognizes or targets a particular cell type, as in a liver cell.
[0122] Further, unless expressly stated to the contrary, “or” refers to an inclusive “or” and not to an exclusive “or”. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).
[0123] Throughout this specification, quantities are defined by ranges, and by lower and upper boundaries of ranges. Each lower boundary can be combined with each upper boundary to define a range. The lower and upper boundaries should each be taken as a separate element.
[0124] As used herein, the term “patient” and “subject” may be used interchangeably. In a preferred embodiment, the subject or patient is a human.
[0125] As used herein, a “patient in need thereof” includes a patient having a primary liver-tropic viral infection.
[0126] Additionally, any examples or illustrations given herein are not to be regarded in any way as restrictions on, limits to, or express definitions of any term or terms with which they are utilized. Instead, these examples or illustrations are to be regarded as being described with respect to one particular embodiment and as being illustrative only. Those of ordinary skill in the art will appreciate that any term or terms with which these examples or illustrations are utilized will encompass other embodiments which may or may not be given therewith or elsewhere in the specification and all such embodiments are intended to be included within the scope of that term or terms. Language designating such nonlimiting examples and illustrations includes, but is not limited to: “for example,” “for instance,” “e.g.,” and “in one embodiment.”
[0127] In this specification, groups of various parameters containing multiple members are described. Within a group of parameters, each member may be combined with any one or more of the other members to make additional sub-groups. For example, if the members of a group are a, b, c, d, and e, additional sub-groups specifically contemplated include any one, two, three, or four of the members, e.g., a and c; a, d, and e; b, c, d, and e; etc.
[0128] The invention may be better understood by reference to the following non-limiting Examples, which are provided as exemplary of the invention. The following examples are presented in order to more fully illustrate the preferred embodiments of the invention and should in no way be construed, however, as limiting the broad scope of the invention.
Example 1
Inherited IL-18BP Deficiency in Human Fulminant Viral Hepatitis
[0129] Fulminant viral hepatitis (FVH) is a devastating and unexplained condition that strikes otherwise healthy individuals during primary infection with common liver-tropic viruses. We report a child who died of FVH upon infection with hepatitis A virus (HAV) at age 11 yr and who was homozygous for a private 40-nucleotide deletion in IL18BP, which encodes the IL-18 binding protein (IL-18BP). This mutation is loss-of-function, unlike the variants found in a homozygous state in public databases. We show that human IL-18 and IL-18BP are both secreted mostly by hepatocytes and macrophages in the liver. Moreover, in the absence of IL-18BP, excessive NK cell activation by IL-18 results in uncontrolled killing of human hepatocytes in vitro. Inherited human IL-18BP deficiency thus underlies fulminant HAV hepatitis by unleashing IL-18. These findings provide proof of principle that FVH can be caused by single-gene inborn errors that selectively disrupt liver-specific immunity. They also show that human IL-18 is toxic to the liver and that IL-18BP is its antidote.
[0130] Rare familial cases of FVH have been reported, including two young siblings from a Turkish family, three young brothers from an Iranian family, and two elderly brothers from a Japa-nese family who developed FVH within weeks of each other following primary HAV infections in their siblings (Durst et al., 2001; Yalniz et al., 2005; Yoshida et al., 2017). In all three families, several relatives of the patients had presented viral hepatitis with a benign course. Inbred strains of mice display differences in susceptibility to mouse hepatitis virus 3 (MHV3; Wege et al., 1982). Upon infection with MHV3, fully susceptible strains (e.g., C57Bl/6J) die from FVH, semisusceptible strains (e.g., C3H/St) develop acute and then chronic hepatitis, and fully resistant strains (e.g., A/J) display no signs of liver disease (MacPhee et al., 1985). These observations suggest that host genetic factors may underlie FVH. The lack of FVH in patients with any of the >350 known primary immunodeficiencies (Picard et al., 2018) suggests that FVH is unlikely to result from inborn errors that broadly disrupt innate and/or adaptive immunity. Instead, previous discoveries that other severe viral diseases striking otherwise healthy individuals, such as epidermodysplasia verruciformis, fulminant EBV disease, herpes simplex encephalitis, severe pulmonary influenza, severe rhi-novirus respiratory disease, and severe varicella zoster virus disease, can result from single-gene inborn errors of protective immunity to specific viruses in specific cell types or tissues (Casanova, 2015a,b; Casanova and Abel, 2018) suggest that FVH may result from inborn errors selectively disrupting immunity to hepatitis viruses in the liver. We tested this hypothesis by searching for genetic etiologies of FVH by whole-exome sequencing (WES) in a small cohort of patients.
Results
[0131] A Private IL18BP Variation in a Patient with Fulminant Viral Hepatitis
[0132] We performed whole-exome sequencing (WES) on leukocyte genomic DNA (gDNA) from three siblings born to Algerian parents living in France: a girl who died from FVH due to HAV at 11 years of age with no signs of previous chronic or acute liver disease of any type, and her two brothers, who had experienced benign HAV infections (Materials and Methods). WES revealed a high rate of homozygosity among the siblings (5.60 to 6.51%), indicating that this family was consanguineous. We therefore hypothesized that a genetic etiology for FVH in this family would display autosomal recessive (AR) inheritance with complete penetrance. We thus searched for very rare (Minor allele frequency (MAF)<0.001) homozygous non-synonymous variants present in the patient, but not in her two siblings (Table 1). Six genes had variants meeting these criteria (Table 2). We prioritized candidate genes for further studies based on (i) the known function of the gene in the liver and/or immunity and (ii) the predicted deleteriousness of the variations. IL18BP, encoding the interleukin-18 binding protein, was the most plausible disease-causing gene, as (i) it encodes a constitutively secreted, naturally circulating neutralizer of the cytokine IL-18 (Aizawa et al., 1999; Novick et al., 1999), the mouse ortholog of which is known to be toxic to the liver (Okamura et al., 1995), and (ii) the patient carried a 40-nucleotide (nt) homozygous deletion centered on an intron-exon boundary within this gene.
[0133] None of the other five variants identified, all of which were missense, had ever been associated with the liver or with immunity (Table 3). Moreover, the IL18BP variant (NG_029021.1:g.7854_7893del; NM_173042.2:c.508-19_528del) was not found in any public database (1000 Genomes, dbSNP, GnomAD, and Bravo) or in our in-house database of more than 4,000 exomes, including 65 from unrelated Algerians and at least 1,000 from North Africans (as confirmed by principal component analysis, as in our patient).
[0134] Homozygosity for a 40-Nucleotide Deletion in IL18BP Segregates with Disease
[0135] We confirmed, by Sanger sequencing, that the familial segregation of the mutant IL18BP allele was consistent with an AR mode of inheritance with complete penetrance, as both parents and one of the healthy siblings were heterozygous for the mutation, whereas the other sibling did not carry the mutation (
[0136] Mutation c.508-19_528Del Causes Aberrant IL18BP mRNA Splicing
[0137] The canonical IL18BP transcript (NM_173042) contains five exons in total, encoding a protein of 194 amino acids (aa), also annotated as IL-18BPa, with an N-terminal signal peptide (1-30 aa) and an Ig-like domain (31-166 aa) responsible for binding to IL-18 (
[0138] Mutation c.508-19_528Del Disrupts IL-18BP Function
[0139] We generated cDNA constructs for expression of the three IL18BP variants (M1-M3) to assess their impact on IL-18BP expression and function in vitro. Human IL-18BP migrates at 25-45 kDa on SDS-PAGE gels in reducing conditions, due to heterogeneous glycosylation (Kim et al., 2000) (
[0140] Expression Patterns of IL-18, IL-18BP, and IL-18R in the Liver
[0141] Human IL-18 was initially identified from a liver cDNA library as an IFN-γ-inducing factor in NK cells and T cells (Ushio et al., 1996). We analyzed the expression patterns of IL-18, IL-18BP, and the two chains of the IL-18 receptor (IL-18R), IL-18R1 and IL-18RAP, in several human cell lines acting as surrogates of liver-resident cells: Hep3B (hepatocytes), HUVEC (endothelial cells), LX-2 (hepatic stellate cells), SV40-fibroblasts, THP1 (monocytes), NKL (NK cells), Jurkat (T cells) and Raji (B cells) cells. We assessed mRNA and protein levels at baseline and following stimulation with various inflammatory cytokines, including IFN-α, IFN-γ, IFN-λ, TNF-α, IL-6, IL-15, IL-18, IL-22, and IL-27 (
[0142] Elevated IL-18 Levels in Liver Tissues of Patients with Fulminant Viral Hepatitis
[0143] It has been suggested that the liver lesions observed during the course of acute hepatitis are not directly due to the cytopathic effects of HAV, but instead result from excessive cytotoxic activity of NK, NKT, and CD8.sup.+ T lymphocytes (Kim et al., 2011; Kim et al., 2018; Lemon et al., 2018). We thus performed immunohistochemical staining on liver sections from the IL-18BP-deficient patient described in this study, an unrelated patient with FVH due to HAV, and an individual without liver inflammation. Only very small numbers of hepatocytes were detected in the liver tissues of the two FVH patients (
[0144] Human IL-18 Induces NK Cell-Mediated Killing of Hepatocytes In Vitro
[0145] IL-18 increases NK and/or T cell-mediated cytotoxicity by inducing (i) the expression of membrane-bound FasL, an activator of cell death, (ii) the secretion of pro-apoptotic cytokines, such as TNF-α and TRAIL, and (iii) the secretion of cytolytic enzymes, such as perforin and granzyme (Kaplanski, 2018; Lebel-Binay et al., 2000; Tsutsui et al., 2000). Moreover, IL-18, together with IL-2, enhances the activation and in vitro cytotoxic activity of human peripheral NK cells (Nielsen et al., 2016; Son et al., 2001). IL-2-stimulated human liver NK cells have also been shown to be strongly cytotoxic, even more so than peripheral blood NK cells, to human HepG2 hepatocytes (Ishiyama et al., 2006). We therefore generated an in vitro model to test IL-18/IL-18BP-regulated hepatotoxicity through the coculture of HepG2 and NK-92 cells, which display a phenotype similar to liver-resident NK cells in humans (Le Bouteiller et al., 2002). NK cells activated with IL-18 killed significantly more hepatocytes than unstimulated NK cells, at various NK cell-to-hepatocyte ratios (
[0146] Discussion
[0147] We have established a causal relationship between AR complete IL-18BP deficiency and lethal FVH due to HAV in an 11-year-old child without preexisting liver disease and with no history of severe infection. Causality is established on both genetic and mechanistic grounds, meeting the criteria for genetic studies in single patients (Casanova et al., 2014). In contrast, it is unclear if the two autoimmune phenotypes of the patient, type I diabetes and Hashimoto's thyroiditis, are also due to IL-18BP deficiency. The child had AR complete IL-18BP deficiency and there are no more than 2.5×10.sup.−8 such patients in the general population. IL-18 is a pleiotropic inflammatory cytokine that was initially discovered in mouse liver and has been reported to activate NK cells and T cells in addition to its potent IFN-γ-inducing activity (Kaplanski, 2018). Serum and hepatic concentrations of IL-18 and IFN-γ were high in patients with various forms of fulminant liver failure (Shinoda et al., 2006; Yumoto et al., 2002). In particular, serum IL-18 was found to be dramatically elevated in patients with acute hepatitis due to HAV infection (Kim et al., 2018). Moreover, mouse IL-18 has been shown to induce intrahepatic inflammatory cell recruitment and severe hepatotoxicity in various liver injury models (Finotto et al., 2004; Kimura et al., 2011; Tsutsui et al., 2000), whereas IL-18BP has been shown to protect against IL-18-mediated fulminant hepatitis in mice (Faggioni et al., 2001; Fantuzzi et al., 2003; Shao et al., 2013). Our findings indicate that uncontrolled IL-18 activity in humans is toxic to the liver, as previously demonstrated in mice, and that human IL-18BP is a potent liver-protective compound, acting as an antidote to IL-18. Recombinant human IL-18BP (Tadekinig Alfa, AB2 Bio Ltd, Switzerland) has been approved for clinical use for indications unrelated to liver conditions and proposed as a treatment for preventing acetaminophen hepatotoxicity (Bachmann et al., 2018). We provide proof-of-principle that FVH can be caused by single-gene inborn errors of immunity to HAV in the liver. Our findings also reveal the essential functions of IL-18 and IL-18BP in humans. By inference from previous human genetic study of other isolated infections, which are characterized by genetic heterogeneity but physiological homogeneity (Casanova, 2015a; Casanova, 2015b; de Jong et al., 2018; Hernandez et al., 2018; Martinez-Barricarte et al., 2018), there might be other genetic etiologies of FVH that impair IL-18BP or enhance IL-18 activity. Furthermore, neutralizing endogenous IL-18, particularly with recombinant IL-18BP, might be beneficial to patients with FVH caused by HAV and possibly other viruses.
[0148] Materials and Methods
[0149] Patient Recruitment and Ethics
[0150] Clinical history and biological specimens were obtained from the referring clinicians, with the consent of the patients and family members participating in the study. All the experiments involving human subjects conducted in this study were in accordance with institutional, local, and national ethical guidelines, and approved by the French Ethics Committee, the French National Agency for the Safety of Medicines and Health Products, and the French Ministry of Research (protocol C09-18), and the Rockefeller University Institutional Review Board (protocol JCA-0700).
[0151] Case Report
[0152] The patient (III.1) was born in France in 2002 to consanguineous Algerian parents. In 2004, she was diagnosed with insulin-dependent diabetes. Tests for anti-islet cell and anti-insulin autoantibodies were positive, but no anti-glutamic acid decarboxylase antibodies were detected (Table 4). In 2009, anti-thyroglobulin autoantibodies were detected, but tests for anti-glutaminase, anti-endomysium and anti-thyroperoxidase antibodies were negative. Celiac disease was therefore excluded. In 2011, the patient was diagnosed with Hashimoto thyroiditis and tests for anti-thyroperoxidase antibodies were positive (Table 4). The patient was treated with levothyroxine. In 2013, the patient presented with fatigue, nausea, and hepatomegaly. She tested positive for HAV IgM on presentation, consistent with primary HAV infection. Serology and PCR results were negative for HBV (other than the presence of anti-HBs due to prior vaccination), HCV, or HEV. The patient was seropositive for CMV and EBV, and negative for HIV, HTLV1 and 2. PCR tests for enterovirus were negative. No liver autoantibodies (anti-NA, anti-SMA, anti-LKM1, anti-LC1, anti-SLA, anti-mitochondria) were detected, excluding autoimmune hepatitis. The patient had no history of exposure to hepatotoxic drugs, and screening tests for such drugs were negative. Liver function tests were as follows: alanine aminotransferase (ALT): 2181 IU/L, aspartate aminotransferase (AST): 2582 IU/L, 7-glutamyl transferase (GGT): 73 IU/L, total bilirubin: 274 μmol/L, conjugated bilirubin: 173 μmol/L, prothrombin time (PT) at 67%, and Factor V at 100% (Table 5). On day 8 of infection, the patient was hospitalized for incoherent speech, jaundice, appetite loss, gingival bleeding, and petechiae. During physical examination at the emergency unit, the patient was unconscious, with icterus, hepatomegaly, and a fever (40° C.). The patient was diagnosed with fulminant viral hepatitis (FVH) due to HAV infection. On day 9, liver function tests revealed high levels of cytolysis, with a PT of 14%, factor V levels at 30%, and a Glasgow coma scale score of 8 (Table 5). The patient underwent liver transplantation, but died a day later of multiple organ failure (day 11). Histological examinations of the native and explanted liver showed 95% hepatocellular necrosis with polymorphic inflammation. The patient had been vaccinated against BCG, Haemophilus influenzae type b, pneumococcus, flu, HBV, and MMR with no adverse effects. She was not tested for immunodeficiency. Her polymorphonuclear cell (PMN) counts were consistently in the normal range (Table 6).
[0153] Both parents were seropositive for HAV (IgM-negative) and had normal transaminase levels at the time of the patient's illness (data not shown). The patient's two siblings (III.2 and III.3) had IgM antibodies against HAV and were admitted to hospital two days after she died, but neither developed FVH. The patient's older brother (III.2), born in 2005, had diarrhea and four vomiting episodes two days before hospitalization. On arrival at the hospital, his clinical examination was normal, with no fever, weight loss, icterus, encephalopathy or hepatosplenomegaly. His laboratory findings on day 1 were as follows: ALT, 42 IU/L; AST, 44 IU/L; GGT, 55 IU/L; Hb, 14.8 g/dL; total bilirubin, 6 μmol/L; CRP≤5; and PT at 100%. Basic metabolic tests gave normal results. The findings on day 2 were as follows: ALT, 35 IU/L; AST, 36 IU/L; GGT, 48 IU/L; total bilirubin, 10 μmol/L; and PT at 89%. Acute HAV infection was diagnosed. On day 3, no vomiting or diarrhea was observed. Hepatic data had returned to normal except for GGT, at 41 IU/L, PT at 100%, and Factor V at 93% (Table 5).
[0154] The patient's other sibling (III.3), born in 2011, had no symptoms before hospitalization. Clinical examination was normal on arrival at the hospital and laboratory findings were as follows: ALT, 1594 IU/L; AST, 755 IU/L; GGT, 293 IU/L; total bilirubin, 10 μmol/L; and PT at 92%. Laboratory findings on day 2 were as follows: ALT, 1298 IU/L; AST, 513 IU/L; GGT, 277 IU/L; total bilirubin, 11 μmol/L. Acute HAV infection was diagnosed. Laboratory findings on day 3 were as follows: ALT, 894 IU/L; AST, 271 IU/L; GGT, 229 IU/L; total bilirubin, 7 μmol/L; PT at 100%, and Factor V at 100% (Table 5). This sibling suffered one episode of diarrhea and vomiting. By day 4, both siblings were discharged from the hospital. None of the parents or siblings has ever had any other symptoms of hepatitis or another liver disease to date.
[0155] Whole-Exome Sequencing (WES) and Genetic Analysis
[0156] Genomic DNA extraction, WES data collection and analyses were performed as previously described (Belkaya et al., 2017; Regateiro et al., 2017). Briefly, allele frequencies were obtained from National Heart, Lung, and Blood Institute (NHLBI) GO Exome Sequencing Project (EVS, ESP6500SI-V2 release on evs.gs.washington.edu/EVS), 1000 Genomes Project (April 2014 data release on browser.1000genomes.org), and the Genome Aggregation Database (GnomAD, February 2017 data release on gnomad.broadinstitute.org/). All variant calls with a genotype quality (GQ)<20, and a depth of coverage (DP)<5 were filtered out. Only indel-inframe, indel-frameshift, start-lost, missense, nonsense, stop-lost, and essential splice-site (splice acceptor and splice donor) variants were retained for further analysis. Given the rare occurrence of FVH in children, we excluded common polymorphisms with an allele frequency (AF) of 0.1% or more in public databases: EVS, 1000 Genomes, and GnomAD, including variants with an AF≥0.1% in each ethnic subpopulation (African, Ashkenazi Jewish, Finnish, non-Finnish European, South Asian, East Asian, and Latino) in GnomAD. In silico predictions of the impact of variants were evaluated with the following algorithms: the gene damage index (GDI, pec630.rockefeller.edu:8080/GDI/) (Itan et al., 2015), CADD (cadd.gs.washington.edu/score) (Kircher et al., 2014) and mutation significance cutoff (MSC, pec630.rockefeller.edu/MSC/) (Itan et al., 2016). MSC scores were generated with a 99% confidence interval based on the CADD 1.3 scores of all disease-causing mutations in the Human Gene Mutation Database, regardless of the gene concerned (Itan et al., 2016).
[0157] Sanger Sequencing of Genomic DNA
[0158] We validated genomic variants in patients and/or their relatives, by amplifying 200-300 base-pair (bp) regions encompassing the mutation from gDNA samples with different sets of site-specific primers (Seq-1 and Seq-2 forward/reverse primer sets) listed in Table S7. The amplicons were then sequenced with BigDye Terminator technology on an ABI 3730 DNA sequencer. SnapGene (Version 3.1.4) was used for sequence analysis.
[0159] Gene Expression Analyses
[0160] EBV-transformed B (EBV-B) cells were generated, as previously described (Durandy et al., 1997), and cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS). Total RNA was isolated with a Qiagen RNeasy kit, using EBV-B cells from healthy controls and the healthy siblings and parents of the patient. For quantitative PCR (qPCR) analysis, cDNA samples generated with oligo-dT primer and the SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific) were used to determine the relative levels of IL18BP expression, by amplification of the cDNA generated with TaqMan Universal PCR Master Mix and various TaqMan probes detecting the transcript variants encoding IL-18BPa: Hs00931914 (Probe 1), Hs00931907 (Probe 2), Hs00931908 (Probe 3), and Hs00934414 (Probe 4) (Thermo Fisher Scientific). We used an Applied Biosystems 7500 Fast Real-Time PCR system with the running program: 50° C. for 2 min; 95° C. for 10 min; 40 cycles of 95° C. for 15 s, 60° C. for 1 min. We calculated qPCR efficiency and the amplification factor for each assay by the standard curve method (Nolan et al., 2006) and used them to determine relative expression levels for IL18BP normalized against endogenous GAPDH (4310884E, Thermo Fisher Scientific) expression in each sample. For relative quantification of the WT and mutant IL18BP transcript variants, we used PowerUp SYBR Green Master Mix (Thermo Fisher Scientific) on an Applied Biosystems 7500 Fast Real-Time PCR system with the following program: 50° C. for 2 min; 95° C. for 2 min; 35 cycles of 95° C. for 15 s, 63° C. for 1 min, 72° C. for 30 s (data collection), followed by the melting curve protocol: 95° C. for 15 s; 63° C. for 1 min; 95° C. for 30 s (1% ramp-rate); 60° C. for 15 s. The primer pairs for each mutant variant (M1, M2, and M3) were designed with the NCBI Primer-BLAST tool (ncbi.nlm.nih.gov/tools/primer-blast). The specificity of these primers was assessed by melting curve analysis and confirmed by Sanger sequencing of the qPCR products. Relative copy numbers were determined by the standard curve method with plasmids containing the WT or mutant IL18BP sequences, followed by normalization against endogenous GAPDH (PrimerBank ID: 378404907c1) levels. The sequences of the SYBR Green qPCR primers are provided in Table 7. All qPCR experiments were performed in duplicate with two independent RNA preparations.
[0161] Human hepatoma cells (Hep3B), SV40-transformed human fibroblasts (SV40-fibroblasts) and human hepatic stellate cells (LX-2, Millipore, USA) were cultured in DMEM supplemented with 10% fetal calf serum (FCS). Human umbilical vein endothelial cells (HUVECs) were cultured in endothelial cell growth medium (Sigma Aldrich). THP1 (monocytes), Jurkat (T cells), Raji (B cells) and NKL (NK cells) cells were cultured in RPMI-1640 supplemented with 10% FCS. Sodium pyruvate (1 mM) was included in the growth media for THP1 and NKL cells. NKL cells were cultured in the presence of recombinant human IL-2 (10 ng/mL; Invitrogen). All cell lines (Hep3B cells at 1×10.sup.6 cells/well, HUVECs at 5×10.sup.5 cells/well, Jurkat cells at 2×10.sup.6 cells/well, LX-2 cells at 1×10.sup.6 cells/well, NKL cells at 2×10.sup.6 cells/well, Raji cells at 2×10.sup.6 cells/well, SV40 fibroblasts at 5×10.sup.5 cells/well, and THP1 cells at 1×10.sup.6 cells/well) were plated in fresh medium in 12-well plates on the day before stimulation with various cytokines: IFN-α, at 5000 U/mL (IntronA, IFN-α2b, Schering-Plough); IFN-γ, at 1000 IU/mL (Imukin, IFN-γ1b, Boehringer); TNF-α, at 10 ng/mL (R&D Systems); IL-6, at 25 ng/mL (R&D Systems); and IL-15, IL-18, IL-22 and IL-29/IFN-λ1, all at 100 ng/mL (R&D Systems). For qPCR analysis, cells were stimulated for 6 h. Total RNA was extracted with the RNeasy Extraction Kit (Qiagen). RNA was reverse-transcribed in High-Capacity RNA-to-cDNA Master Mix (Applied Biosystems). We then performed qPCR in TaqMan assays specific for IL18 (Hs01038788), IL18BP (Hs00931914), IL18R1 (Hs00977691), and IL18RAP (Hs00977695). GAPDH (Hs99999905) was used as an endogenous control for IL18BP, IL18R1 and IL18RAP, whereas HPRT1 (Hs99999909) was used for IL18. Relative expression analyses were performed by the dCt method, according to the kit manufacturer's instructions.
[0162] Expression of alternative splice forms of IL18BP was assessed by PCR amplification with different primer sets (F.sub.Ex1-2 and R.sub.IL18BPa; F.sub.Ex1-2 and R.sub.IL18BPbd; F.sub.Ex1-2 and R.sub.IL18BPc; F.sub.Ex2-3 and R.sub.IL18BPa; F.sub.Ex2-3 and R.sub.IL18BPbd; F.sub.Ex2-3 and R.sub.IL18BPc) on cDNAs generated using total RNA from HepG2 cells, THP1 cells, and primary human hepatocytes isolated from human liver chimeric mice (Michailidis et al., 2019, manuscript submitted). The primer sequences are provided in Table 7. For IL-18 and IL-18BP ELISAs (DuoSet, R&D Systems) cells were stimulated for 24 h with the concentrations indicated above. Supernatants were obtained and kept at −80° C. ELISA was performed in accordance with the kit manufacturers' instructions.
[0163] Flow Cytometry
[0164] Unstimulated and stimulated cells were washed with PBS including 2% FCS and 2 mM EDTA. Adherent cell lines were detached with trypsin (Invitrogen) prior to PBS washing. Only certain cell lines, HUVEC, LX-2, NKL and THP1, which had induced IL18R1 mRNA expression upon stimulation with various cytokines, were stimulated for 24 h, as described above in the section of gene expression analyses, and further analyzed for surface IL-18R1 expression by flow cytometry. Cells were then fixed with Fix Buffer I (BD Biosciences) for 10 minutes at 37° C. and washed twice. Staining was performed using a primary antibody against IL-18R1 (R&D Systems) for 1 hour at room temperature, followed by washing and secondary staining with Goat IgG (H+L) APC-conjugated Antibody (R&D Systems) for 30 minutes at room temperature. Cells were washed twice and acquired on a Gallios flow cytometer (Beckman Coulter). Analyses were performed with the FlowJo software (FlowJo). Of note, surface IL-18RAP expression on the cell lines above was not assessed, as we did not validate any antibody specific for human IL-18RAP detection by flow cytometry using control transfected cell lines (data not shown).
[0165] 3′ RACE and Exon Trapping
[0166] Rapid amplification of 3′ cDNA ends (3′ RACE) was performed as previously described (Scotto-Lavino et al., 2006). Briefly, cDNA was synthesized with an adapter primer (AP-1) and SuperScript II Reverse Transcriptase (Thermo Fisher Scientific) from total RNA isolated from the EBV-B cells of a healthy control and the patients' siblings. The first PCR amplification of 3′ partial cDNA ends was performed with a forward primer (F.sub.Ex1) specific to IL18BP exon 1, and a universal amplification primer (UAP-1) binding to AP-1. The PCR products were purified and used for nested PCR with an IL18BP-specific primer (F.sub.Ex1-2) spanning the junction of exons 1 and 2, and an abridged universal amplification primer (AUAP). The final amplicons generated from the WT and heterozygous sibling samples were ligated into the pGEM-T-Easy vector (Promega) and the colonies were sequenced. For in vitro 3′ terminal exon-trapping experiments, 1558 bp DNA fragments starting with a 3′-fragment of IL18BP intron 3 (43 bp) were amplified from the genomic DNA samples of the WT sibling and the patient, with pTAG4-IL18BP forward and reverse primers. These fragments were between three EcoRI and XhoI sites of the pTAG4 vector. The pTAG4 exon-trapping vector (Krizman and Berget, 1993) was kindly provided by Dr. Anders Lade Nielsen. COS7 cells were transfected with one WT and one mutant clone in the presence of X-tremeGENE 9 DNA Transfection Reagent (Roche). After 16 h, total RNA was extracted with the RNeasy Mini Kit (QIAGEN), treated with DNase (TURBO DNA-free Kit, Invitrogen), and used for cDNA synthesis with the SuperScript III First-Strand Synthesis System (Life Technologies) and an adapter primer (AP-2) (Blechingberg et al., 2007). The first PCR was performed with a specific primer (F.sub.AD1-2) spanning the junction of AD exons 1 and 2, and the UAP-2, which binds to AP-2. This reaction was followed by nested PCR with a specific primer (F.sub.AD2-Ex4) encompassing the junction of AD exon 2 and IL18BP exon 4, and a reverse primer (Run.sub.UTR) binding to the end of the IL18BP 3′ UTR. The nested PCR products were inserted into the pGEM-T-Easy vector and the colonies were sequenced. Successful and equivalent cDNA synthesis was confirmed by the amplification by PCR of HPRT1, as the housekeeping gene control. SnapGene was used to analyze the DNA sequences of the clones. All clones carrying PCR artifacts or sequences not matching the IL18BP canonical transcript were excluded from subsequent analyses. Digital images were captured by the Amersham Imager 600 (GE Healthcare, Life Sciences). The primer sequences are provided in Table 7.
[0167] Generation of IL18BP Constructs and Site-Directed Mutagenesis (SDM)
[0168] The human canonical IL18BP cDNA ORF clone (CCDS8206) was obtained from OriGene and inserted into the pEF-BOS-EX mammalian expression plasmid (Murai et al., 1998), which was kindly provided by Dr. Shigekazu Nagata. The forward primer (IL18BP BamHI) included a BamHI site and a Kozak consensus sequence; whereas the reverse primer (IL18BP-His XbaI) contained an in-frame coding sequence for a C-terminal 6×-His tag and an XbaI site. Site-directed mutagenesis (SDM) to generate the IL18BP variants present in GnomAD (p.V23I, p.R121Q, and p.P184L) was performed by a modified overlap-extension PCR-based method (Shimada, 1996). IL18BP BamHI/SDM-Reverse primers and SDM-Forward/IL18BP-His XbaI primers were used to amplify the mutated IL18BP in two separate reactions, using the pEF-BOS-EX-IL18BP-His WT plasmid as the template. Each PCR product was purified and the different products were mixed without primers for overlap-extension PCR. SDM for the p.Q192H allele was performed by one-step PCR with the IL18BP BamHI and Q192H-His-XbaI primers. Novel IL18BP transcript variants (M1, M2, and M3) were generated with the IL18BP BamHI and SDM-Reverse primers or the SDM-Forward and mutant reverse primers (containing an in-frame coding sequence for a C-terminal 6×-His tag, a stop codon and an XbaI site) in two separate reactions, using the pEF-BOS-EX-IL18BP-His WT plasmid and control gDNA, respectively, as templates. The M1 and M2 transcripts were cloned including an 18 nt poly-A insert downstream of the 6×-His tag, followed by a stop codon. All constructs were confirmed by DNA sequencing. The primer sequences are provided in Table 7.
[0169] Transient Expression of IL-18BP and Immunoblotting
[0170] COS7 cells were used to seed six-well plates at a density of 2×10.sup.5 cells/well, in DMEM supplemented with 10% FBS. They were transfected with pEF-BOS-EX-IL18BP-His constructs (500 ng per well) in the presence of the X-tremeGENE 9 DNA transfection reagent (Roche Applied Sciences). The culture medium was removed six hours after transfection, and the cells were incubated for three days in serum-free OPTIMEM. Culture supernatants were concentrated by centrifugation on Amicon centrifugal protein filters (3 kDa; EMD Millipore) according to the manufacturer's instructions, and were stored at −80° C. for later use. Total protein concentration in the COS7 supernatants was determined with a Pierce BCA protein assay kit (Thermo Fisher Scientific) and total protein (15 μg protein per lane) was then subjected to SDS/PAGE (12% polyacrylamide gel) under reducing conditions. Immunoblotting was performed with primary antibodies against the His-Tag (MA1-21315, 0.5 μg/mL, Thermo Fisher Scientific) and IL-18BP (AF119 at 0.5 μg/mL, R&D Systems). Digital images were captured by the Amersham Imager 600 (GE Healthcare, Life Sciences).
[0171] IL-18BP Bioassay
[0172] The bioactivity of human IL-18BP was assessed by measuring inhibition of the IFN-γ-inducing activity of human IL-18 in NK-92 cells. Briefly, NK-92 cells were cultured in RPMI supplemented with 10% FBS and 200 IU/ml IL-2 (Chiron). Before the IL-18BP bioassay, NK-92 cells were rested for 24 h in the absence of IL-2. Rested NK-92 cells, at a density of 0.5×10.sup.6 cells/mL in flat-bottomed 96-well plates, were stimulated for 24 h with 100 μg/mL IL-12 (R&D Systems), 10 ng/mL IL-18 (R&D Systems), and various concentrations of recombinant IL-18BP-His (125-500 ng/mL, Sino Biologicals) or with concentrated supernatants (100 μg/mL of total protein) from COS7 cells transiently transfected with IL-18BP constructs. IL-18 and IL-18BP were mixed together and incubated for 1 h at 37° C. before their addition to cells. At 24 h, the supernatants were used for ELISA, to assess IFN-γ production (Human IFN gamma Ready-Set-Go, Thermo Fisher Scientific). All IL-18BP bioassays were performed in duplicate.
[0173] Immunohistochemistry
[0174] Immunohistochemical staining was performed on 4 μm-thick sections of formalin-fixed paraffin-embedded (FFPE) tissue samples, with Bond autostainer (Leica, Newcastle upon Tyne, UK), after antigen retrieval and endogenous peroxidase inhibition with H.sub.2O.sub.2 (3%). We used primary antibodies against CD3 (polyclonal rabbit, CD3epsilon, A0452, Dako, Agilent), CD4 (monoclonal IgG1k mouse, NCL-L-CD4-368, Leica), CD8 (monoclonal IgG1k mouse, C8/144B, Dako), CD20 (monoclonal IgG2ak mouse, L26, Dako), CD57 (monoclonal IgM mouse, NK-1, Leica), CD68 (monoclonal IgG2a mouse, 514H12, Leica), CD163 (monoclonal IgG1 mouse, NCL-L-CD163, Leica), perforin (monoclonal IgG1 mouse, 5B10, Leica), NKp46 (polyclonal IgG goat, AF1850, R&D Systems), and Hep Par-1 (monoclonal IgG1k mouse, OCH1E5, Dako). These antibodies were detected and the signal was amplified with the Bond Polymer Refine Detection kit (DS9800, Leica) and hematoxylin counterstaining. We also used primary antibodies against IL-18BP (monoclonal mouse, clone 13603, R&D), IL-18BP (polyclonal rabbit, HP37434, Sigma), and IL-18 (polyclonal rabbit, HPA0003980, Sigma). These antibodies were detected and the signal was amplified with the Bond Intense R kit (DS9263, Leica) and hematoxylin counterstaining. Native explanted livers are usually fixed in formalin at room temperature during 3 to 8 days, while most other tissue samples are fixed during 6 to 72 hours. This long immersion in formalin may be responsible for alteration of some epitopes and for increased background signals when performing immunohistochemistry. Moreover, the most efficient kits available for immunohistochemistry include a streptavidin-biotin amplification step. This may also explain for increased background signals during immunohistochemistry staining, as hepatocytes contain endogenous biotin. Although IL-18BP staining was validated on control transfected cell lines, the staining observed in healthy or diseased livers was not significantly different from the staining detected with the diluent without the primary antibody as negative control (data not shown). Normal liver corresponds to liver parenchyma of patients who did not receive chemotherapy and underwent surgery for metastasis of colon carcinoma. The sampling was at least 2 cm away from the nearest metastasis.
[0175] Coculture Experiments and HAV Infection
[0176] HepG2 cells (kindly provided by Dr. Yosef Shaul, Weizmann Institute of Science, Israel) were used to seed collagen-coated 96-well plates at 1.5×10.sup.4 cell/well, in DMEM supplemented with 10% FBS and 0.1 mM non-essential amino acids (NEAA). NK92 cells (1-2×10.sup.6 cell/mL in 24-well plates) were stimulated with IL-18 (200 ng/mL) and/or IL-18BP (1000 ng/mL) in the absence of IL-2 for 18 h. IL-18 and IL-18BP were mixed together and incubated for 1 h at 37° C. before their addition to cells. On the day of coculture, HepG2 cells, at 80-90% confluence, were washed once with serum-free RPMI, and stained with calcein-AM (0.5 μM, Trevigen) for 30 min at 37° C. (Bugide et al., 2018; Somanchi et al., 2015). Calcein-labeled HepG2 cells were washed twice with complete RPMI. Activated NK cells, washed and resuspended in complete RPMI, were added at a density of 1-3×10.sup.5 cells/well to HepG2 cells, and the two cell types were cocultured at 37° C. for 4 h. PBMCs from healthy donors were isolated by Ficoll-Paque density gradient, as previously described (Hernandez et al., 2018). Isolated PBMCs (4×10.sup.6 cell/mL in 24-well plates) were stimulated with IL-18 (200 ng/mL) and/or IL-18BP (1000 ng/mL) in the presence of IL-2 (400 IU/mL) for 24 h. Stimulated PBMCs, washed and resuspended in complete RPMI, were cultured at various densities (2-4×10.sup.5 cells/well) with HepG2 cells for 4 h.
[0177] HepG2 and Huh7.5 cells were infected with HAV (HM-175/18f) at various dilutions and maintained in DMEM supplemented with 3% FBS and 0.1 mM NEAA for several days. At 3 days post-infection, when HepG2 and Huh7.5 cells reached to maximum infection rates, 40% and 100%, respectively, both mock- and HAV-infected hepatocytes were detached and reseeded on collagen-coated 96-well plates at 1.5×10.sup.4 cell/well. Surplus infected cells were lysed for RNA isolation, and their supernatants were stored for future use. On the following day, calcein-labeled mock- or HAV-infected HepG2 cells were cultured with NK-92 cells (3×10.sup.5 cells/well), which were activated with IL-18 and/or IL-18BP, as described above. For Huh7.5 cells, NK-92 cells were stimulated with IL-18 (200 ng/mL) and/or IL-18BP (1000 ng/mL) in the presence of IL-2 (20 IU/mL) for 18 h and cultured at 3×10.sup.5 cells/well with calcein-labeled mock- or HAV-infected Huh7.5 cells. After 4 h of coculture, supernatants were collected and stored at −80° C. for later use. Cells were washed twice with PBS and fixed by incubation with 4% PFA for 20 min at room temperature in the dark. The nuclei were then stained with DAPI (D1306 at 1/5000, Thermo Fisher Scientific). For high-content imaging analyses, ImageXpress Micro XLS (Molecular Devices, Sunnyvale, Calif.) and MetaXpress PowerCore software were used. The total-cell integrated intensity of calcein-positive cells was determined for each well and adjusted by subtracting the background fluorescence (unstained hepatocyte cells). As an alternative to fluorescence imaging, albumin levels in hepatocyte-NK cell cocultures were determined by ELISA, as previously described (de Jong et al., 2014). Relative fluorescence and albumin levels were determined by normalization against the mean value for hepatocytes cocultured with effector cells (NK-92 cells or PBMCs) without pretreatment, set to 100. All coculture experiments were performed in quadruplicate.
[0178] For HAV immunostaining, infected hepatocytes were fixed with 4% PFA for 30 min at room temperature. Fixed cells were washed twice with PBS and incubated with 100 mM Glycine for 15 min. Cells were then permeabilized for 10 min using PBS with 0.1% Triton-X100, followed by blocking for 1 h with PBS+5% Goat serum (Jackson ImmunoResearch, Code 005-000-121). Cells were stained with primary antibodies against HAV (Mouse anti-HAV VP1, K2-4F2, Creative Biolabs; Mouse Anti-HAV, 7E7, Mediagnost) at 1/1000 for overnight at 4° C., followed by staining with Goat-anti-mouse AlexaFluor 594 at 1/1000 and DAPI at 1/5000 for 1 h at room temperature. All antibody dilutions were carried out in PBS+5% Goat serum. Finally, cells were imaged using a Nikon Eclipse TE300 fluorescent microscope, and images were processed with ImageJ.
[0179] Total RNA and supernatants harvested from infected HepG2 and Huh7.5 cells were utilized for expression analysis of IL18 and IL18BP by qPCR and ELISA, as described above, however no mRNA and protein induction of IL18 or IL18BP was detected in HAV-infected HepG2 or Huh7.5 hepatocytes (data not shown).
[0180] Statistical Analysis
[0181] The results of the experiments were plotted as means±SEM. GraphPad Prism Software (www.graphpad.com) was used to calculate mean values and SEM, and for one-way ANOVA. Statistical significance is indicated by asterisks (*, P<0.05, **, P<0.01, and ***, P<0.001), with P values greater than 0.05 considered non-significant (n.s.).
[0182] Data Availability
[0183] The WES data are available in the Sequence Read Archive (ncbi.nlm.nih.gov/sra) with the BioProject accession number PRJNA543035.
[0184] Tables
[0185] The tables referenced in this Example 1 are provided below. Table 1 summarizes the genetic analysis of the WES data of the patient and two siblings. Table 2 lists the homozygous rare nonsynonymous variations present only in the patient. Table 3 describes the characteristics of genes with homozygous rare nonsynonymous variations present only in the patient. Table 4 provides other clinical findings of the patient prior to fulminant viral hepatitis. Table 5 shows the analytical findings in the patient and her siblings following HAV infection. Table 6 presents the immunological phenotyping data of the patient. Table 7 lists the primers used in this study.
TABLE-US-00002 TABLE 1 Genetic analysis of the WES data of the patient and two siblings WES data analysis Number of annotated variations (Number of mutated genes) III.1 (Patient) III.2 (Sibling) III.3 (Sibling) Total 142,213 (19,550) 141,383 (19,650) 141,145 (19,665) Nonsynon- 12,140 (6,148) 12,168 (6,232) 12,232 (6,234) ymous & essential splicing MAF < 0.1% 241 (230) 240 (222) 271 (246) Homozygous 9 (9) 12 (12) 7 (7) Present only 6 (6) — — in the patient
Only nonsynonymous (indel-inframe, indel-frameshift, start-lost, missense, nonsense, stop-lost) and essential splice-site (splice acceptor and splice donor) variants were selected. Variants with a minor allele frequency (MAF) of 0.1% or greater in public databases: EVS, 1000 Genomes, and GnomAD, including variants with an AF≥0.1% in each ethnic subpopulation (African, Ashkenazi Jewish, Finnish, non-Finnish European, South Asian, East Asian, and Latino) in GnomAD were excluded. Finally, under the autosomal recessive model of inheritance, only homozygous variants were selected, and six genes with homozygous variants present only in the patient were considered to be candidate disease-causing genes. The number of genes mutated is shown in parentheses.
TABLE-US-00003 TABLE 2 Homozygous rare nonsynonymous variations present only in the patient Variation Zygosity Gene GDI.sup.& NI.sup.# Type Change III.1 III.2 III.3 MAF CADD/MSC IL18BP 2.05 1.764 deletion NM_173042.2:c.508-19_528del hom het wt 0 28.2/12.19 ADAMTS1 4.70 0.100 missense NM_006988:p.Lys648Arg hom wt wt 2.06E−04 25/17.33 SLC6A19 6.44 0.152 missense NM_001003841.2:p.Val551Met hom het het 9.39E−05 25.1/14.87 TM7SF2 9.21 3.610 missense NM_003273.3:p.Ala36Gly hom het wt 3.24E−05 16.63/16.48 ZNF324 1.65 0.143 missense NM_014347.2:p.Ile385Met hom het het 0 23.2/12.19 ZNF814 2.16 0.005 missense NM_001144989.1:p.His407Asp hom het het 3.54E−05 15.57/12.19 Total 6 genes were identified with rare (MAF < 0.001) nonsynonymous variants present at homozygous state only in the patient. .sup.&GDI: Gene Damage Index, where the range is between 0.0001 for least damaged human gene and 42.91 for most damaged human gene. .sup.#NI: McDonald-Kreitman neutrality index (NI) measurement, where NI < 1 indicates a purifying selection.
TABLE-US-00004 TABLE 3 Characteristics of genes with homozygous rare nonsynonymous variations present only in the patient Gene description Expression pattern Known function Clinical significance Interleukin 18 Broad expression in spleen, appendix, Inhibitor of the proinflammatory Unknown binding protein (IL18BP) lymph nodes, and 23 other tissues cytokine, IL18 ADAM metallopeptidase with Broad expression in ovary, placenta, Role in growth, fertility, Unknown thrombospondin type 1 motif 1 and 17 other tissues and organ (ADAMTSI) morphology and function Solute carrier family 6 member 19 Broad expression in small intestine, Transportation of most Biallelic mutations (SLC6A19) duodenum, and kidney neutral amino acids in this gene cause across the apical Hartnup disorder membrane of epithelial cells Transmembrane 7 Broad expression in adrenal, fat, Role in cholesterol biosynthesis Unknown superfamily member 2 and 20 other tissues (TM7SF2) Zinc finger protein 324 (ZNF324) Ubiquitous expression in brain, May be involved in Unknown ovary, and 25 other tissues transcriptional regulation Zinc finger protein 814 (ZNF814) Ubiquitous expression in prostate, May be involved in Unknown spleen, and 25 other tissues transcriptional regulation
Information related to the expression pattern, known function and clinical significance of these six genes in humans were obtained from the NCI Gene database.
TABLE-US-00005 TABLE 4 Other clinical findings of the patient prior to fulminant viral hepatitis July October March October April September November Clinical findings.sup.# 2004 2005 2007 2009 2011 2012 2013 Thyroiditis anti-TPO NA Negative Negative NA Positive NA NA (positive >60 IU/mL) (<45) (<60) (145) anti-PO NA NA NA Negative Positive NA NA (positive >10 IU/mL) (51) anti-TG NA Negative Negative Positive NA NA NA (positive >116 IU/mL) (<90) (<60) Free T4 NA NA NA NA Positive Normalization upon (normal: 1-1.07 ng/dL) (2.66) treatment TSH NA NA NA NA Positive Normalization upon (normal: 0.4-4 mIU/L) (119.22) treatment Celiac disease (excluded) RLA IgA anti-tTG NA Negative Negative Negative NA Negative Negative (positive >150 cpm) (<47) (<45) (<66) (<44) (<129) EMA NA Negative Negative Negative NA NA NA Langerhans anti-IC Positive NA NA NA NA NA NA Diabetes anti-Insulin Positive NA NA NA NA NA NA (positive >0.7%) (1.31) anti-IA2 Negative NA NA NA NA NA NA (positive >80 cpm) (<29) anti-GAD65 Negative NA NA NA NA NA NA (positive >180 cpm) (<84) Glycemia High Normalization upon treatment (normal: 4-6 mmol/L) (37) HbA1c High Normalization upon treatment (normal: 4-5.6%) (8.9) .sup.#TPO: Thyroid peroxidase, PO: Peroxidase, TG: Thyroglobulin, T4: Thyroxine, TSH: thyroid-stimulating hormone, RLA: Radioligand-receptor assay, tTG: Tissue transglutaminase, EMA: Endomysial antibodies (IgA), IC: Islet cells, IA2: Islet antigen 2, GAD65: Glutamic acid decarboxylase, HbA1c: Hemoglobin A1c, NA: Not available
TABLE-US-00006 TABLE 5 Analytical findings in the patient and her siblings following HAV infection Patient (III.1) Sibling (III.2) Sibling (III.3) Hospitalization Hospitalization.sup.# Hospitalization.sup.# (Day 8-11) (Day 1-3) Day Day (Day 1-3) Day Day Day Days Day 1 Day 8 Day 9.sup.$ Day 10* Day 11.sup.& Day 1 Day 2 Day 3 5 15 Day 1 Day 2 Day 3 4 5 15 IgM Positive >60 ND ND ND >60 ND ND ND ND >60 ND ND ND ND ND anti-HAV (no (IU/L) value) ALT 2181 1481 1117 275 403 42 35 29 27 23 1594 1298 894 757 531 96 (<40 IU/L) AST 2582 1982 1863 1393 1182 44 36 34 33 39 755 513 271 149 108 52 (<40 IU/L) GGT 73 28 22 27 28 55 48 41 38 33 293 277 229 206 162 55 (<25 IU/L) Factor V 100 48 30 42 29 ND ND 93 100 ND 100 ND 100 100 ND ND (>80%) PT 67 9 14 82 53 100 89 100 100 100 92 92 100 100 100 98 (>70%) .sup.#Two siblings (III.2 and III.3) were hospitalized 2 days after the patient died. .sup.$Day 9: Values were prior to liver transplantation. *Day 10: Post-transplantation values are shown. .sup.&Day 11: Patient died.
TABLE-US-00007 TABLE 6 Immunological phenotyping data of the patient PBMC Day 1 Day 8 Day 9 Day 10 PMNs 6.00 3.76 5.2 3.76 (normal: 1.5-8 10.sup.9/L) Lymphocytes 2.21 1.44 2.26 2.38 (normal: 1.5-5 10.sup.9/L) Monocytes 0.44 0.41 0.08 0.12 (normal: 0.1-1 10.sup.9/L)
TABLE-US-00008 TABLE 7 List of primers used in this study Name Sequence (5′-3′) Application Seq-1 Fwd CAGCCTGTGAACTAATGCC Sanger sequencing (SEQ ID NO: 3) Seq-1 Rev GAATTTGGTGAGAGAAGGGA Sanger sequencing (SEQ ID NO: 4) Seq-2 Fwd CAAGGAGAGGCCTCCAG Sanger sequencing (SEQ ID NO: 5) Seq-2 Rev ACTCCAGGTAGACAGGTAG Sanger sequencing (SEQ ID NO: 6) IL18BP SYBR CAGCTCTGGGCTGGGCTGAG SYBR Green qPCR Fwd* (SEQ ID NO: 7) IL18BP SYBR GGGGTGTGTTGCGCATCCAC SYBR Green qPCR Rev* (SEQ ID NO: 8) M1 SYBR Fwd GGAACGTGGGAGCACAGGTA SYBR Green qPCR (SEQ ID NO: 9) M1 SYBR Rev TTGAGCGTTCCCCTGCCAGA SYBR Green qPCR (SEQ ID NO: 10) M2 SYBR Fwd GGAACGTGGGAGCACAGGTA SYBR Green qPCR (SEQ ID NO: 11) M2 SYBR Rev CTTGAGCGTTCCCCCAGAGC SYBR Green qPCR (SEQ ID NO: 12) M3 SYBR Fwd CCTGCACAGCACCAACTTCTC SYBR Green qPCR (SEQ ID NO: 13) M3 SYBR Rev GAGACATGGGAGTGGGAGCCA SYBR Green qPCR (SEQ ID NO: 14) GAPDH SYBR GGAGCGAGATCCCTCCAAAAT SYBR Green qPCR Fwd.sup.# (SEQ ID NO: 15) GAPDH SYBR GGCTGTTGTCATACTTCTCA SYBR Green qPCR Rev.sup.# TGG (SEQ ID NO: 16) AP-1 CCAGTGAGCAGAGTGACGA Exon trapping GGACTCGAGCTCAAGCTTT TTTTTTTTTTTTTT (SEQ ID NO: 17) UAP-1 CCAGTGAGCAGAGTGACG Exon trapping (SEQ ID NO: 18) AUAP GAGGACTCGAGCTCAAGC Exon trapping (SEQ ID NO: 19) F.sub.Ex1 ATGACCATGAGACACAACTGGA Exon trapping (SEQ ID NO: 20) F.sub.Ex1-2 CTGGACACCAGACCTCAGCC Exon trapping/ (SEQ ID NO: 21) PCR F.sub.Ex2-3 CCACTGAATGGAACGCTGAG PCR (SEQ ID NO: 22) R.sub.IL18BPa TTAACCCTGCTGCTGTGGA PCR (SEQ ID NO: 23) R.sub.IL18BPbd TCAAGGTTGTGCTGCTGCT PCR (SEQ ID NO: 24) R.sub.ILI8BPc TCACAGGCTGCTCTGGCA PCR (SEQ ID NO: 25) pTAG4- CTGGAATTCCTTCTGCGGCCTT Exon trapping IL18BP CTCATG Fwd (SEQ ID NO: 26) pTAG4- GCACTCGAGGATGGATCTTTTC Exon trapping IL18BP TAAATGTT Rev (SEQ ID NO: 27) AP-2 GCGGAATTCGGATCCCTCGAGTT Exon trapping TTTTTTTTTTTTTTTTT (SEQ ID NO: 28) UAP-2 GCGGAATTCGGATCCCTCGAGTT Exon trapping (SEQ ID NO: 29) FA.sub.D1-2 AGGGCCAGCTGTTGGGCTCG Exon trapping (SEQ ID NO: 30) F.sub.AD2-Ex4 CGTCGGCCTCCGAACGCCGGGA Exon trapping (SEQ ID NO: 31) R.sub.UTR CTCCATTGAATAATCCTTTATGA Exon trapping GGACC (SEQ ID NO: 32) HPRT1 Fwd CTGGCGTCGTGATTAGTGATGA Exon trapping TG (SEQ ID NO: 33) HPRT1 Rev TTGAGCACACAGAGGGCTACA Exon trapping ATG (SEQ ID NO: 34) IL18BP GTTGGATCCGCCACCATGAGA Cloning BamHI CACAACTGGACACCA (Kozak) (SEQ ID NO: 35) IL18BP- ATATCTAGATTAATGATGAT Cloning His XbaI GATGATGATGACCCTGCTGC TGTGGACTGC (SEQ ID NO: 36) V23I SDM CTCCTGTGTGCCCACATCGT Mutagenesis Fwd CACTCTCCTGGTC (SEQ ID NO: 37) V23I SDM GACCAGGAGAGTGACGATGT Mutagenesis Rev GGGCACACAGGAG (SEQ ID NO: 38) R121Q SDM AGGGGAGCACCAGCCAGGAA Mutagenesis Fwd CGTGGGAGCAC (SEQ ID NO: 39) R121Q SDM GTGCTCCCACGTTCCTGGC Mutagenesis Rev TGGTGCTCCCCT (SEQ ID NO: 40) P184L SDM CACCCAAGAAGCCCTGCTC Mutagenesis Fwd TCCAGCCACAG (SEQ ID NO: 41) P184L SDM CTGTGGCTGGAGAGCAGGG Mutagenesis Rev CTTCTTGGGTG (SEQ ID NO: 42) Q192H-His ATATCTAGATTAATGATGA Mutagenesis XbaI TGATGATGATGACCCTGAT GCTGTGGACTGC (SEQ ID NO: 43) M1-His ATATCTAGATTAATGATGA Cloning XbaI TGATGATGATGGGATGTTT TTTTTTTTTTTTTTTCTCC ATTGAATAATC (SEQ ID NO: 44) M2-His ATATCTAGATTAATGATGAT Cloning XbaI GATGATGATGGGATGTTTTT TTTTTTTTTTTTTCTCCATT GAATAATC (SEQ ID NO: 45) M3-His ATATCTAGATTAATGATGAT Cloning XbaI GATGATGATGCTGAAGAGGC AGCATTTCA (SEQ ID NO: 46) M1 SDM CCAGCTCTGGCAGGGGAAC Mutagenesis Fwd GCTCAAGCCTG (SEQ ID NO: 47) M1 SDM CGTTCCCCTGCCAGAGCTG Mutagenesis Rev GGCCAGGACGA (SEQ ID NO: 48) M2 SDM CCAGCTCTGGGGGAACGCT Mutagenesis Fwd CAAGCCTGGTT (SEQ ID NO: 49) M2 SDM GAGCGTTCCCCCAGAGCTGG Mutagenesis Rev GCCAGGACGA (SEQ ID NO: 50) M3 SDM CCAGCTCTGGCTCCCACTCC Mutagenesis Fwd CATGTCTCTG (SEQ ID NO: 51) M3 SDM GGAGTGGGAGCCAGAGCTG Mutagenesis Rev GGCCAGGACGA (SEQ ID NO: 52) *Khalid KE, Nsairat HN, Zhang JZ (2016) The presence of interleukin 18 binding protein isoforms in Chinese patients with rheumatoid arthritis. AIMS Med Sci 3: 103-113 .sup.#PrimerBank ID: 378404907c1
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Example 2
IL-10RB Mutation in a Multiplex Family with Fulminant Hepatitis Due to Hepatitis A Virus
[0244] In studies in line with those undertaken in Example 1, we evaluated a distinct family with fulminant viral hepatitis. In this family, a mutation in the IL-10 Receptor B (IL-10RB), was identified and associated with FVH in the patients of the family.
[0245] Interleukin 10 receptor, beta subunit (IL-10 RB) is a subunit for the interleukin-10 receptor, also designated IL-10 Receptor subunit 2 (IL10R2) and cluster differentiation antigen CD Ag CDw210b. IL-10RB has the GeneID 3588 and UnitprotKB/SwissProt identifier Q08334. IL-10RB protein amino acid sequence (SEQ ID NO: HGNC 5965 or NP_000619.3)
[0246] is provided below. The IL-10RB protein belongs to the cytokine receptor family and is an accessory chain essential for the active interleukin 10 receptor complex. Coexpression of IL-10RB and IL-10 receptor subunit 1 protein (also denoted IL-10RA) is required for IL10-induced signal transduction.
[0247] Familial segregation with IL-10RB segregation is depicted in
[0248] We evaluated population genetics of homozygous coding missense, homozygous loss-of-function IL-10RB mutations taken from GnomAD and previous reported loss-of-function IL-10RB mutations (
[0249] The IL-10RB amino acid sequence (NCBI Ref sequence NP_000619.3) (SEQ ID NO:2) is provided below. The Tryptophan and W100 site of the W100G mutation is in bold and underlined. DNA sequence for IL-10RB human is known (NCBI Ref sequence NG_012089.1)
TABLE-US-00009 10 20 30 MAWSLGSWLG GCLLVSALGM VPPPENVRMN 40 50 60 SVNFKNILQW ESPAFAKGNL TFTAQYLSYR 70 80 90 IFQDKCMNTT LTECDFSSLS KYGDHTLRVR 100 110 120 AEFADEHSD VNITFCPVDD TIIGPPGMQV 130 140 150 EVLADSLHMR FLAPKIENEY ETWTMKNVYN 160 170 180 SWTYNVQYWK NGTDEKFQIT PQYDFEVLRN 190 200 210 LEPWTTYCVQ VRGFLPDRNK AGEWSEPVCE 220 230 240 QTTHDETVPS WMVAVILMAS VFMVCLALLG 250 260 270 CFALLWCVYK KTKYAFSPRN SLPQHLKEFL 280 290 300 GHPHHNTLLF FSFPLSDEND VFDKLSVIAE 310 320 DSESGKQNPG DSCSLGTPPG QGPQS
[0250] Individuals and patients previously reported to carry IL-10RB mutations and their particular mutations are depicted below in Table 8.
TABLE-US-00010 TABLE 8 IL-10RB Patients Carrying at Least One Missense Mutation Num- ber of Patients' Onset of Muta- pa- Molecular country disease tion tients mechanism of origin (days) References S31F 1 Homozygous Iran 60 Yazdani, 2019 Shouval, 2014; S58R 1 Compound USA <30 Shouval, 2017 heterozygous Neven, 2013; Y59C 1 Homozygous Italy 14 Pigneur, 2013 C66Y 1 Homozygous Ban- 45 Kotlartz, 2012 gladesh France/ Saudi W100G 2 Homozygous Arabia 84/? Pigneur, 2013 G193R 1 Homozygous Turkey 10 Engelhardt, 2013 W204C 2 Compound France/ 14/? Neven, 2013; heterozygous/ China Pigneur, 2013; Homozygous Gong, 2019
[0251] Numerous reported IL-10RB mutations, along with this familial W100G mutation, were evaluated in an expression system. HEK293T cells were transfected with various IL-10RB missense mutation alleles and characterized (
[0252] A summary of the overexpression studies is provided in Table 9. The W100G mutant shows normal IL-22 and IL-29 response and altered IL-10 response, wherein STAT3 phosphorylation and SOCS3 expression is near wild type levels after IL-10 stimulation but STAT1 phosphorylation is significantly reduced and CXCL9 expression is not observed with IL-10 stimulation. The IL-10 response pathway is significantly altered with the IL-10RB W100G mutation.
TABLE-US-00011 TABLE 9 Summary of results on the overexpression system Read IL10RB overexpression system Stimulation out WT W100G S31F S58R Y59C C66Y S193R W204C E141X W159X IL10 WB- +++ + − − − + − + − − pSTAT1 WB- +++ +++ − − − +++ − +++ − − pSTAT3 qPCR- +++ − ND ND − ND ND ND − − CXCL9 qPCR- +++ +++ ND ND − ND ND ND − − SOCS3 IL22 WB- +++ +++ − +++ +++ + − + − − pSTAT1 WB- +++ +++ − +++ +++ + − + − − pSTAT3 qPCR- +++ +++ − +++ +++ + − + − − CXCL9 IL29 WB- +++ +++ − +++ ++ + − + − − pSTAT1 WB- +++ +++ − +++ ++ + − + − − pSTAT3 qPCR- +++ +++ − +++ + + − + − − CXCL9
[0253] Further characterization of the instant W100G mutation and other reported IL10RB missense mutations was conducted. IL10RB mRNA was measured by qPCR in patient, another IL10RB-deficient patient (IL-10RB.sup.KO) and control IL-10RB SV40-fibroblasts cells (
[0254] IL-10, IL-22 and IL-29 responses were further evaluated in different mutant alleles of IL-10RB by overexpression experiments. For these studies, IL10RB.sup.KO cells were transfected with different mutated alleles of IL10RB. Following treatment with one of the interleukins, CXCL9 or SOCS3 transcription was assessed by qPCR. In
[0255] In further such studies, the control, patient W100G and IL-10RB.sup.KO cells, transduced with an empty or IL-10RB-WT vector were evaluated for response to IL-10, IL-22 or IL-29 treatment. Transcription levels of CXCL9, SOCS3 and IL-10RB were assessed by qPCR (
[0256] The responses to IL-10, IL-22 and IL-29 of various IL-10RB alleles, mapped to the extracellular domain on the IL-10RB protein, are depicted in
[0257] These studies demonstrate that fulminant viral hepatitis can result following hepatitis virus infection in a patient or individual harboring mutation of IL-10 Receptor subunit, particularly IL-10RB, such that IL-10 mediated responses are blocked or significantly reduced. The instant patient IL-10 RB W100G mutation leads to altered IL-10 mediated responses, particularly demonstrating failure to generate CXCL9 response and phosphorylation of STAT1 and STAT3. Altered IL-10 response as provided herein results in an altered and ineffective viral response and leads to fulminant viral hepatitis.
[0258] Whole-exome sequencing (WES) of the patient of this study (denoted patient 2) revealed numerous homozygous mutations in this patient, including the IL-10RB W100G mutation, as shown in Table 10.
REFERENCES
[0259] Engelhardt, K. R., Shah, N., Faizura-Yeop, I., Kocacik Uygun, D. F., Frede, N., Muise, A. M., Shteyer, E., Filiz, S., Chee, R., Elawad, M., Hartmann, B., Arkwright, P. D., Dvorak, C., Klein, C., Puck, J. M., Grimbacher, B., and E. O. Glocker. 2013. Clinical outcome in IL-10- and IL-10 receptor-deficient patients with or without hematopoietic stem cell transplantation. Journal of allergy and clinical immunology 131:825-830. [0260] Gong, Y. Z., Ning, H. J., Ma, X., Zhu, D., Wang, F. P., Zhang, R., Zhang, Y. L., Zhong, X. M. 2019. [Clinical and genotypic characteristics of infantile inflammatory bowel disease]. Zhonghua Er Ke Za Zhi 57:520-525. [0261] Kotlarz, D., Beier, R., Murugan, D., Diestelhorst, J., Jensen, O., Boztug, K., Pfeifer, D., Kreipe, H., Pfister, E. D., Baumann, U., Puchalka, J., Bohne, J., Egritas, O., Dalgic, B., Kolho, K. L., Sauerbrey, A., Buderus, S., Güngör, T., Enninger, A., Koda, Y. K., Guariso, G., Weiss, B., Corbacioglu, S., Socha, P., Uslu, N., Metin, A., Wahbeh, G. T., Husain, K., Ramadan, D., Al-Herz, W., Grimbacher, B., Sauer, M., Sykora, K. W., Koletzko, S., and C. Klein. 2012. Loss of interleukin-10 signaling and infantile inflammatory bowel disease: Implications for diagnosis and therapy. Gastroenterology 143:347-355. [0262] Neven, B., Mamessier, E., Bruneau, J., Kaltenbach, S., Kotlarz, D., Suarez, F., Masliah-Planchon, J., Billot, K., Canioni, D., Frange, P., Radford-Weiss, I., Asnafi, V., Murugan, D., Bole, C., Nitschke, P., Goulet, O., Casanova, J. L., Blanche, S., Picard, C., Hermine, O., Rieux-Laucat, F., Brousse, N., Davi, F., Baud, V., Klein, C., Nadel, B., Ruemmele, F., and A. Fischer. 2013. A Mendelian predisposition to B-cell lymphoma caused by IL-10R deficiency. Blood 122:3713-3722. [0263] Pigneur, B., Escher, J., Elawad, M., Lima, R., Buderus, S., Kierkus, J., Guariso, G., Canioni, D., Lambot, K., Talbotec, C., Shah, N., Begue, B., Rieux-Laucat, F., Goulet, O., Cerf-Bensussan, N., Neven, B., and F. M. Ruemmele. 2013. Phenotypic characterization of very early-onset IBD due to mutations in the IL10, IL10 receptor alpha or beta gene: A survey of the GENIUS working group. Inflammatory bowel diseases 19:2820-2828. [0264] Shouval, D. S., Biswas, A., Goettel, J. A., McCann, K., Conaway, E., Redhu, N. S., Mascanfroni, I. D., Al Adham, Z., Lavoie, S., Ibourk, M., Nguyen, D. D., Samsom, J. N., Escher, J. C., Somech, R., Weiss, B., Beier, R., Conklin, L. S., Ebens, C. L., Santos, F. G., Ferreira, A. R., Sherlock, M., Bhan, A. K., Müller, W., Mora, J. R., Quintana, F. J., Klein, C., Muise, A. M., Horwitz, B. H., and S. B. Snapper. 2014. Interleukin-10 receptor signaling in innate immune cells regulates mucosal immune tolerance and anti-inflammatory macrophage function. Immunity 40:706-719. [0265] Shouval, D. S., Konnikova, L., Griffith, A. E., Wall, S. M., Biswas, A., Werner, L., Nunberg, M., Kammermeier, J., Goettel, J. A., Anand, R., Chen, H., Weiss, B., Li, J., Loizides, A., Yerushalmi, B., Yanagi, T., Beier, R., Conklin, L. S., Ebens, C. L., Santos, F. G. M. S., Sherlock, M., Goldsmith, J. D., Kotlarz, D., Glover, S. C., Shah, N., Bousvaros, A., Uhlig, H. H., Muise, A. M., Klein, C., and S. B. Snapper. 2017. Enhanced TH 17 Responses in Patients with IL10 Receptor Deficiency and Infantile-onset IBD. Inflammatory bowel diseases 23:1950-1961. [0266] Yazdani, R., Moazzami, B., Madani, S. P., Behniafard, N., Azizi, G., Aflatoonian, M., Abolhassani, H., and A. Aghamohammadi. 2019. Candidiasis associated with very early onset inflammatory bowel disease: First IL10RB deficient case from the National Iranian Registry and review of the literature. Clinical immunology 205:35-42.
TABLE-US-00012 TABLE 10 Homozygous Mutations Present on Patient 2 Polyphen Sift_ 2HVAR_ CADD_ Known Chr Pos Ref Alt Function Gene AAChange Pred Pred Phred MSC GDI PID? 21 34649025 T G missense IL10RB p.Trp100Gly D, D D, D, 29.8 23.8 10.34 Yes D, D 2 208725979 T C missense PLEKHM3 p.Glu653Gly D, D P 29.7 2.31 3.26 No 16 88926306 C G missense TRAPPC2L p.Phe100Leu D P, P 25.9 23.84 5.19 No 6 41303876 C T missense NCR2 p.Thr35Met D, D, D D, D, D 23.3 2.31 1.78 No 20 47269910 C T missense PREX1 p.Glu779Lys T, T B, B 20.9 2.31 13.43 No 7 15725797 ATGG A indel- MEOX2 p.His77del 20.5 4.78 5.44 No inframe 4 8307852 G T missense HIRA3 p.Val451Leu T B 15.35 2.31 2.85 No 2 238737852 T A splicing RBM44 10.96 2.31 4.67 No 2 238737860 A G splicing RBM44 p.Arg868Arg 10.79 2.31 4.67 No 13 38320594 TA T splicing TRPC4 9.94 2.31 1.08 No 2 238737854 T G splicing RBM44 8.267 2.31 4.67 No 16 12009530 C CCCG indel- GSPT1 p.Gly16dup 7.76 2.31 10.44 No inframe 7 131241029 G GGGCGAC indel- PODXL p.Ser29_ 5.726 2.31 3.35 No inframe Pro30dup 20 32664864 C CCAG indel- RALY p.Ala230_ 4.967 2.31 5.17 No inframe Gly231insSer 19 49657889 T TTCC indel- HRC p.Glu202dup 4.004 2.31 10.86 No inframe 13 25355965 GT G splicing RNF17 3.637 2.31 9.75 No 3 158537415 CTTTTT C splicing MFSD1 2.493 2.31 5.61 No 17 78081526 A AGCAGCGG splicing GAA 0.341 0.01 7.92 No
While there have been described what are presently believed to be the preferred embodiments of the present invention, those skilled in the art will realize that other and further changes and modifications may be made thereto without departing from the spirit of the invention, and it is intended to claim all such modifications and changes as come within the true scope of the invention.