EP4630033A1 - Novel human antiviral genes related to the eleos and lamassu prokaryotic systems - Google Patents

Abstract

Viral infection is a common threat to prokaryotic and eukaryotic life, which has resulted in the evolution of a myriad of antiviral systems. Some of these eukaryotic systems are thought to have evolved from prokaryotic antiphage proteins, with which they may display sequence and structural homology. Here, the inventors show that homologs of recently discovered antiphage systems are widespread in eukaryotes. They demonstrate that such homologs can retain a function in immunity by unveiling that eukaryotic proteins of the anti-transposon piRNA pathway display domain homology with the antiphage system Mokosh. The inventors further utilize this conservation to discover novel human antiviral genes related to the Eleos and Lamassu prokaryotic systems.

Description

NOVEL HUMAN ANTIVIRAL GENES RELATED TO THE ELEOS AND LAMASSU PROKARYOTIC SYSTEMS

FIELD OF THE INVENTION:

The present invention is in the filed of medicine, in particular virology.

BACKGROUND OF THE INVENTION:

A common denominator of prokaryotic and eukaryotic life is the threat posed by viruses. In both domains, evolutionary pressures resulting from viral infections led to the selection of antiviral mechanisms. In chordates, including humans, the first line of defense against infection relies on the steady-state expression of restriction factors, as well as on the interferon-driven upregulation of a panel of antiviral effectors termed interferon-stimulated genes (ISGs) (1). Prokaryotes implement diverse antiviral systems to thwart bacteriophages (phages), including restriction-modification and CRISPR-Cas systems (2-5). Each system may be composed of a unique gene or of multiple genes whose products act in concert. Genes of a given system are closely located on the genome, allowing tight coregulation of gene expression and genetic exchange via horizontal gene transfer (6, 7). Recently, more than 100 novel antiviral prokaryotic systems were uncovered (2-7). Among them, multiple bacterial antiviral systems were identified as the evolutionary origin of eukaryotic antiviral proteins, including CBASS (homolog of the eukaryotic cGAS/STING pathway), gasdermins, viperins or Avs (related to the NLR family) (8-12).

SUMMARY OF THE INVENTION:

The present invention is defined by the claims. In particular, the present invention relates to novel human antiviral genes related to the Eleos and Lamassu prokaryotic systems.

DETAILED DESCRIPTION OF THE INVENTION:

Viral infection is a common threat to prokaryotic and eukaryotic life, which has resulted in the evolution of a myriad of antiviral systems. Some of these eukaryotic systems are thought to have evolved from prokaryotic antiphage proteins, with which they may display sequence and structural homology. Here, the inventors show that homologs of recently discovered antiphage systems are widespread in eukaryotes. They demonstrate that such homologs can retain a function in immunity by unveiling that eukaryotic proteins of the anti-transposon piRNA pathway display domain homology with the antiphage system Mokosh. The inventors further utilize this conservation to discover novel human antiviral genes related to the Eleos and Lamassu prokaryotic systems.

Accordingly, the present invention relates to a method of treating a viral infection in a subject in need thereof comprising administering to the subject a therapeutically effective amount of i) at least one polypeptide related to the Eleos or Lamassu prokaryotic systems or ii) at least one polynucleotide encoding for said polypeptide.

In some embodiments, the viral infection comprises infection by a RNA virus or a DNA virus. In some embodiments, the subject is infected by one or more viruses selected from the group consisting of Arenaviridae, Astroviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Closter oviridae, Comoviridae, Cystoviridae, Flaviviridae, Flexiviridae, Hepevirus, Leviviridae, Luteoviridae, Mononegavirales, Mosaic Viruses, Nidovirales, Nodaviridae, Orthomyxoviridae, Picobirnavirus, Picornaviridae, Potyviridae, Reoviridae, Retroviridae, Sequiviridae, Tenuivirus, Togaviridae, Tombusviridae, Totiviridae, Tymoviridae, Hepadnaviridae, Herpesviridae, Paramyxoviridae or Papillomaviridae viruses. Relevant taxonomic families of RNA viruses include, without limitation, Astroviridae, Birnaviridae, Bromoviridae, Caliciviridae, Closteroviridae, Comoviridae, Cystoviridae, Flaviviridae, Flexiviridae, Hepevirus, Leviviridae, Luteoviridae, Mononegavirales, Mosaic Viruses, Nidovirales, Nodaviridae, Orthomyxoviridae, Picobirnavirus, Picornaviridae, Potyviridae, Reoviridae, Retroviridae, Sequiviridae, Tenuivirus, Togaviridae, Tombusviridae, Totiviridae, and Tymoviridae viruses. In some embodiments, the viral infection comprises infection by one or more viruses selected from the group consisting of adenovirus, rhinovirus, hepatitis, immunodeficiency virus, polio, measles, Ebola, Coxsackie, Rhino, West Nile, small pox, encephalitis, yellow fever, coronavirus, Dengue, influenza (including human, avian, and swine), lassa, lymphocytic choriomeningitis, junin, machuppo, guanarito, hantavirus, Rift Valley Fever, La Crosse, California encephalitis, Crimean-Congo, Marburg, Japanese Encephalitis, Kyasanur Forest, Venezuelan equine encephalitis, Eastern equine encephalitis, Western equine encephalitis, severe acute respiratory syndrome (SARS), parainfluenza, respiratory syncytial, Punta Toro, Tacaribe, pachindae viruses, adenovirus, Dengue fever, influenza A and influenza B (including human, avian, and swine), junin, measles, parainfluenza, Pichinde, punta toro, respiratory syncytial, rhinovirus, Rift Valley Fever, severe acute respiratory syndrome (SARS), Tacaribe, Venezuelan equine encephalitis, West Nile and yellow fever viruses, tick-borne encephalitis virus, Japanese encephalitis virus, St. Louis encephalitis virus, Murray Valley virus, Powassan virus, Rocio virus, louping-ill virus, Banzi virus, Ilheus virus, Kokobera virus, Kunjin virus, Alfuy virus, bovine diarrhea virus, and Kyasanur forest disease.

In some embodiments, the subject is infected by a herpesvirus. As used herein, the term “Herpesviridae” is a large family of DNA viruses that cause infections and certain diseases in animals, including humans. Nine herpesvirus types are known to primarily infect humans, at least five of which - herpes simplex viruses 1 and 2 (HSV-1 and HSV-2, also known as HHV- 1 and HHV-2; both of which can cause orolabial herpes and genital herpes), varicella zoster virus (or HHV-3; the cause of chickenpox and shingles), Epstein-Barr virus (EBV or HHV-4; implicated in several diseases, including mononucleosis and some cancers), and human cytomegalovirus (HCMV or HHV-5) - are extremely common among humans.

As used herein, the term "subject" or "subject in need thereof", is intended for a human or non-human mammal. Typically the patient is affected or likely to be infected by a virus. In some embodiments, the subject can be human or any other animal (e.g., birds and mammals) susceptible to viral infection (e.g. domestic animals such as cats and dogs; livestock and farm animals such as horses, cows, pigs, chickens, etc.). Typically said subject is a mammal including a non-primate (e.g., a camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, and mouse) and a primate (e.g., a monkey, chimpanzee, and a human). In some embodiments, the subject is a non-human animal. In some embodiments, the subject is a farm animal or pet. In some embodiments, the subject is a human. In some embodiments, the subject is a human infant. In some embodiments, the subject is a human child. In some embodiments, the subject is a human adult.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

As used herein, the term “polypeptide” has its general meaning in the art and refers to a polymer of amino acids of any length. The polymer can comprise modified amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art.

In some embodiments, the polypeptide comprises domains similar to prokaryotic Eleos. In some embodiments, the polypeptide is a GTPases immunity-associated protein (GIMAP). In some embodiments, the polypeptide comprises an amino acid sequence having at least 90% of identify with the amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO:2. SEQ ID NO : 1 >sp | Q96F15 | GIMA5_HUMAN GTPase IMAP family member 5 OS=Homo sapiens OX=9606 GN=GIMAP5 PE=1 SV=1

MGGFQRGKYGTMAEGRSEDNLSATPPALRI ILVGKTGCGKSATGNSILGQPVFESKLRAQ

SVTRTCQVKTGTWNGRKVLWDTPSI FESQADTQELYKNIGDCYLLSAPGPHVLLLVIQL

GRFTAQDTVAIRKVKEVFGTGAMRHWILFTHKEDLGGQALDDYVANTDNCSLKDLVREC

ERRYCAFNNWGSVEEQRQQQAELLAVIERLGREREGSFHSNDLFLDAQLLQRTGAGACQE

DYRQYQAKVEWQVEKHKQELRENESNWAYKALLRVKHLMLLHYEI FVFLLLCSILFFI I F

LFI FHYI

SEQ ID NO : 2>sp | Q6P9H5 | GIMA6_HUMAN GTPase IMAP family member 6 OS=Homo sapiens OX=9606 GN=GIMAP6 PE=2 SV=1

MEEEEYEQI PQENPPEELSQDPVLELSGGLREKEQKTPRRLRLILMGKTGSGKSATGNSI

LGRDVFESKLSTRPVTKTSQRRSREWAGKELEVIDTPNILSPQVSPEVADAICQAIVLSA

PGPHAVLLVTQLGRFTDEDQQWRRLQEVFGVGVLGHTILVFTRKEDLAGGSLEDYVRET

NNQALAWLDVTLARRHCGFNNRAQGEEQEAQLRELMEKVEAIMWENEGDYYSNKAYQYTQ

QNFRLKELQERQVSQGQGSEDVPGEESWLEGLSQIQKESEEAHRCLLGKADL

In some embodiments, the subject is administered with a therapeutically amount of at least 2 polypeptides related to the Lamassu prokaryotic system. In some embodiments, the subject is administered with an amount of i) a first polypeptide having an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO: 3 and ii) a second polypeptide having an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NON.

SEQ ID NO : 3>sp | Q99895 | CTRC_HUMAN Chymotrypsin-C OS=Homo sapiens OX=9606 GN=CTRC PE=1 SV=2

MLGITVLAALLACASSCGVPSFPPNLSARWGGEDARPHSWPWQI SLQYLKNDTWRHTCG

GTLIASNFVLTAAHCI SNTRTYRVAVGKNNLEVEDEEGSLFVGVDTIHVHKRWNALLLRN

DIALIKLAEHVELSDTIQVACLPEKDSLLPKDYPCYVTGWGRLWTNGPIADKLQQGLQPV

VDHATCSRIDWWGFRVKKTMVCAGGDGVI SACNGDSGGPLNCQLENGSWEVFGIVSFGSR RGCNTRKKPWYTRVSAYIDWINEKMQL

SEQ ID NO : 4>sp | Q96C19 | EFHD2 HUMAN EF-hand domain-containing protein D2 OS=Homo sapiens OX=9606 GN=EFHD2 PE=1 SV=1

MATDELATKLSRRLQMEGEGGGETPEQPGLNGAAAAAAGAPDEAAEALGSADCELSAKLL

RRADLNQGIGEPQSPSRRVFNPYTEFKEFSRKQIKDMEKMFKQYDAGRDGFIDLMELKLM

MEKLGAPQTHLGLKNMIKEVDEDFDSKLSFREFLLI FRKAAAGELQEDSGLCVLARLSEI

DVSSEGVKGAKSFFEAKVQAINVSSRFEEEIKAEQEERKKQAEEMKQRKAAFKELQSTFK

As used herein, the term “polynucleotide” as used herein refers to polymers of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded deoxyribonucleic acid (“DNA”), as well as triple-, double- and single-stranded ribonucleic acid (“RNA”). It also includes modified, for example by alkylation, and/or by capping, and unmodified forms of the polynucleotide. More particularly, the term “polynucleotide” includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA and mRNA, whether spliced or unspliced, any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing normucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids “PNAs”) and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA. In some embodiments, the polynucleotide comprises an mRNA. In other aspect, the mRNA is a synthetic mRNA. In some embodiments, the synthetic mRNA comprises at least one unnatural nucleobase. In some embodiments, all nucleobases of a certain class have been replaced with unnatural nucleobases (e.g., all uridines in a polynucleotide disclosed herein can be replaced with an unnatural nucleobase, e.g., 5-methoxyuridine). In some embodiments, the polynucleotide (e.g., a synthetic RNA or a synthetic DNA) comprises only natural nucleobases, i.e., A, C, T and G in the case of a synthetic DNA, or A, C, T, and U in the case of a synthetic RNA.

As used herein, the term "encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as, for example, a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase “nucleotide sequence that encodes a protein or a RNA” may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

In some embodiments, the polynucleotide encodes for a polypeptide that comprises at least one domain similar to prokaryotic Eleos. In some embodiments, the polynucleotide encodes for a GTPases immunity-associated protein (GIMAP). In some embodiments, the polynucleotide encodes for a polypeptide comprising an amino acid sequence having at least 90% of identify with the amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO:2.

In some embodiments, the subject is administered with an amount of i) a first polynucleotide that encodes for a polypeptide having an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO: 3 and ii) a second polynucleotide that encodes for polypeptide having an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NON. In some embodiments, a bicistronic polynucleotide that encodes both for i) a first polypeptide having an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NON and ii) a second polypeptide having an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NON.

In some embodiments, the polynucleotide of the present invention is a messenger RNA (mRNA).

In some embodiments, the polynucleotide is inserted in a vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or a viral vector. Typically, the vector is a viral vector which is an adeno-associated virus (AAV), a retrovirus, bovine papilloma virus, an adenovirus vector, a lentiviral vector, a vaccinia virus, a polyoma virus, or an infective virus. Typically, the vector of the present invention include "control sequences", which refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell. Another nucleic acid sequence, is a "promoter" sequence, which is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3 '-direction) coding sequence. Transcription promoters can include "inducible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), "repressible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc ), and "constitutive promoters”.

In some embodiments, the polypeptide or polynucleotide of the present invention can be conjugated to at least one other molecule. Typically, said molecule is selected from the group consisting of polynucleotides, polypeptides, lipids, lectins, carbohydrates, vitamins, cofactors, and drugs.

Typically, the active ingredient of the present invention (i.e. the polypeptide or polynucleotide) is combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. The term "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

In some embodiments, the polypeptide or polynucleotide of the present invention is formulated with lipidoids. The synthesis of lipidoids has been extensively described (see Mahon et al., Bioconjug Chem. 2010 21 : 1448-1454; Schroeder et al., J Intern Med. 2010 267:9-21; Akinc et al., Nat Biotechnol. 2008 26:561-569; Love et al., Proc Natl Acad Sci USA. 2010 107: 1864- 1869; Siegwart et al., Proc Natl Acad Sci US A. 2011 108: 12996-3001). While these lipidoids have been used to effectively deliver double stranded small interfering RNA molecules in rodents and non-human primates (see Akinc et al., Nat Biotechnol. 2008 26:561-569; Frank- Kamenetsky et al., Proc Natl Acad Sci USA. 2008 105: 11915-11920; Akinc et al., Mol Ther. 2009 17:872-879; Love et al., Proc Natl Acad Sci USA. 2010 107: 1864-1869; Leuschner et al., Nat Biotechnol. 2011 29: 1005-1010), the present disclosure describes their formulation and use in delivering polynucleotides.

In some embodiments, the polypeptide or polynucleotide of the present invention is formulated using one or more lipid-based structures that include but are not limited to liposomes, lipoplexes, or lipid nanoparticles (Paunovska, Kalina, David Loughrey, and James E. Dahlman. "Drug delivery systems for RNA therapeutics." Nature Reviews Genetics (2022): 1-16). Liposomes are artificially-prepared vesicles which can primarily be composed of a lipid bilayer and can be used as a delivery vehicle for the administration of pharmaceutical formulations. Liposomes can be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which can be hundreds of nanometers in diameter and can contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which can be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) which can be between 50 and 500 nm in diameter. Liposome design can include, but is not limited to, opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as, but not limited to, endocytosis. Liposomes can contain a low or a high pH in order to improve the delivery of the pharmaceutical formulations. As a non-limiting example, liposomes such as synthetic membrane vesicles are prepared by the methods, apparatus and devices described in US Patent Publication No. US20130177638, US20130177637, US20130177636, US20130177635, US20130177634, US20130177633, US20130183375, US20130183373 and US20130183372. In some embodiments, the liposomes are formed from 1, 2-di oleyloxy -N,N- dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, Wash.), l,2-dilinoleyloxy-3 -dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), and MC3 (as described in US20100324120) and liposomes which can deliver small molecule drugs such as, but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, Pa.). The polypeptide of polynucleotide of the present invention can be encapsulated by the liposome and/or it can be contained in an aqueous core which can then be encapsulated by the liposome (see International Pub. Nos. W02012031046, W02012031043, W02012030901 and W02012006378 and US Patent Publication No. US20130189351, US20130195969 and US20130202684).

In some embodiments, the polynucleotide of the present invention is formulated with stabilized plasmid-lipid particles (SPLP) or stabilized nucleic acid lipid particle (SNALP) that have been previously described and shown to be suitable for oligonucleotide delivery in vitro and in vivo (see Wheeler et al. Gene Therapy. 1999 6:271-281; Zhang et al. Gene Therapy. 1999 6: 1438- 1447; Jeffs et al. Pharm Res. 2005 22:362-372; Morrissey et al., Nat Biotechnol. 2005 2: 1002- 1007; Zimmermann et al., Nature. 2006 441 : 111-114; Heyes et al. J Contr Rel. 2005 107:276- 287; Semple et al. Nature Biotech. 2010 28: 172-176; Judge et al. J Clin Invest. 2009 119:661- 673; deFougerolles Hum Gene Ther. 2008 19: 125-132; U.S. Patent Publication No US20130122104). The original manufacture method by Wheeler et al. was a detergent dialysis method, which was later improved by Jeffs et al. and is referred to as the spontaneous vesicle formation method. The liposome formulations are composed of 3 to 4 lipid components in addition to the polynucleotide. As an example a liposome can contain, but is not limited to, 55% cholesterol, 20% disteroylphosphatidyl choline (DSPC), 10% PEG-S-DSG, and 15% 1,2- dioleyloxy-N,N-dimethylaminopropane (DODMA), as described by Jeffs et al. As another example, certain liposome formulations contain, but are not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cationic lipid, where the cationic lipid can be 1,2- distearloxy-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or 1,2- dilinolenyloxy-3 -dimethylaminopropane (DLenDMA), as described by Heyes et al.

In some embodiments, the polynucleotide of the present invention is formulated in a lipid nanoparticle such as those described in International Publication No. W02012170930. Lipid nanoparticle formulations typically comprise a lipid, in particular, an ionizable cationic lipid, and further comprise a neutral lipid, a sterol and a molecule capable of reducing particle aggregation, for example a PEG or PEG-modified lipid. The lipid can be selected from, but is not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2- DMA, DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids and amino alcohol lipids. In some embodiments, the lipid is a cationic lipid such as, but not limited to, DLin-DMA, DLin-D- DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA and amino alcohol lipids. The amino alcohol cationic lipid can be the lipids described in and/or made by the methods described in US Patent Publication No. US20130150625. As a non-limiting example, the cationic lipid can be 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-dien-l- yloxy]methyl}propan-l-ol (Compound 1 in US20130150625); 2-amino-3-[(9Z)-octadec-9-en- l-yloxy]-2-{[(9Z)-octadec-9-en-l-yloxy]methyl}propan-l-ol (Compound 2 in US20130150625); 2-amino-3 -[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]-2-

[(octyloxy)methyl]propan-l-ol (Compound 3 in US20130150625); and 2-(dimethylamino)-3- [(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy ] -2- { [(9Z, 12Z)-octadeca-9, 12 -di en- 1 - yloxy]methyl}propan-l-ol (Compound 4 in US20130150625); or any pharmaceutically acceptable salt or stereoisomer thereof. Nanoparticle formulations of the present disclosure can be coated with a surfactant or polymer in order to improve the delivery of the particle. In some embodiments, the nanoparticle is coated with a hydrophilic coating such as, but not limited to, PEG coatings and/or coatings that have a neutral surface charge. The hydrophilic coatings can help to deliver nanoparticles with larger payloads such as, but not limited to, polynucleotides within the central nervous system. As a non-limiting example nanoparticles comprising a hydrophilic coating and methods of making such nanoparticles are described in US Patent Publication No. US20130183244.

As used herein, the “percent identity” between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions/total number of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below. The percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology. 48 (3): 443- -53). The percent identity between two nucleotide or amino acid sequences may also be determined using for example algorithms such as EMBOSS Needle (pair wise alignment; available at www.ebi.ac.uk). For example, EMBOSS Needle may be used with a BLOSUM62 matrix, a “gap open penalty” of 10, a “gap extend penalty” of 0.5, a false “end gap penalty”, an “end gap open penalty” of 10 and an “end gap extend penalty” of 0.5. In general, the “percent identity” is a function of the number of matching positions divided by the number of positions compared and multiplied by 100. For instance, if 6 out of 10 sequence positions are identical between the two compared sequences after alignment, then the identity is 60%. The % identity is typically determined over the whole length of the query sequence on which the analysis is performed. Two molecules having the same primary amino acid sequence or nucleic acid sequence are identical irrespective of any chemical and/or biological modification. According to the invention a first amino acid sequence having at least 90% of identity with a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Fig. 1. Distant homologs of antiphage systems can be detected in eukaryotes. (A-E) Representation of DefenseFinder hits for the antiphage systems viperin (A), CBASS (B), Mokosh (C), Eleos (D) and Lamassu (E) in their genomic context. Prokaryotic and eukaryotic proteins are colored in blue and green, respectively, and labeled with their NCBI identification number. Homology between eukaryotes and prokaryotes are highlighted by colored shades, mapping on a given HMM protein profile. Bottom plots quantify the average coverage of the HMM profile for all eukaryotic (green) and prokaryotic (blue) hits. Total number of unique hits are indicated. (F) Bioinformatics pipeline used in this study.

Fig. 2. The protective piRNA pathway of the animal germline involves domains shared with the antiphage system Mokosh.

(A-B) Phylogenetic analysis combining prokaryotic MkoA (A) and MkoB (B) with their eukaryotic hits (see Material and Methods). Branches are colored according to the kingdom (blue for prokaryotes, green for eukaryotes). Human hits of interest are highlighted in red. (C) Conservation of the RNA helicase and PLD-like nuclease domains of prokaryotic antiphage MkoA and MkoB (blue) in the human homologs MOV10L1 and PLD6 (green). (D) Structural comparison of a Mokosh from E. coli K12, in which MkoA and MkoB are fused (yjhR protein, depicted in blue), with the helicase domain of human MOV10L1 (top) or the PLD domain of human PLD6 (bottom). Optimal local alignment between the two structures (as determined by foldseek) is represented in yellow. (E) Structural comparisons of human MOV10L1 (top) and PLD6 (bottom) with the yjhR protein of A. coli. For MOV10L1 (respectively PLD6) homologs, the yellow domain corresponds to the protein’s optimal alignment to the MkoA (respectively MkoB) domain of yjhR. Structures were predicted using AlphaFold.

Fig. 3. GIMAPs, proteins related to the antiphage Eleos system, are antiviral.

(A) Phylogenetic analysis combining prokaryotic LeoBC with their eukaryotic hits. Branches are colored according to the kingdom (blue for prokaryotes, green for eukaryotes). Positions of human GIMAPs are indicated in red. (B) Conservation of the P-loop NTPase domain of prokaryotic antiphage LeoBC (blue) in human GIMAP5 and 6 (green). (C) Structural comparison of prokaryotic LeoBC with human GIMAP6. TM-score = 0.4. Structures were predicted using AlphaFold. (D,E) 293T cells were transfected with plasmids encoding for GIMAP5 or GIMAP6 and infected with HSV-1 (D) or SINV (E) coding for GFP. Expression of GIMAPs and GFP was quantified by flow cytometry at 48h (HSV-1) or 24h (SINV) postinfection. GIMAP-, no expression; GIMAP+, mild expression; GIMAP++, high expression as described in fig. S6. Two-sided independent t-test comparing GIMAP- and GIMAP++, (D) p = 0.008 (GIMAP5) and p = 0.001 (GIMAP6), (E) p = 0.00005 (GIMAP5) and p = 0.0001 (GIMAP6).

Fig. 4. CTRC, a structural human homolog of the antiphage protein LmuA, is antiviral.

(A) Conservation of the trypsin and SMC N domains of prokaryotic antiphage LmuA and B (blue) in human CTRC and FHAD1 (green). (B) Phylogenetic analysis combining prokaryotic LmuA with its eukaryotic hits (see Material and Methods). Branches are colored according to the kingdom (blue for prokaryotes, green for eukaryotes). Human CTRC is indicated in red. Fusion proteins hit by both eukaryotic profiles are indicated by yellow dots. Fusions found in the Cyprinidae family are flagged with a green star. (C) Structural comparison of LmuA from a Lamassu of E. coli with human CTRC, TM-score = 0.69. Structures were computed using AlphaFold (not shown). (D,E) 293T cells were transfected with plasmids encoding for EFHD2 or CTRC and infected with HSV-1. Expression of CTRC and EFHD2 (D) and virus-encoded GFP (E) was quantified by flow cytometry at 48h post infection. Two-sided independent t-test comparing EFHD2-CTRC- to EFHD2+CTRC+, (E) p = 0.0007. (F) FHAD1 or CTRC levels are knock-down by shRNA transduction in blood-derived human myeloid cells (n=3 donors), then infected with HSV-1. Infection is monitored through GFP measurement by live videomicroscopy (see Methods). Bottom lane corresponds to cells treated with soluble interferon alpha/beta receptor B18 (B18R) that blocks the type-I interferon response by quenching interferons in the media.

EXAMPLE:

Material & Methods

Database of eukaryotic and prokaryotic genomes

Eukaryotic genomes were downloaded from genbank in January 2022. Isoforms were removed from the protein fasta files, when possible, by keeping the longest one. Pseudogenes were removed as well, when annotated as such. CDS were renamed to take into account the relative position to easily assess whether two proteins are co-localized in the genome. In total, 4,616 eukaryotic genomes representing 2407 species were screened. 22,920 prokaryotic complete genomes were downloaded from Refseq in July 2022. List of genomes are available at https://github.com/mdmparis/cury_mordret_et_al_supplementary_data. Identification of homologous antiphage systems in eukaryotic genomes

DefenseFinder vl.0.9 with models vl.2.2 was run with default parameters on the custom database of 4,616 eukaryotic genomes, generating 14,701 candidate systems composed of a total of 17,932 associated genes. The output hits of DefenseFinder was examined to remove potential bacterial genomic contaminations. Each candidate gene was searched against the UniProt90 (https://www.uniprot.org) database using Diamond (v2.0.9 -b 10 -c 1 — max-target- seqs 5 — outfmt 6) (31), retrieving the 5 best hits per query. We used the uniprot id-mapping API to fetch the common ncbi taxon id of each of the UniProt90 families. This step fetched a taxon id for 78% of the hits. For the remaining 22% of the hits whose protein’s id did not match any UniProt90 family, we fetched their taxonomy by mapping the id to UniProtKB (12%) or to UniParc (10%). Each candidate protein was deemed a contamination if at least one of the 5 best hits on the UniProt90/UniProtKB/UniParc database came from a prokaryote, and its associated systems were subsequently removed from the pipeline. This approach led us to remove a total of 746 (4.2%) genes and 484 systems (3.3%) from our list, leaving us with 14,217 eukaryotic system candidates composed of 17,186 genes.

Homology search of Mokosh, Eleos, Lamassu

We looked for homologs in eukaryotic genomes starting for the following primary HMM profiles: LmuA effector Protease, LmuB_SMC_Hydrolase_protease, MkoA, MkoB, LeoA, LeoBC (available at https://github.com/mdmparis/defense-fmder-models). For each profile, the pipeline for homology search was similar. First, we searched the primary profile against our custom database of eukaryotic and prokaryotic proteins using hmmsearch (version 3.3.2), at an e-value threshold of le-3 (32). Prokaryotic hits were then marked as "paired" if they were found in proximity to their defense system partner(s), according to the rules of DefenseFinder, and as "isolated" otherwise. We selected a subset of these sequences, containing approximately 200 paired and 300 isolated prokaryotic hits, to perform downstream phylogenetic analyses. For both the paired and isolated groups, we picked half of the proteins among the top ranking hits to the reference hmm profile, and the other half by sampling uniformly along the list (not shown).

The subset of sequences was then filtered to remove duplicated proteins (using usearch vl 1.0.667 with the option -cluster fast and -id 1) and aligned with mafft (version 7.505 with the option —auto) (33, 34). The resulting alignment was trimmed with clipkit (version 1.3.0) using the kpic-gappy mode to keep informative and constant sites and removes sites with more than 90% of gaps (35). Phylogenetic trees were computed using IQTree (version 2.2.0.3) using model VT+F+I+G4 and 1000 ultrafast bootstraps (36), and visualized on the iTOL webserver (https://itol.embl.de). The trees were further annotated to display whether hits were paired or isolated, and whether the sequences were eukaryotic or prokaryotic. Clades were manually curated to include eukaryotic proteins distant from prokaryotic proteins with non-immune function (not shown) . Their eukaryotic members were aligned using rnafft. We performed a manual curation of the alignments by removing sequences that did not align well with the rest, recomputed the alignments, and manually truncated the N and C termini. These alignments were used to build secondary hmm profiles using hmmbuild (from hmmer version 3.3.2). All alignments and profiles are available at https://github.com/mdmparis/cury_mordret_et_al_supplementary_data.

Each of these secondary profiles was then used to search against the eukaryotic protein database using hmmsearch. We visualized the distribution of scores, and manually determined a score threshold to keep or discard these secondary hits (not shown). For each secondary profile, we composed a new subset of representative genes, composed of 300 isolated secondary hits, 100 paired eukaryotic secondary hits, all fusions (i.e. genes matched by the secondary hmm profiles of two different partners of the same system), all secondary hits in the human genome, and all the paired prokaryotic hits found in the previous search of the associated primary profile. We removed duplicates and performed the alignment in two steps: first, we aligned the prokaryotic sequences of the subset together using the procedure described above. The final alignment of bacterial proteins was performed using the option-maxiterate 1000 and —localpair (i.e. rnafft- linsi). We then aligned the eukaryotic proteins to the bacterial alignment with maff-linsi parameters —add and —keeplength (37). We trimmed the alignment with clipkit, and generated trees as described above.

Structural comparisons

Structures were either downloaded from the alphafold.ebi.ac.uk database, or computed using the ColabFold notebook (not shown) (38-40). We created custom databases of structures and searched them using the easy-search command of foldseek version 3.915ef7d (41) (https://github.com/mdmparis/cury_mordret_et_al_supplementary_data.) with default parameters. Structures were imported, realigned based on the Foldseek alignment, and visualized in the PyMol version 2.5.4 (42) (Schrodinger, LLC). Cell culture

Human embryonic kidney 293T cells (293T cells) and Vero cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies) including 10% fetal calf serum and lOOU/ml penicillin/streptomycin (Gibco). Prior to transfection, 293T cells were plated in medium without antibiotics.

Viral stocks and plaque assay

Herpes Simplex virus 1 (HSV-1, KOS strain) was built by fusing VP26 fused to GFP (43). Sindbis virus was generated from the pTE32J infectious clone, as described elsewhere (44). Briefly, viral RNA was generated from in vitro transcription of linearized infectious clones, purified and electroporated in Vero cells. Viral stocks were amplified on 293T cells (HSV) or Vero cells (SINV) and titrated by plaque assay. Briefly, 200,000 Vero cells were seeded in 24- well plates and infected with 10-fold dilutions of sample for Ih at 37°C. Cells were then overlaid with DMEM containing 2% FCS, 100 U/ml penicillin-streptomycin and 0.8% agarose. After 3 days (SINV) or 4 days (HSV-1), cells were fixed with 10% formalin and visualized by crystal violet staining.

Plasmids, transfection and viral infections

Human proteins of interest were synthetized through Genscript and integrated in a pcDNA3.1(+) backbone. GIMAP5 and 6 were tagged with a flag tag and a HA tag at the N- terminus, respectively, whilst EFHD2 was equipped with a flag tag (N-terminus), CTRC with a HA tag (C-terminus) and FHAD1 with a V5 tag (C-terminus). 100 ng of each plasmid was transfected in 293T cells using Lipofectamine 2000 (Thermo Fisher Scientific) following the manufacturer’s instruction. One day after plasmid transfection, cells were infected with HSV- GFP or SINV-GFP at multiplicity of infection 0.5. Viral supernatant was collected at the indicated time points for viral particle titration, or cells were collected at 48h post-infection for flow cytometry analysis.

Flow cytometry

Cells were collected in a V-shape bottom 96-well plate (Corning), and resuspended in 50 ul PBS supplemented with DAPI. FIX & PERM kit (Thermo Fisher Scientific, GAS004) was used according to the manufacturer’s instructions. Proteins of interest were visualized using an anti- Flag-PE (BioLegend, 637310), an anti-HA-Alexa647 (BioLegend, 682404) and an anti-Flag- PE-Cy7 (Thermo Fisher Scientific, 25-6796-42) at a 1 :300 dilution. Analysis were performed on a FACSVerse Cell Analyser (BD Biosciences) and results were analyzed with FlowJo (Tree Star).

Data visualization

Trees were drawn with ITOL (https://itol.embl.de). Schematics of Eukaryotic organisms were taken from https://beta.phylopic.org/. Graphs were plotted using matplotlib (https://matplotlib.org) and seaborn (http://seaborn.pydata.org/index.html). Web logos of alignment were built using webserver of weblogo (45).

Results

Eukaryotic genomes encode distant homologs of antiphage systems

We hypothesized that components of some of the recently-discovered antiphage systems may be conserved in eukaryotes, and thus could be identified through protein homology search. We used DefenseFinder, a pipeline developed to map known antiphage systems in prokaryotic genomes (2). This pipeline currently detects 132 types of antiphage systems, and relies on protein sequence homology to detect a system’s individual components. Each component is searched using a protein profile allowing for low homology detection (hidden Markov model, HMM). Because most antiphage systems are organized in operons, DefenseFinder also takes into account genomic proximity on the chromosome (colocalization) of genes from a given system. Our analysis thus relies on two complementary criteria: 1) for single-gene systems, high sequence homology with a protein profile; 2) for multi-gene systems, a more relaxed sequence homology of each component coupled with the constraint of genes’ colocalization. These two criteria are expected to limit the potential detection of antiphage homologs in eukaryotes as 1) protein homology may be limited and 2) coregulation of eukaryotic gene expression does not canonically rely on genomic colocalization. Nonetheless, genes of eukaryotic homologs of antiphage systems can be genomically colocalized, as recently observed for viperin and its kinase partner in metazoans, including humans (9, 13).

DefenseFinder analysis of a database of 4,616 genomes spanning the kingdom of eukaryotes detected 14,217 hits, which were filtered to remove bacterial genomic hits. This approach was successful in detecting hits of eukaryotic immune proteins with known antiphage homologs, such as viperin (Fig. 1A, 1,164 homologs). Viperin’s antiphage activity relies on the generation of modified nucleotides that act as chain terminators of viral replication. In mammals, viperin is an ISG that stops infection by a similar mechanism (9, 14). We also identified 34 instances of homology with CBASS, which is the prokaryotic homolog of the cGAS/STING pathway, a key antiviral immune pathway in animals (Fig. IB). cGAS detects viral DNA and signals through STING to induce an antiviral response (7, 75). In animals, STING is considered to be the canonical partner of cGAS. There is more diversity in bacteria, in which cGAS proteins can be associated with other effectors such as phospholipases (S). Our search documents gene colocalization between cGAS and phospholipases in arthropods, suggesting that other versions of the CBASS antiphage system may be present in eukaryotes.

Because we detected known eukaryotic homologs of antiphage pathways, we then assessed if a similar approach could be leveraged to unravel additional conserved systems. We identified numerous homologs of the two-gene systems Mokosh (MkoA, MkoB), Eleos (LeoA, LeoBC) and Lamassu (LmuA, LmuB). 178 homologs of the Mokosh system were detected in 165 eukaryotic genomes (Fig. 1C). The antiphage system termed Eleos in this work, after the Greek goddess of compassion, corresponds to the combination of LeoA and LeoBC, which was previously described as an antiphage system with dynamin-like domains (3, 76). 415 homologs of Eleos were identified in 317 eukaryotic genomes (Fig. ID). Finally, 624 homologs of Lamassu were detected, notably in rotifers, arthropods and chordates (88% of the hits, Fig. IE) (3, 6). These data suggest that unknown homologs of bacterial immune systems can be detected in eukaryotic genomes by using protein homology searches that are calibrated on bacterial proteins.

This initial analysis identified distant homologs of antiphage systems in eukaryotes. It however relies on a criterion of genomic colocalization, which prevents the detection of homologous multi-gene systems in which all components exist in a eukaryotic genome, but would not be genomically colocalized. Among the full systems detected by DefenseFinder in eukaryotic genomes, we focused on Mokosh, Eleos and Lamassu, and scanned our database of genomes for their individual components (Fig. IF). The number of eukaryotic hits obtained with this analysis varied drastically between profiles, ranging from 1,080 for MkoB to 1,095,192 for MkoA (not shown). Since protein domains can typically be shared between immune and non- immune proteins (77), we expect the resulting hits to be a mix of immune and non-immune proteins. We hypothesized that eukaryotic proteins with immune roles would be phylogenetically closer to prokaryotic proteins with immune functions - defined, here, as DefenseFinder hits in prokaryotic genomes. We devised a procedure (see Material and Methods) to create a set of HMM profiles from groups of eukaryotic proteins, based on their phylogenetic proximity to prokaryotic immune proteins (not shown). We used these new HMM profiles to look for eukaryotic proteins potentially involved in immunity. These new profiles detected various numbers of hit, from 1,270 (MkoB) to 68,655 (LmuA) proteins in the eukaryotic genomes. With respect to our initial analysis relying on bacterial HMM profiles, our query using refined eukaryotic profiles is expected to retrieve a more specific set of hits, as it putatively encompasses the phylogenetic relationship to prokaryotic immune proteins. It is also expected to be more sensitive, as the profile should capture eukaryotic sequence diversity. As such, only 17.4% of hits are common between the two methods (not shown). Hits uncovered using eukaryotic profiles are described in the following sections (not shown), with a focus on humans (not shown).

Domains with structural homology to the antiphage Mokosh system participate in the anti-transposon piRNA pathway of the animal germline

As a proof-of-concept, we first determined if our comparative immunology approach allows to identify known human defense genes. Mokosh is an antiphage system composed of proteins with at least two domains: an RNA helicase (encoded by MkoA) and a phospholipase D domain (PLD, present in MkoB) (Fig. 1C and 2C) (3). Our analysis revealed a single human hit of MkoB: the mitochondrial cardiolipin hydrolase PLD6 (only identified using the eukaryotic HMM profile, not shown). This protein is involved in the piRNA pathway, a key defense mechanism of the animal germline (Fig. 2A,C) (7S). piRNAs prevent the expression of transposable elements, which are genomic sequences originating, in part, from ancient events of retrovirus integration (not shown). PLD6 is a nuclease responsible for the degradation of piRNA precursors, that are ultimately converted into mature piRNAs to target transposable elements (not shown). It does so by interacting with the RNA helicase MOV10L1, which unwinds piRNA precursors 18-20). Our analysis shows that these two proteins, MOV10L1 and PLD6, share domains with the prokaryotic proteins MkoA and MkoB, respectively (Fig. 2A-C). To explore prokaryote-eukaryote structural conservation, we aligned the conserved domains of human PLD6 and MOV10L1 with a predicted Mokosh protein from E. coli. in which MkoA and MkoB are fused in a single protein (Fig. 2D). The PLD domain of PLD6 shows homology to E. coll Mokosh (template modeling score, TM-score of 0.63), as does the RNA helicase domain of MOV10L1 (TM-score of 0.74) (Fig. 2D,E). We expanded structural comparisons to other animals, in which the piRNA pathway relies on PLD6 and MOV10L1, and similarly showed that domains of both proteins have clear structural homologies with prokaryotic MkoB and MkoA (not shown). While the RNA helicase and PLD domains are conserved in MOV10L1 and PLD6, respectively, the proteins also include additional domains that are not present in bacterial Mokosh systems (not shown). This data suggests that the antiphage system Mokosh may have been the origin of these key proteins of the piRNA pathway, highlighting another case of a possible bacterial origin of a eukaryotic protective mechanism.

Note that our search uncovered 15 additional hits of MkoA, 27% of which have been described to be involved in antiviral immunity (not shown). The ISG helicase with zinc finger domain 2 (HELZ2) is antiviral against dengue virus by regulating lipid metabolism through its interaction with the aryl hydrocarbon receptor (27). RNA helicase zinc finger NFXl-type containing 1 (ZNFX1) is a dsRNA sensor that triggers a type-I interferon response upon RNA virus infection (22). Altogether, this data provides a proof-of-principle that comparative immunology across domains of life is a fruitful method to identify defense genes in eukaryotes.

GIMAPs, human proteins related to antiphage Eleos, are antiviral.

We then asked if our approach could be leveraged to identify novel antiviral factors. Eleos is a two gene-system (LeoA and LeoBC) coding for proteins with dynamin-like domains, from the P-loop NTPase superfamily (3). The search for human homologs of LeoBC identified two protein families: EH domain-containing proteins (EHDs) and GTPases immunity-associated proteins (GIMAPs) (Fig. 3A,B). We focused on GIMAPS, a group of 8 human paralogs, as they are transcriptionally induced by interferon, and have a documented role in T lymphocyte survival as well as in resistance to infection by the parasite Toxoplasma gondii (23-25). We hypothesized that GIMAPs could also be involved in antiviral immunity. Structural comparison between a LeoBC of Bacillus sp. HY001, and GIMAP6 revealed moderate structural similarities of the P-loop NTPase domain (predicted structures, TM-score of 0.45, Fig. 3C). Expression of human GIMAP5 and 6 was induced in human embryonic kidney 293T cells by plasmid transfection, and cells were infected with GFP-encoding herpes simplex virus 1 (HSV- 1), a DNA virus. GIMAP expression, as well as HSV-1 infection, were monitored at the single cell level using intracellular immunostaining and flow cytometry (not shown). Cells expressing GIMAP5 and 6 display 1.4- and 2-fold reduction in infection by herpes simplex virus 1, respectively (Fig. 3D). Viral replication measured by the intensity of virus-encoded GFP, was reduced by up to 40% (not shown). To interrogate if GIMAP antiviral activity is herpes-specific, GIMAP-expressing cells were infected with Sindbis virus (SINV), an RNA virus. Viral infection and replication, measured by the expression of SINV-encoded GFP, were reduced by up to 2 folds upon expression of GIMAP5 and 6 (Fig. 3E). We concluded that human proteins with domains similar to prokaryotic Eleos are ISGs that exert an antiviral activity against DNA and RNA viruses.

Distant human homologs of antiphage Lamassu are antiviral

Finally, we explored if novel human genes with no known links to immunity could be identified using comparative immunology across domains of life. One unexpected aspect of our analysis is the detection, in eukaryotic genomes, of gene colocalization for hits of the different components of a same system. Lamassu is composed of LmuB, which contains a SMC domain predicted to bind DNA, and LmuA, a predicted effector that can be a trypsin protease (Fig. 4A) (3). 35% of chordates genomes encode LmuA and LmuB colocalized (not shown). A protein fusion of trypsin domain (LmuA) and coil-coiled domain (LmuB) can be detected in the Cyprinidae family of fish (Fig. 4B). The existence of fusion proteins in fish, as well as the conservation of genomic colocalization, suggest that the two proteins participate in the same pathway also in eukaryotes. In humans, out of 109 hits to LmuA and 28 proteins to LmuB, one genomic locus encodes both LmuA and LmuB colocalized.

Chymotrypsin-C (CTRC) protease is detected as a hit in our LmuA search, and the human CTRC gene is located next to Forkhead-associated phosphopeptide binding domain 1 (FHAD1), whose protein product is a hit in our LmuB analysis (Fig. 4A-C). FHAD1 and CTRC genes are separated by EF-hand domain-containing protein D2 (EFHD2, Fig. 4A). This protein performs pleiotropic functions, including regulation of immune activation in myeloid cells (26). CTRC is a protease secreted by pancreatic exocrine cells (27). It is additionally expressed, as is EFHD2, by macrophages and dendritic cells, responsible for immune surveillance and priming of the adaptive immune response (fig. S9). Intracellular levels of CTRC and EFHD2 were analyzed by immunostaining and flow cytometry in 293T cells transfected concomitantly with two plasmids encoding both proteins. As the transfection efficiency does not reach 100%, each cell will receive either no plasmid, only EFHD2 or CTRC plasmid, or both plasmids. Because CTRC is canonically secreted, one should not expect to detect intracellular accumulation of the protein in CTRC -transfected cells. Indeed, cells containing only CTRC protein represent 1% of total transfected cells (Fig. 4D). However, cells expressing CTRC can be detected when co-expressing EFHD2 (18% of total transfected cells, Fig. 4D). This suggests that EFHD2 affects the secretion of CTRC and triggers its intracellular retention. 293T cells expressing CTRC+EFHD2, but not EFHD2 alone, display a two-fold reduction in infection with HSV-1 (Fig. 4E). No clear antiviral effect was detected against SINV (fig. S10C,D). Addition of FHAD1 did not improve the antiviral capacity of EFHD2-CTRC in this experimental system, which could be related to the low expression of the protein compared to CTRC and EFHD2 (not shown). Thus, the human protein CTRC, with distant homology to antiphage Lamassu, is a restriction factor that displays antiviral activity against a herpesvirus. To interrogate the role of endogenous FHAD1 and CTRC in thwarting HSV-1 infection, we used myeloid cells derived from human blood, in which gene expression is knock-down by shRNA transduction and infection with HSV-1 -GFP is monitored by live video-microscopy (Fig. 4f and Data not shown). Downregulating endogenous CTRC levels in human myeloid cells translates into a 3.5-26-fold increase in HSV-1 infection compared to control cells (Fig. 4f). Decreasing levels of FHADl similarly increases viral infection by 1.6-6.5 folds (Fig. 4f). Addition of B18R (an interferon inhibitor) in the culture media does not prevent increased infection of FHADl and CTRC knock-down cells (Fig. 4f). This demonstrates that the endogenous proteins FHADl and CTRC are antiviral and suggests that they act independently of the type-I interferon response. Altogether, these results document the conservation of microsynteny of likely homologs of Lamassu across eukaryotes and demonstrate that human proteins likely homolog of the antiphage Lamassu system protect against HSV-1.

Discussion:

In this work, we unravel the conservation of recently discovered bacterial antiphage systems in eukaryotes, and utilize it to discover novel immune proteins in humans. These data - together with previously documented conservation events between prokaryotes and eukaryotes - show that proteins related to defense mechanisms in bacteria are involved at multiple levels of eukaryotic immune pathways, including pathogen detection (cGAS/STING), signal transduction (TIR domains), activity against transposons (PLD6/MOV10L1) and antiviral effectors (GIMAPs, EFHD2-CTRC) (8, 12, 18, 28). Bacterial antiphage systems could thus have served as providers of building blocks for the evolution of immunity in eukaryotes. Importantly, the fact that certain hits in our search display antiviral activity does not imply that all hits in our search will have a role in immunity. Instead, a significant proportion may have been co-opted for unrelated cellular functions, or may present a dual role within and outside of immunity. Evolutionary and mechanistic studies will help delineate each protein’s contribution to eukaryotic immune pathways. We did not perform an exhaustive analysis of antiphage homologs, but rather chose to focus on specific systems. It is thus likely that additional homologs of antiphage systems will be identified.

Our work shows that three antiphage systems share domains with eukaryotic proteins participating in immunity. MOV10L1 and PLD6, reminiscent of Mokosh, play a key role in the piRNA pathway which protects the animal germline against transposable elements. Human proteins displaying domain conservation with Eleos (GIMAP5-6) and Lamassu (CTRC- EFHD2) can shield cells from HSV-1 infection. Interestingly, GIMAPs and CTRC-EFHD2 can block herpesviruses, which is part of the Duplodnaviridae, the realm of virus that includes bacteriophages (29). Connexions between phages and herpesviruses were indeed documented by a recent work describing the antiphage system Avs, that can be triggered in vitro by human herpesvirus-8 proteins (77).

Our data also documents a diversification of the proteins’ protective roles, which can be rewired to address other eukaryotic threats. The Mokosh system could have been co-opted to block transposable elements integrated in the genome. Similarly, GIMAP5 and 6 can also target RNA viruses, a broad antiviral tropism also documented for other members of the dynamin superfamily such as the ISGs MxA and MxB 30). There is thus a certain level of modularity in the evolutionary co-optation of antiphage systems, which may participate in various branches of eukaryotic immunity, including responses against bacteria or fungi. In addition to unraveling striking prokaryote-eukaryote conservation, our work proposes a comparative immunology approach to discover mechanisms of defense across domains of life, including phyla like plants or protists. We anticipate that the refinement of our method, as well as the expansion of knowledge on antiphage systems, will provide a more comprehensive understanding of prokaryotes’ contribution to eukaryotic immunity and lead to the identification of novel immune pathways across eukaryotes.

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Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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Claims

CLAIMS: A method of treating a viral infection in a subject in need thereof comprising administering to the subject a therapeutically effective amount of i) at least one polypeptide related to the Eleos or Lamassu prokaryotic systems or ii) at least one polynucleotide encoding for said polypeptide. The method of claim 1 wherein the viral infection comprises infection by a RNA virus or a DNA virus. The method of claim 2 wherein the subject is infected by one or more viruses selected from the group consisting of Arenaviridae, Astroviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Closter oviridae, Comoviridae, Cystoviridae, Flaviviridae, Flexiviridae, Hepevirus, Leviviridae, Luteoviridae, Mononegavirales, Mosaic Viruses, Nidovirales, Nodaviridae, Orthomyxoviridae, Picobirnavirus, Picornaviridae, Potyviridae, Reoviridae, Retroviridae, Sequiviridae, Tenuivirus, Togaviridae, Tombusviridae, Totiviridae, Tymoviridae, Hepadnaviridae, Herpesviridae,

Paramyxoviridae or Papillomaviridae viruses. The method of claim 3 wherein the subject is infected by a herpesvirus. The method according to any one of claims 1 to 4 wherein the subject is a human or a non-human mammal. The method according to any one of claims 1 to 5 wherein the polypeptide comprises at least one domain similar to prokaryotic Eleos. The method of claim 6 wherein the polypeptide is a GTPases immunity-associated protein (GIMAP). The method of claim 7 wherein the polypeptide comprises an amino acid sequence having at least 90% of identify with the amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO:2. The method according to any one of claims 1 to 5 wherein the subject is administered with a therapeutically amount of at least 2 polypeptides related to the Lamassu prokaryotic system.

10. The method of claim 9 wherein the subject is administered with an amount of i) a first polypeptide having an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:3 and ii) a second polypeptide having an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:4.

11. The method according to any one of claims 1 to 5 wherein the polynucleotide encodes for a polypeptide that comprises at least one domain similar to prokaryotic Eleos.

12. The method of claim 11 wherein the polynucleotide encodes for a GTPases immunity- associated protein (GIMAP).

13. The method of claim 12 wherein the polynucleotide encodes for a polypeptide comprising an amino acid sequence having at least 90% of identify with the amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO:2.

14. The method according to any one of claims 1 to 5 wherein the subject is administered with an amount of i) a first polynucleotide that encodes for a polypeptide having an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO: 3 and ii) a second polynucleotide that encodes for polypeptide having an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:4.

15. The method according to any one of claims 1 to 5 wherein the subject is administered with an amount of a bicistronic polynucleotide that encodes both for i) a first polypeptide having an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:3 and ii) a second polypeptide having an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:4.

16. The method according to any one of claims 11 to 15 wherein the polynucleotide is a messenger RNA (mRNA).