Patent Application
20110189155
The invention relates to the large form of human 2′,5′-OligoAdenylate Synthetase (OAS3) as a medicament for preventing infection with or treating positive-sense single-stranded RNA viruses.
The invention relates also the large form of human 2′,5′-OligoAdenylate Synthetase (OAS3) as a marker for determining the genetic susceptibility to infection with positive-sense single-stranded RNA viruses.
Flaviviridae and Togaviridae are two positive-sense single-stranded (ss)RNA virus families comprising pathogens that can affect human and animal health world wide. Examples of these viruses include members of dengue, yellow fever (YF), Japanese encephalitis (JE) and tick-borne encephalitis (TBE) antigenic complexes of the Flavivirus genus (Flaviviridae family), members of Eastern Equine Encephalitis (EEE)/Venezuelan Equine Encephalitis (VEE), Semliki Forest (SF), and Sindbis (SIN) groups of Alphavirus genus (Toagaviridae family), and members of Hepacivirus genus (Flaviviridae family) such as Hepatitis C(HCV) and Hepatitis G (HGV) viruses.
Dengue virus, (DV; DEN antigenic complex of flavivirus genus) is endemic in most urban centers of the tropics since a dramatic increase in urbanization created ideal conditions for increased transmission of mosquito-borne dengue disease. The four serotypes of dengue virus (DV-1 to DV-4) are transmitted to humans by the mosquito vector Ae. aegypti. DV infection results in a spectrum of illnesses, ranging form a flu-like disease (dengue fever, DF) to dengue hemorrhagic fever (DHF) that can progress to dengue shock syndrome (DSS) and death. To date, dengue illness is the most important arbovirosis in humans with an estimated 100 millions cases and over 500,000 cases of DHF/DSS occurring each year, including about 25,000 fatal cases, mainly in children under the age 15. Epidemics with a high frequency of DHF/DSS continue to expand geographically in Asia and South America. Despite increased health and economic impacts, the pathogenesis of dengue disease is currently poorly understood. There is no available anti-dengue vaccine or specific therapy for the treatment of dengue virus infection.
West Nile virus (WNV; JE antigenic complex of flavivirus genus) circulates in natural transmission cycles involving mosquitoes (Culex species) and birds, and horses and human are incidental hosts. Zoonotic WNV became a major health concern in North America, the Middle East, and Europe, due to the emergence of a highly neuroinvasive strain in Israel in 1998 (variant Isr98/NY99 from clade Ia of the WN virus lineage I) (Ceccaldi et al., FEMS Microbiol. Lett., 2004, 233, 1-6). WNV infects the central nervous system and causes viral encephalitis in a large range of animal species. In Humans, clinical infections can range in severity from uncomplicated West Nile fever to fatal meningo-encephalitis. The emergence of WNV has been associated with a dramatic increase in the severity of infection in humans drawing the attention to viral encephalitis as a public health concern. Within the last 10 years, WNV has spread across the Western Hemisphere and the Caribbeans. The US outbreaks of WNV have involved thousands of patients causing severe neurological diseases (meningoencephalitis and poliomyelitis-like syndrome) and hundreds of associated deaths. Although mosquito-borne transmission of WNV is the predominant mode, WNV infection transmitted by blood transfusion, organ donation and transplacental transmission to the foetus were also recognized. There is no available vaccine or anti-viral therapy for WNV-related disease.
Chikungunya virus (CHIKV; SF group of alphavirus) is widespread throughout Africa, Southeast Asia, India and Western Pacific, and numerous epidemics have been reported in these areas. Clinically, infection by CHIKV results in fever, rash and intense, invalidating and sometimes persistent arthralgia. In 2005-06, CHIK virus has spread to the south of the Indian Ocean, particularly on La Réunion Island (France), where the outbreak has involved hundreds of thousand of patients. More recently the virus emerged in the east of Italy, where more than 200 people were infected.
Hepatitis C virus (HCV) infection is common worldwide; it is estimated that about 3% of the world's population have HCV and there are about 4 million carriers in Europe alone. Between 20 and 30% of these individuals will develop hepatic cirrhosis and its long-term sequelae such as hepatocellular carcinoma. Treatment of HCV infection with pegylated interferon alpha (IFN-α) in combination with ribavirin may achieve a sustained response in patients infected with HCV viral genotypes 2 or 3 (80%) but the response rate is much lower in patients infected with HCV viral genotype 1 (42%).
Innate antiviral mechanisms mediated by Type-I interferons (IFN-α/β) are potentially the most important pathways of host cell defense limiting viral replication. Indeed, IFN-α/13 are able to trigger the activation of a specific signal transduction pathway leading to the induction of IFN-stimulated genes (ISGs) that are responsible for the establishment of an antiviral state. The ISGs believed to affect RNA virus replication in single cells are the RNA-specific Adenosine Desaminase (ADAR), the proteins of the myxovirus resistance (Mx) family, the double-stranded RNA-dependent protein kinase (PIER), and the 2′,5′-oligoadenylate synthetase (2′,5′-OAS or OAS) family associated to endoribonuclease RNase L.
The OAS/RNase L system is a RNA decay pathway known to play an important role in the established endogeneous antiviral pathway. Binding of enzymatically active OAS to activator double-stranded (ds) viral RNA results in the production of 2′- to 5′-linked oligoadenylates (2-5A). Latent monomeric RNase L is enzymatically activated through homodimerization induced by binding to 2-5A oligomers. Once activated RNase L degrades single-stranded RNA molecules including mRNA and viral RNA, suppressing viral replication (Silverman, J. Virol. 81: 12720, 2007).
Human OAS is a family of enzymes encoded by three closely linked genes on chromosome 12q24.2, with the following order: small (OAS1, p40/46), medium (OAS2, p69/71), and large (OAS3, p100) OAS isoforms (Hovnanian et al., Genomics, 1998, 52, 267-277; Rebouillat, D. and Hovanessian, A. G., Journal of Interferon and Cytokine Research, 1999, 19, 295-308; Rebouillat et al., Genomics, 2000, 70, 232-240; Justesen et al., Cellular and Molecular Life Sciences, 2000, 57, 1593-1612; Rebouillat; Hovanessian, A. G., Cytokine and Growth Factor Reviews, 2007, 18, 351-361). Each OAS gene consists of a conserved OAS unit composed of five translated exons (exons A-E). OAS1 has one unit, whereas OAS2 and OAS3 have two and three units, respectively, and all three genes encode active 2′,5′-Oligoadenylate Synthetase. Another gene, OASL (OAS-Like) encodes a single-unit of OAS-like protein, which however, lacks 2′-5′ synthetase activity (Hartmann et al., Nucleic Acids Res., 1998, 26, 4121-4128; Rebouillat et al., Eur. J. Biochem., 1998; 257, 319-330). Within each size class, multiple members arise as a result of alternate splicing of the primary transcript. The OAS proteins share a conserved unit/domain of about 350 amino acids (OAS unit); OAS1 (p40/p46), OAS2 (p69/71) and OAS3 (p 100) contains one, two and three tandem copies of the OAS unit, respectively. Each OAS protein accumulates in different cellular locations, require different amounts of dsRNA to be activated, and catalyse the formation of differently sized 2-5A products. Whether OAS1 functions as a tetramer, and OAS2 is only active as a dimer, OAS3 has been observed only as a monomer. In addition, the large form of human OAS is presumably not involved in RNase L activation (for review, Rebouillat, D. and Hovanessian, A. G., Journal of Interferon and Cytokine Research, 1999, 19, 295-308).
The first direct evidence for the involvement of OAS family in the antiviral effect exhibited by IFN was provided by transfection of 2′,5′-oligoadenylate synthetase (OAS) cDNA into cells. Overexpression of OAS1 or OAS2 leads to resistance of cells to picornavirus replication (Hovanessian, A. G., Cytokine and Growth Factor Reviews, 2007, 18, 351-361). The importance of OAS1 for clearing WNV infection in vivo was also supported by the finding that murine Oas1b, the orthologous gene of human OAS1, may play a key role into the susceptibility/resistance phenotype of mice to WNV-induced encephalitis (Mashimo, T. et al., Proc. Natl. Acad. Sci. USA, 2002, 99, 11311-11316; Lucas et al., Immunol. Cell. Biol., 2003, 81, 230-236; Kajaste-Rudnitski et al., Journal of Biological Chemistry, 2006, 281, 46244637; International PCT Application WO 02/081741). Analysis of the OAS genetic polymorphism in human demonstrated that genetic markers in OAS genes were the most strongly associated with enzyme activity. Given that OAS1 is an excellent candidate for a human gene that influences host susceptibility to viral infection (Bonnevie-Nielsen et al., Am. J. Hum. Genet., 2005, 76, 623-633), genetic variations in human OAS1 as well as OASL genes were associated to the risk of viral encephalitis, type 1 DM, HCV related disease and other virus infection. With a particular emphasis on HCV disease, a series of OAS1 genotypes linked with the outcome of HCV infection has been reported (International PCT Application WO 03/089003 and WO 2005/040428).
Whether antiviral activity of OAS family is selective for positive-sense ssRNA viruses is a critical issue that it remains to be investigated. It has been reported ectopic expression of OAS1 leads to resistance to encephalomyocarditis virus (picornavirus) replication, but not VSV virus replication. However, OAS is considered to display no role in the elimination of HCV in patients treated with IFN-α, as opposed to other ISGs such as IFN-inducible RNA-dependent protein kinase (PKR) and myxovirus resistance 1 (M×A) genes. Transcription levels of OAS, PKR and Mx were up-regulated after DV infection in non-human primate model (Sariol et al., Clinical and Vaccine Immunology, 14, 2007, 756-766). The analysis of gene transcription pattern associated to Dengue Shock Syndrome showed that OAS3 gene transcripts were less abundant in dengue patients with DSS than in those with non-DSS (Simmons et al J. Infect. Dis., 195:1097, 2007). However, the role of these ISGs in the pathogenesis of DV infection remains poorly understood.
It has been reported that CHIKV is highly sensitive to the antiviral action of type I interferons (IFN-α/β), (Couderc et al., PloS Pathogens, 2007, 4, e29). In the case of alphaviruses, a body of evidence exists to suggest that IFN-mediated inhibition of virus growth does not require RNase L (Ryman et al., J. Virol., 2005, 79, 1487-1499; for review, Silverman, J. Virol., 2007, 81, 12720-9). Whether any members of human ISGs such as OAS family could exert antialphaviral activity is a critical issue that it remained to be investigated.
For the first time, the inventors demonstrate a role for OAS3 in the established endogenous antiviral pathway against positive-sense ssRNA viruses such as alphaviruses (CHIKV, SINV, and SFV). They show that OAS3 acts on the stages of CHIKV growth in blocking viral protein synthesis and viral RNA replication inside the infected human epithelial cells. Very little information is available on the human genetic susceptibility to alphavirus infection. Screening the OAS3 gene for polymorphism in healthly Caucasian individuals identified a single-nucleotide polymorphism (SNP) at the first position of codon CGA-844 where the substitution T for C resulted in a non-sense mutation (OAS3.R844X). The SNP at position OAS3.R844X is expected to result in a truncated form of OAS3 protein, lacking about 20% from the carboxy-terminus. Ectopic expression of mutant OAS3 resulted in a lower efficiency of CHIKV inhibition as compared to full-length OAS3 protein. The notion that genetic polymorphism of OAS3 could control its antialphaviral activity suggests a role of human OAS genes in the pathogenesis of alphavirus-related disease such as Chikungunya fever.
For the first time, the inventors demonstrate that OAS3 exerts antiflaviviral activity in human cells infected with DV (hepatoma cells) and WNV (hepatoma and epithelial cells). Inspection of the complete OAS3 gene in DSS patients and non-DSS dengue patients identified the SNP rs2285993 at the 3rd position of codon AGG-381 where the substitution G to C resulted to amino acid change from Arg to Ser. Our genetic data suggest that variant Ser-381 is associated with a dominant protection against the risk of DSS in dengue That patients. The inventors demonstrate here that OAS3 with Ser-381 is a potent inhibitor of DV growth in human hepatocytes.
The inventors also demonstrate that live-attenuated vaccine strain 17D-204 of YFV (STAMARIL, Sanofi-Pasteur) has inherent resistance to OAS3-mediated antiviral pathway in infected human epithelial and hepatoma cells. However IFN-α was able to establish an antiviral state against vaccine strain 17D-204 of YFV in human cells.
These findings are useful for the development of OAS3-based prophylaxis and therapy against positive-sense ssRNA viruses of major medical importance including CHIKV, WNV and DV. They are also useful for the development of new OAS3-based molecular tools for the prediction of human susceptibility to the infection with alphaviruses, flaviviruses and other positive-sense ssRNA viruses of major medical importance, and in particular for the prediction of severe forms of disease in dengue, West Nile and Chikungunya patients.
A subject of the invention is an isolated 2′,5′-oligoadenylate synthetase 3 protein or an isolated polynucleotide encoding said 2′-5′-oligoadenylate synthetase 3 protein, as a medicament.
The 2′,5′-oligoadenylate synthetase activity of the OAS3 protein of the invention may be assayed by chromatographic or electrophoretic methods to determine the end-point amounts of oligoadenylates formed (St Laurent et al., Cell, 1983, 33, 95-102; Johnston et al., In: Interferon 3: Mechanisms of Production and Action, 1984, 189-298, Friedman, R. M., Ed, Elsevier, Amsterdam; Justesen et al., Proc. Natl. Acad. Sci., USA, 1980, 77, 4618-4622; Justesen et al., Nucleic Acids Res., 1980, 8, 3073-3085; Justesen, J. and Kjelgaard, N. O., Anal. Biochem., 1992, 207, 90-93).
A protein having 75% identity with residues 1 to 1087 of SEQ ID NO: 2 is a protein whose sequence may include up to (25×10.87=271) changes when the protein sequence is aligned and compared to residues 1 to 1087 of with SEQ ID NO: 2. One change refers to the deletion, substitution or insertion of one amino acid as compared to residues 1 to 1087 of SEQ ID NO: 2. For example, a 1080 amino acid sequences having 50 changes with the first 1080 residues of SEQ ID NO: 2 has 1080−50/10.87=94.75% identity with SEQ ID NO: 2. For example, a 1090 amino acid sequences having 50 changes with residues 1 to 1087 of SEQ ID NO: 2 has 1087−50/10.87=95.4% identity with SEQ ID NO: 2.
The invention encompasses modified OAS3 protein including one or more modifications selected from the group consisting of: the mutation (insertion, deletion, substitution) of one or more amino acids in the OAS3 amino acid sequence, the addition of an amino acid fusion moiety, the substitution of amino acid residues by non-natural amino acids (D-amino-acids or non-amino acid analogs), the modification of the peptide bond, the cyclization, the addition of chemical groups to the side chains (lipids, oligo- or -polysaccharides), and the coupling to an appropriate carrier. These modifications which are introduced by procedures well-known in the art, result in a modified OAS3 protein which is still active for 2′-5′-oligoadenylate synthetase and inhibition of positive-sense single-stranded RNA virus replication activities.
According to a preferred embodiment of the invention, said 2′-5′-aligoadenylate synthetase 3 (OAS3) is human 2′-5′-oligoadenylate synthetase 3.
According to another preferred embodiment of the invention, said OAS3 protein has at least 70% amino acid sequence identity or 80% amino acid sequence similarity, preferably at least 80% amino acid sequence identity or 90% amino acid sequence similarity to residues 1 to 1087 of SEQ ID NO: 2.
According to a more preferred embodiment of the invention, said OAS3 protein comprises a Serine at position 381 of SEQ ID NO: 2. Preferably, said OAS3 protein comprises or consists of an amino acid sequence selected in the group consisting of: SEQ ID NO: 2 and SEQ ID NO: 7.
According to another preferred embodiment of the invention, said OAS3 polynucleotide is coding for a protein as defined above, more preferably it comprises or consists of a nucleotide sequence selected in the group consisting of: SEQ ID NO: 1 which encodes the protein SEQ ID NO: 2 and SEQ ID NO: 6 which encodes the protein SEQ ID NO: 7.
According to another preferred embodiment of the invention, said polynucleotide is inserted in an expression vector.
A vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. A vector which can be used in the present invention includes, but is not limited to, a viral vector, a plasmid, a RNA vector or a linear or circular DNA or RNA molecule which may consists of a chromosomal, non chromosomal, semi-synthetic or synthetic nucleic acids. Preferred vectors are those capable of autonomous replication (episomal vector) and/or expression of nucleic acids to which they are linked (expression vectors). Large numbers of suitable vectors are known to those of skill in the art and commercially available.
Viral vectors include retrovirus, adenovirus, parvovirus (e.g. adeno-associated viruses or AAVs), coronavirus, negative strand RNA viruses such as ortho-myxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g. measles and Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include: avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).
Preferably said vectors are expression vectors, wherein the sequence encoding the OAS3 protein of the invention is placed under control of appropriate transcriptional and translational control elements to permit production or synthesis of said protein. Therefore, said polynucleotide is comprised in an expression cassette. More particularly, the vector comprises a replication origin, a promoter operatively linked to said encoding polynucleotide, a ribosome-binding site, an RNA-splicing site (when genomic DNA is used), a polyadenylation site and a transcription termination site. It also can comprise an enhancer. Selection of the promoter will depend upon the cell in which the polypeptide is expressed. Suitable promoters include tissue specific and/or inducible promoters. Examples of inducible promoters are: eukaryotic metallothionine promoter which is induced by increased levels of heavy metals, heat shock promoter which is induced by increased temperature. Examples of tissue specific promoters are skeletal muscle creatine kinase, prostate-specific antigen (PSA), α-antitrypsin protease, human surfactant (SP) A and B proteins and β-casein. Vectors can comprise selectable markers, for example: neomycin phosphotransferase, histidinol dehydrogenase, dihydrofolate reductase, hygromycin phosphotransferase, herpes simplex virus thymidine kinase, adenosine deaminase, glutamine synthetase, and hypoxanthine-guanine phosphoribosyl transferase for eukaryotic cell culture; TRP1, URA3 and LEU2 for S. cerevisiae; tetracycline, rifampicin or ampicillin resistance in E. coli.
The choice of the vector depends on their use (stable or transient expression) or and on the host cell; viral vectors and “naked” nucleic acid vectors are preferred vectors for expression in mammal cells (human and animal). Use may be made, inter alia, of viral vectors such as adenoviruses, retroviruses, lentiviruses and AAVs, into which the sequence of interest has been inserted beforehand.
The subject-matter of the present invention is also a pharmaceutical composition characterized in that it comprises at least one OAS3 protein or one OAS3 polynucleotide, preferably inserted in an expression vector, as defined above, and at least one acceptable vehicle, carrier, additive and/or immunostimulating agent.
Any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical composition of the present invention, the type of carrier varying depending on the mode of administration. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline buffer, lactose, mannitol, glutamate, a fat or a wax and the injectable pharmaceutical composition is preferably an isotonic solution (around 300-320 mosmoles). For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed. Biodegradable microspheres (e.g. polylactic galactide) may also be employed as carriers for the pharmaceutical compositions of this invention. Suitable biodegradable microspheres are disclosed, for example in U.S. Pat. Nos. 4,897,268 and 5,075,109. The additive may be chosen among antiaggregating agents, antioxidants, dyes, flavor enhancers, or smoothing, assembling or isolating agents, and in general among any excipient conventionally used in the pharmaceutical industry. Any of the variety of immunostimulating agent may be employed in the compositions of the present invention to enhance the immune response.
The pharmaceutical composition may be in a form suitable for oral administration. For example, the composition is in the form of tablets, ordinary capsules, gelatine capsules or syrup for oral administration. These gelatine capsules, ordinary capsules and tablet forms can contain excipients conventionally used in pharmaceutical formulation, such as adjuvants or binders like starches, gums and gelatine, adjuvants like calcium phosphate, disintegrating agents like cornstarch or algenic acids, a lubricant like magnesium stearate, sweeteners or flavourings. Solutions or suspensions can be prepared in aqueous or non-aqueous media by the addition of pharmacologically compatible solvents. These include glycols, polyglycols, propylene glycols, polyglycol ether, DMSO and ethanol.
The OAS3 protein or the OAS3 polynucleotide (isolated or inserted in a vector) are introduced into cells, in vitro, ex vivo or in vivo, by any convenient means well-known to those in the art, which are appropriate for the particular cell type, alone or in association with at least either an appropriate vehicle and/or carrier. For example, the OAS3 protein/polynucleotide may be associated with a substance capable of providing protection for said sequences in the organism or allowing it to cross the host-cell membrane. The OAS3 protein may be advantageously associated with liposomes, polyethyleneimine (PEI), and/or membrane translocating peptides (Bonetta, The Scientist, 2002, 16, 38; Ford et al., Gene Ther., 2001, 8, 1-4; Wadia and Dowdy, Curr. Opin. Biotechnol., 2002, 13, 52-56; Langel, U. In Handbook of cell penetrating peptides (2nd Ed.), 2006, Lavoisier, FRANCE); in the latter case, the sequence of the OAS3 protein is fused with the sequence of a membrane translocating peptide (fusion protein). Polynucleotide encoding OAS3 (isolated or inserted in a vector) may be introduced into a cell by a variety of methods (e.g., injection, direct uptake, projectile bombardment, liposomes, electroporation). OAS3 protein can be stably or transiently expressed into cells using appropriate expression vectors as defined above.
In one embodiment of the present invention, the OAS3 protein/polynucleotide is substantially non-immunogenic, i.e., engenders little or no adverse immunological response. A variety of methods for ameliorating or eliminating deleterious immunological reactions of this sort can be used in accordance with the invention. In a preferred embodiment, the OAS3 protein is substantially free of N-formyl methionine. Another way to avoid unwanted immunological reactions is to conjugate protein/polynucleotide to polyethylene glycol (“PEG”) or polypropylene glycol (“PPG”) (preferably of 500 to 20,000 daltons average molecular weight (MW)).
Another subject of the present invention is an OAS3 protein or a polynucleotide coding for said OAS3 protein as defined above for preventing or treating an infection with a positive-sense single-stranded RNA virus.
According to a more preferred embodiment, said virus is of the Alphavirus genus. More preferably, it is selected from the group consisting of: Chikungunya (CHIK), Sindbis (SIN), Semliki Forest (SF), Eastern Equine Encephalitis (EEE), Western Equine Encephalitis (WEE), Venezuelan Equine Encephalitis (VEE), Ross River (RR), O'Nyong Nyong (ONN) and Banna Forest (BF) viruses.
According to another more preferred embodiment, said virus is of the Flavivirus genus. More preferably, said virus is selected from the group consisting of: Dengue, Japanese Encephalitis, Kyasanur Forest Disease, Murray Valley Encephalitis, St. Louis Encephalitis, Tick-Borne Encephalitis, West Nile, Yellow Fever and Omsk hemorrhagic fever (OHF) virus.
According to another more preferred embodiment, said virus is of the Hepacivirus genus. More preferably, said virus is the Hepatitis C virus.
The subject-matter of the present invention is also products containing at least an OAS3 protein or an OAS3 polynucleotide, preferably inserted in an expression vector, as defined above and a second product which is different from the first one, said second product being selected from the group consisting of: antiviral, anti-inflammatory and immunomodulatory drugs, as a combined preparation for simultaneous, separate or sequential use in the prevention or the treatment of a positive-sense single-stranded RNA virus infection.
The subject-matter of the present invention is also a method for preventing or curing a positive-sense single-stranded RNA virus infection in an individual in need thereof, said method comprising the step of administering to said individual a composition as defined above, by any means.
In general, the composition may be administered by parenteral injection (e.g., intradermal, intramuscular, intravenous or subcutaneous), intranasally (e.g. by aspiration or nebulization), orally, sublingually, or topically, through the skin or through the rectum.
The amount of OAS3 (protein/polypeptide) present in the composition of the present invention is a therapeutically effective amount. A therapeutically effective amount of OAS3 (protein/polypeptide) is that amount necessary so that OAS3 protein performs its role of inhibiting positive-sense single-stranded RNA virus replication without causing, overly negative effects in the subject to which the composition is administered. The exact amount of OAS3 (protein/polypeptide) to be used and the composition to be administered will vary according to factors such as the positive-sense single-stranded RNA virus species and the individual species (human, animal) being treated, the mode of administration, the frequency of administration as well as the other ingredients in the composition.
Preferably, the composition is composed of from about 10 μg to about 10 mg and more preferably from about 100 μg to about 1 mg, of OAS3 (protein/polypeptide). By “about”, it is meant that the value of said quantity Gig or mg) of OAS3 can vary within a certain range depending on the margin of error of the method used to evaluate such quantity.
For instance, during an oral administration of the composition of the invention, individual to be treated could be subjected to a 1 dose schedule of from about 10 μg to about 10 mg of OAS3 (protein/polypeptide) per day during 3 consecutive days. The treatment may be repeated once one week later.
For parenteral administration, such as subcutaneous injection, the individual to be treated could be subjected to a 1 dose of from about 10 μg to about 10 mg and more preferably from about 100 μg to about 1 mg, of OAS3 (protein/polypeptide). The treatment may be repeated once one week later.
A subject of the invention is also a method in vitro for evaluating the susceptibility of an individual to an infection with a positive-sense single-stranded RNA virus as defined above, comprising: the detection of a polymorphism in the OAS3 gene in a nucleic acid sample obtained from said individual.
The nucleic acid sample may be genomic DNA, total mRNA or cDNA.
The polymorphism is detected by any method known in the art that allows the detection of mutation in nucleic acid sequences as those described for example In Current Protocols in Human Genetics, 2008, John Wiley & Sons, Inc. Examples of genotyping assays include with no limitation: RAPD, RFLP, AFLP, sequence specific oligonucleotide hybridization, SnapShot PCR, Ligase detection reaction, PCR and Maldi-TOF, Pyrosequencing. This assay may use the OAS3 specific primers of Table II and III and in particular the primers SEQ ID NO: 11, 12, 62 to 86 and 118 to 142.
According to a preferred embodiment of said method, said positive-sense single-stranded RNA virus is an alphavirus such as Chikungunya virus or a flavivirus such as Dengue virus.
According to another preferred embodiment of said method, said polymorphism is the mutation of the Arg844 codon to a stop codon (R844X); said polymorphism detected in the Caucasian population, is associated with an increased susceptibility to positive-sense single-stranded RNA virus infection, particularly Chikungunya virus infection. The R844X mutation may be detected by PCR-RFLP using the pair of primers (SEQ ID NO: 11 and SEQ ID NO: 12), followed by digestion of the 183 bp PCR product with BglII; the presence of two fragments of 115 bp and 68 bp indicates the presence of OAS3.R844X mutation.
According to another preferred embodiment of said method, said polymorphism is a single nucleotide polymorphism (SNP) at the third position of codon 381. Preferably, said SNP is a G to C substitution of codon AGG-381 that changes amino acid from Arg to Ser (R381S). Genetic data suggest that variant Ser-381 is associated with a dominant protection against the risk of DSS in dengue patients (Table VI). This SNP may be detected by any appropriate genotyping assay as defined above. For example, this assay may comprise the direct sequencing of genomic DNA amplified with a pair of OAS3 specific primers such as the pair of PCR primers specific to OAS3 exon 6 that is presented in Table III (SEQ ID NO: 70 and 126).
A subject of the invention is also an isolated OAS3 protein comprising or consisting of the sequence SEQ ID NO: 7 or SEQ ID NO: 30.
A subject of the invention is also an isolated OAS3 protein fragment comprising or consisting of the sequence SEQ ID NO: 9; this OAS3 fragment which includes residues 1 to 843 of SEQ ID NO: 7 comprises the first and the second OAS domains of OAS3 but lacks most of the third (C-terminal) OAS domain.
A subject of the invention is also:
A subject of the invention is also a recombinant vector, preferably an expression vector comprising a polynucleotide having a sequence selected in the group consisting of: SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 28 and SEQ ID NO: 29.
A subject of the invention is also a host cell transfected or transformed by a polynucleotide comprising or consisting of a sequence selected in the group consisting of: SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 28 and SEQ ID NO: 29.
According to a preferred embodiment of the invention, it is an HeLa-Tet-Off cell line expressing a recombinant human OAS3, named HeLa-Tet-Off/OAS3#C417-1, deposited Feb. 26, 2008, at the Collection Nationale de Cultures de Microorganismes, 25 rue du Docteur Roux, 75724 Paris Cedex 15, under the accession number I-3927.
According to another preferred embodiment of the invention, it is an a-Tet-Off cell line expressing a truncated recombinant human OAS3, named HeLa-Tet-Off/OAS3/delta/1C, deposited Apr. 17, 2008, at the Collection Nationale de Cultures de Microorganismes, 25 rue du Docteur Roux, 75724 Paris Cedex 15, under the accession number I-3968.
According to a preferred embodiment of the invention, it is an HepG2-Tet-Off cell line expressing a recombinant human OAS3, named HeLa-Tet-Off/OAS3#F8, deposited May 15, 2009, at the Collection Nationale de Cultures de Microorganismes, 25 rue du Docteur Roux, 75724 Paris Cedex 15, under the accession number I-4158.
These cell lines which are derived from HeLa or HepG2 cells which can be infected with various viruses are useful for assaying the susceptibility of a virus to OAS3 mediated antiviral activity.
A subject of the invention is also a non-human transgenic animal comprising a polynucleotide as defined above.
A subject of the invention is also a transgenic plant comprising a polynucleotide as defined above.
The OAS3 protein/polynucleotide of the invention are prepared using well-known recombinant DNA and genetic engineering techniques. For example, a sequence comprising the OAS3 ORF is amplified from a DNA template, by polymerase chain reaction with specific primers. The PCR fragment is then cloned in an expression vector by using appropriate restriction sites. The OAS3 protein is expressed in a host cell or a transgenic animal/plant modified by the expression vector, under conditions suitable for the expression of the OAS3 protein, and the OAS3 protein is recovered from the host cell culture or from the transgenic animal/plant.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Harries & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York), specifically, Vols. 154 and 155 (Wu et al. eds.) and Vol. 185, “Gene Expression Technology” (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Current Protocols in Human Genetics (John Wiley & Sons, Inc, 2008), specifically Chapter 12 “Vectors For Gene Therapy” and Chapter 13 “Delivery Systems for Gene Therapy”).
a) Cell cultures
The HeLa.Tet-Off cell line was purchased from BD BIOSCIENCES CLONTECH. HeLa.Tet-Off cells are maintained in 5% CO2 at 37° C., in DMEM (INVITROGEN), supplemented with 10% heat-inactivated fetal calf serum (FCS), 4 mM L-glutamine, 100 UI/ml penicillin, 10 μg/ml streptomycin and 200 μg·mL−1 G418 (INVITROGEN).
The HeLa.Tet-Off/OAS3 cell line (CNCM I-3927) is maintained in DMEM, 10% FCS, 4 mM L-glutamine, 100 UI/ml penicillin, 10 μg/ml streptomycin, 200 μg·mL−1 G418, 100 hygromycin B (BD BIOSCIENCES CLONTECH), and 2 μg·mL−1 tetracycline (SIGMA-ALDRICH). The cells are grown in monolayers; the expected cell density is of 80% to 100%; the population doubling time is of about two days. The cells are harvested by trypsinization; they are sub-cultured 1/10 every week. The cells have a limited lifespan (15 to 20 passages). The cells are frozen in DMEM supplemented with 20% FCS, 10% DMSO, 4 mM glutamine, 100 UI/ml penicillin, 10 μg/ml streptomycin, 200 μg·mL−1 G418, 100 μg·mL−1 hygromycin B, and 2 μg·mL−1 tetracycline.
The HeLa.Tet-Off/OAS3/delta/1C (OAS3ΔC-term) cell line (CNCM I-3968) is maintained in DMEM, 10% FCS, 20 mM L-glutamine, 10,000 UI/ml penicillin, 10 μg/ml streptomycin, 200 μg·mL−1 G418, 100 μg·mL−1 hygromycin B (BD BIOSCIENCES CLONTECH), and 10 μg·mL−1 tetracycline (SIGMA-ALDRICH). The cells are grown in monolayers; the expected cell density is of 90%; the population doubling time is of about two days. The cells are harvested by trypsinization; they are sub-cultured 1/10 every week. The cells have a limited lifespan (25 passages). The cells are frozen in DMEM supplemented with 20% FCS, 10% DMSO, 4 mM glutamine, 10,000 UI/ml penicillin, 10 μg/ml streptomycin, 200 μmL−1 G418, 100 μg·mL−1 hygromycin B, and 10 μg·mL−1 tetracycline.
b) Establishing HeLa.Tet-Off cell lines expressing OAS3 proteins
Two overlapping cDNA fragments of human OAS3 cloned separately in the PCR4-TOPO vector (INVITROGEN), were used as templates for expression of the full-length OAS3 protein (aa 1 to 1,087; SEQ ID NO: 24 and
The OAS3 sequence was modified by PCR to be flanked on the 3′ open reading frame end by the additional 10-residue sequence EQKLISKEDL (SEQ ID NO: 10) followed by a stop-codon with the couple of primers pTet-OAS3-univ1 and pTet-OAS3-rev2 for OAS3 and the couple of primers pTet-OAS3-univ1bis and pTet-OAS3-rev2bis for OAS3{1-843} (Table II).
Enzyme recognition site are underlined. Sequence complementary to a stop codon are shown in bold. The OAS3 sequences (SEQ ID NO: 23 and 25) are flanked by the NotI and EcoRV restriction enzymatic sites at the downstream and upstream ends, respectively. The PCR products were digested with NotI and EcoRV and then inserted into the unique sites NotI and EcoRV of pTRE2hyg expression vector (BD BIOSCIENCES CLONTECH) to generate pTRE2hyg-OAS3 (
Production of clinical isolate Chikungunya virus strain 06-49 (CHIKV 06-49; Schuffenecker et al., PLoS Medicine 3, 2006, e263) on mosquito Aedes pseudoscutellaris (AP61) cell monolayers and virus titration by focus immunodetection assay (Desprès et al., Virology, 1993, 196, 209-219) were performed as previously described (Bréhin et al., Virology, 2008, 371, 185-195). Infectivity titers were expressed as focus forming units (FFU) on AP61 cells. In order to assay its antiviral effect, human IFN-α (BIOSOURCE) was directly added to culture medium at 1,000 IU·mL−1.
Cellular proteins were subjected to immunoblot analysis as previously described (Bréhin et al., Virology, 2008, 371, 185-195). Viral protein expression was detected using anti-CHIKV HMAF or anti-CHIK.E2 MAb 3E4 (Bréhin et al., Virology, 2008, 371, 185-195). OAS3 protein expression was detected using anti-OAS3 N-term (Santa-Cruz) or C-term (ABGENT) antibodies.
Cells were detached and then fixed with 3.2% paraformaldehyde in PBS. Fixed cells were permeabilized, stained with anti-CHIKV HMAF and analyzed by flow cytometry as described previously (Bréhin et al., Virology, 2008, 371, 185-195).
First, the susceptibility of parental HeLa.Tet-Off cell line to CHIKV infection, was evaluated (
The stable HeLa.Tet-Off/OAS3#C417-1 cell clone that up-regulates OAS3 protein expression under the control of the Tet-Off expression system was selected to assess the antiviral activity of the large form of OAS. The recombinant OAS3 protein is composed of three adjacent OAS units (domain I, II, and III) including three potential active catalytic sites (
The experimental procedures are as described in example 1.
To investigate the effect of OAS3 protein on CHIKV growth in human epithelial cells, HeLa.Tet-Off/OAS3 cells were infected 18 h with CHIKV.06-49 at 1 MOI. As determined by flow cytometry using MAb 3E4, uninduced HeLa.Tet-Off/OAS3 and HeLa.Tet-Off cells displayed similar susceptibility to CHIKV infection (
To further assess the efficiency with which HeLa.Tet-Off/OAS3 cells inhibit CHIKV growth, induced cells were exposed to increasing input of CHIKV-06.49 (
Kinetic studies showed that OAS3-mediated inhibition of CHIKV was effective during virus life cycle (
Measurements of Lactate dehydrogenase (LDH), a cytoplasmic enzyme that is released into the culture medium upon cell lysis and therefore is a measure of membrane integrity, showed no significant loss of viability of infected HeLa/Tet-Off/OAS3 cells within the first 24 h of infection as compared to infected parental cells. Thus, the resistance to CHIKV infection was directly related to the antiviral activity of the OAS3 rather than elimination of virus-infected cells through apoptosis induction.
The experimental procedures are as described in example 1. In addition virus production and titration were performed as described below.
Production of low passaged West Nile Virus (WNV) IS-98-ST1 strain (GenBank accession number AF 481864) on mosquito Aedes pseudoscutellaris (AP61) cell monolayers and virus titration by focus immunodetection assay (Desprès et al., Virology, 1993, 196, 209-219) were performed as previously described (Bréhin et al., Virology, 2008, 371, 185-195; International Application WO 02/081741). Infectivity titers were expressed as focus forming units (FFU) on AP61 cells. Sindbis virus strain AR339 (SINV AR339) and Semliki Forest Virus strain SF 64 (SFV 64) were propagated on African green monkey kidney (VERO) cell line and infectivity titers were expressed as plaque forming units (PFU) on VERO cells. In order to assay its antiviral effect, human IFN-α. (BIOSOURCE) was directly added to culture medium at 1,000 IU·mL−1.
The ability of the large form of OAS to inhibit growth of SINV strain AR339, SFV strain SF 64 and WNV strain IS-98-ST1 in human epithelial cells was examined (
The experimental procedures are as described in example 1. In addition quantitative RT-PCR was performed as described below.
Real-Time RT-PCR analysis of viral RNA accumulation was performed with an ABI Prism 7700 sequence detection using the SYBR® Green PCR essentially as described previously (Kajaste-Rudnistki et al., J. Biol. Chem., 2006, 281, 4624-4637). The primers for CHIKV E2 gene are Chik/E2/9018/+ and Chik/E2/9235/− (Table II). GAPDH mRNA was used as an endogenous sequence control for the normalization of each sample. The primers GAPDH-For and GAPDH-Rev are listed in Table II. For the quantification of viral RNA copies, an in vitro CHIK RNA transcript encoding the N-terminal region of E2 was performed to build the standard curve as previously described (Vazeille et al., PLoS One, 2007, 2(11): e1168).
In an effort to resolve the molecular basis of the antiviral action of OAS3, it was determined whether inhibition of CHIKV growth was due to a lack of accumulation of structural virus proteins. Total proteins were extracted from Hela.Tet-off and induced HeLa.Tet-Off/OAS3 cells at the 18-h timepoint post-infection and capsid protein (C), envelope glycoproteins pE2 (precursor of E2), E2, and E1 were detected by immunoblot using anti-CHIKV HMAF, whereas envelope glycoproteins pE2 (precursor of E2) and E2 were detected specifically by immunoblot using anti-CHIKV E2 MAb 3E4. As shown in
It was determined whether the inefficiency of viral protein synthesis was due to a lack of accumulation of viral RNA. Total RNA was extracted from Hela.Tet-off and induced HeLa.Tet-Off/OAS3 cells at the 8 h time-point post-infection and production of genomic and subgenomic viral RNAs was analyzed using RT-PCR assay with primers designed on the basis of CHIKV E2 gene. As determined by real-time RT-PCR analysis, there was a 2 log reduction in viral RNA level recovered from OAS3-expressing cells when compared to that found in parental cells (
The experimental procedures are as described in example 1. In addition, genotyping of OAS3 polymorphism was assayed as described below.
The SNP OAS3.R844X was genotyped by PCR-RFLP assay using the couple of primers OAS3.R844X-F(SEQ ID NO: 11) and OAS3.R844X-R (SEQ ID NO: 12) which are presented in Table II. The 183 bp-long PCR product was subjected to digestion with Bgl II and the detection of two fragments of 115 and 68 bp indicated the presence of OAS3.R844X. 2) Results
The OAS3 gene was screened for polymorphisms. Re-sequencing total 16 exons, flanking introns and 2 kb 5′ to the OAS3 gene in fourty-eight healthy Caucasians individuals identified a single-nucleotide polymorphism (SNP) at the first position of codon CGA-844 where the substitution T for C resulted in non-sense mutation (OAS3.R844X) (Table I). This SNP was further screened in 180 healthy Caucasian individuals and identified two heterozygotes which gave allele frequency of 0.5%. Because OAS3.R844X possibly truncates about 20% of the OAS3 protein from the carboxy terminus (
The HepG2.Tet-Off cell line was purchased from CLONTECH (# 632106). HepG2.Tet-Off cells are maintained in 5% CO2 at 37° C., in DMEM (GIBCO, # 41965), supplemented with 10% heat-inactivated fetal calf serum (FCS), 4 mM L-glutamine, 100 UI/ml penicillin G sodium, 100 μg/ml streptomycin sulfate (GIBCO, # 15140-122), 0.1 mM non-essential amino acids, and 100 μg·mL−1 G418 (GIBCO, # 10131-019).
The HepG2.Tet-Off/OAS3#F8 cell line (CNCM I-4158) is maintained in DMEM, 10% FCS, 4 mM L-glutamine, 100 UI/ml penicillin, 100 μg/ml streptomycin, 100 μg·mL−1 G418, 100 μg·mL−1 hygromycin B (CLONTECH, # 631309), and 2 μg·mL−1 tetracycline (SIGMA-ALDRICH, # T-7660).
An inducible HepG2.Tet-Off/OAS3#F8 clone expressing the full-length human OAS3 protein (SEQ ID NO: 24) was selected as described in example 1 for the inducible HeLa.Tet-Off/OAS3#C417-1. The HepG2.Tet-Off/OAS3#F8 cell line was deposited May 15, 2009, at the Collection Nationale de Cultures de Microorganismes, 25 rue du Docteur Roux, 75724 Paris Cedex 15, under the accession number I-4158
This cell line is maintained under repressing condition in the presence of 2 μg·mL−1 Tet.
Production of low passaged West Nile Virus (WNV) IS-98-ST1 strain (GenBank accession number AF 481864; Lucas et al., Virology J., 2004, 1, 9-) on mosquito Aedes pseudoscutellaris (AP61) cell monolayers and virus titration by focus immunodetection assay (Desprès et al., Virology, 1993, 196, 209-219) were performed as previously described (Bréhin et al., Virology, 2008, 371, 185-195). Production of DV1 strain FGA/NA d1d and virus titration were performed as previously described (Duarte dos Santos et al. Virology, 2000, 274, 292-). Infectivity titers were expressed as focus forming units (FFU) on AP61 cells. In order to assay its antiviral effect, human IFN-α (BIOSOURCE) was directly added to culture medium at 1,000 IU·mL−1.
Virus-infected cells were fixed with methanol/acetone at −20° C. for 20 min. Immunofluorescence assay was performed using FITC-conjugated anti-dengue E mAb 4E1 or cy3-conjugated anti-WNV.E MAb E24. Nuclei were stained with DAPI.
First, the susceptibility of parental HepG2.Tet-Off cell line to DV infection was evaluated (
Then, the ability of IFN-α to establish an antiviral state in HepG2.Tet-Off cells was investigated. Pretreatment of HepG2.Tet-off cells with 1,000 IU·mL−1 human IFN-α5 hours prior DV infection (10 MOI) resulted in about 0.7 log reduction in virus titer at 40 h p.i. (
To investigate the effect of OAS3 protein on DV growth in Hep.G2 cells, induced (−Tet) and uninduced (+Tet) HepG2.Tet-Off/OAS3 cells and parental HepG2.Tet-Off cells were infected with increasing input of DV-1 virus strain FGA/NA d1d. At 40 h p.i., immunofluorescence assay using anti-dengue E mAb was performed and the percentage of HepG2 cells positive for viral antigen was determined (
Thus, OAS3-expressing HepG2 cells show resistance to DV infection.
The effect of OAS3 protein on WNV growth in Hep.G2 cells was also analyzed by immunofluorescence assay. Parental HepG2.Tet-Off and induced HepG2.Tet-Off/OAS3#F8 cell clone were infected with West Nile virus strain IS-98-ST1 at multiplicity of infection 1 AP61FFU/cell or mock-infected. At 40 h post-infection viral antigen (WNV E glycoprotein) was detected by immunofluorescence using cy3-conjugated mAb E24. The results (
Attenuated Yellow Fever Virus (YFV) vaccine strain (STAMARIL®, AVENTIS-PASTEUR) was propagated on African green monkey kidney (VERO) cell line and infectivity titers were expressed as plaque forming units (PFU) on VERO cells. In order to assay its antiviral effect, human IFN-α(BIOSOURCE) was directly added to culture medium at 1,000 IU·mL−1. Immunofluorescence assay was performed as described in example 6, using anti-French Neurotropic Virus HMAF and FITC-conjugated goat anti-mouse Ig.
Examples 2, 3, 4 and 6 provided the first evidence that OAS3-dependent antiviral activity mediated by IFN-α/β represents a major human cell defence strategy against alphaviruses such as Chikungunya virus and flaviviruses such as West Nile and dengue viruses in human cells.
It was investigated whether OAS3 displays antiviral activity against yellow fever virus (YFV). For this, live-attenuated 17D-204 vaccine strain of YFV (STAMARIL, Aventis-Pasteur) were twice propagated on African green monkey kidney (VERO) cell line and infectivity titers were expressed as Plaque Forming Units (PFU) on VERO cells. HeLa cells were infected with YFV 17D at 1 PFU/cell and then treated with 1,000 IU/ml human IFN-α or mock-treated (control) at various time-points post-infection. Virus progeny productions were determined at 72 h post-infection. As shown in
The resistance of YFV 17D to OAS-3 mediated antiviral effect was confirmed by immunofluorescence assay in parental HepG2.Tet-Off and HepG2.Tet-Off/OAS3#F8 cell clone infected with YFV 17D (
The observation that live-attenuated strains of YFV show unexpected resistance to antiflaviviral action of OAS3 opens a new avenue for elucidating the mechanism of flavivirus attenuation.
a) Patients and controls
750 patients (male:female ratio=0.99) with symptomatic dengue viral (DV) infection during the 3 year period (June 2000-2003) were enrolled in the study from two medical centers in Bangkok, Ramathibodi Hospital and Siriraj Hospital, Mahidol University and 1 hospital in Khon-Kaen province, Thailand. Their ages ranged from 1 to 25 years with a mean of 9.6 years. Patients with suspicion of dengue viral infection based on clinical features including high fever, severe headache, retro-orbital pain, myalgia, arthalgia, nausea and vomiting, and rash were admitted to the hospitals for clinical observation and treatment. The diagnosis of dengue virus infection was later confirmed by a comparable IgG and IgM enzyme-linked immunosorbent assay titer on a late acute and/or convalescent sera. Differential diagnosis of DF (Dengue fever) and DHF (Dengue Hemorrhagic Fever) was established based on the absence (DF) or presence (DHF) of evidence for increased vascular permeability manifested by hemoconcentration or pleural effusion. Specifically, the diagnosis of DHF was made based on all of the four following characteristics: 1) high continuous fever lasting 2-7 days, 2) hemorrhagic tendency such as a positive tourniquet test, petechii, purpura or hematemesis, 3) thrombocytopenia (platelet count≦100,000/μl and 4) evidence of plasma leakage due to increased vascular permeability manifested by hemoconcentration (an increased in hematocrit of 20% or more) or pleural effusion. Some dengue patients who could not be classified as DF or DHF because of unclear clinical symptoms were assigned an unknown DF/DHF status. The severity of DHF was categorized by four grades according to WHO criteria. Grades III and IV were DHF with narrowing pulse pressure with a characteristically elevated diastolic pressure to profound shock. Secondary infection was defined as a dengue-specific IgM/IgG ratio<1.8. The project protocol and objectives of the study were carefully explained to the patients and their parents or relatives. Informed consent was individually obtained from all subjects. The protocol has been approved by the ethical committee of each hospital.
The controls consisted of 296 blood donors from Ramathibodi hospital and 216 blood donors from Siriraj Hospital and 184 ethnic matched healthy controls from Khon-Kaen region (male:female ratio=1). Both cases and control groups came from Bangkok and Central part of Thailand. Whole blood samples were collected from patients and controls on EDTA, and DNA was extracted using a standard phenol/chloroform extraction method.
During 2004-2006, we enrolled additional 254 patients (male:female ratio=1.07) from Ramathibodi and Khon-Kaen hospital using the same clinical and viral diagnosis criteria.
Polymorphisms were identified by direct sequencing of PCR amplified genomic DNA, on individual DNA samples (24 dengue patients and 32 Thai controls). Primers used for sequencing OASs exons, part of introns and 5′ and 3′ regions are shown in Table III.
Sequencing results were analyzed by Genalys Software (Takahashi et al., J. Bioinform. Comput Biol., 2003, 1, 253-265). OAS3-R381S was genotyped by TaqMan assay using ABI Prism 7000 Sequence Detection System, with recommended protocols. The codon 381 of the more common variant of OAS3 sequence is AGG which corresponds to a arginine. The polymorphism OAS3-R381S, named rs2285933, has a G to C substitution at the third nucleotide of codon 381 of OAS3 (AGG to AGC) that changes amino acid from arginine to serine. The 2 variants of OAS3-(R381 and S381) mRNA, ORF and amino acid sequences correspond to SEQ ID NO: 28, 29, 30 (R381) and SEQ ID NO: 27, 1 and 2 (S381), respectively.
We performed association study using allele count by Pearson chi-square test implemented in the Haploview program (Barrett et al., Bioinformatics, 2005, 21, 263-265). This program allows us to perform permutation test to obtain an empirical P value. We performed logistic regression analysis coding the genotype results according to the genetic model tested using STATA version 9.
During 2000-2003, we have recruited 580 dengue patients from two hospitals (Siriraj, S I and Ramathibodi, R A) in Bangkok, and 170 from another hospital in Khonkaen province (KK), north-east of Thailand. We collected DNA samples from 512 blood donors from the same hospitals in Bangkok and 184 healthy volunteer from the north-east region of Thailand to represent gene frequency in general populations. Genetic study of part of these patients and controls has been reported previously (Sakuntabhai et al., Nature Genetics, 2005, 37, 507-513). Viral diagnosis was confirmed by serologic tests, and patients were classified into 4 groups according to WHO criteria: DF (no evidence of plasma leakage), and DHF1 (evidence of plasma leakage), DHF2 (with spontaneous bleeding) and DSS (with shock) (Table IV).
There were 51 patients who could not be classified as having DF or DHF according to WHO criteria (unclassified; UC). Proportion of patients with each severity category was significantly different among hospitals (P=1.5×10−8). This reflects difference in type of hospital (secondary care for KK or medical school for SI and RA) and inclusion criteria (fever less than 3 days in KK and suspected dengue cases in SI and RA).
There was an highly significant increased risk of plasma leakage (DHF1/DHF2/DSS versus DF) and spontaneous bleeding (DHF2/DSS versus DF/UC/DHF1) in patients who had a predominant IgG response compared to patients with predominant IgM response to dengue infection (P=1.1×10−7 and P=7.6×10−5, respectively) but only marginal significance for shock (DSS versus the rest, P=0.038) (Table V).
These findings are consistent with the notion that individuals with IgG response to DV are predisposed to the more severe forms of the disease.
For screening of tagged polymorphisms, tests for association were performed using allele count on the risk of plasma leakage (DHF1/DHF2/DSS versus DF), risk of spontaneous hemorrhage (DHF2/DSS versus DF/UC/DHF1), risk of shock (DSS versus non-shock DV patients) in patients from the 2 hospitals in Bangkok (Table VI).
As shown in Table VI, none of the polymorphisms showed evidence of association with the risk of disease. However, one non-synonymous polymorphism of OAS3 had highly significantly different allele count distributions depending on the severity of the disease. This variant (OAS3-R381S: rs2285933) is a G to C substitution at the 3rd nucleotide of codon 381 of OAS3, that changes amino acid from arginine to serine. In both hospitals, the C allele of this polymorphism was more frequent in DF, DHF1 and DHF2, than in DSS. There was no significance difference in allele frequency between each group of patients and controls between the 2 hospitals. In combined analyses, the C allele had a frequency of 14% in general population, 20% in DF, and 15-16% in DHF1 and DHF2 and 7% in DSS. The difference between DSS and non-shock DV cases was highly significant (P=7.10−4). We performed 10,000 permutations and obtained an empirical P value of 0.0032.
In order to find out mode of inheritance of the OAS3-R381S, we compared the likelihood of the models based on 3 different mode of inheritance: additive, dominant and recessive of the C allele using logistic regression on the risk of DSS. While recessive model of the C allele did not show significant association, highly significant level was obtained with dominant model of the C allele (<10−5 P<10−5, OR=0.30 95CI=0.17-0.53) and additive (17.93, P<10−5). By log likelihood ratio test, the dominant model is significantly fitter than the additive model (P=0.04). We therefore used dominant model for the rest of our analyses.
We performed a replication study of the OAS3-R381S in patients from the 3rd hospital (KK) during the same epidemic (2001-2003). The C allele showed less frequency in DSS group compared with DV patients without shock similar to previous results (8% versus 17% for C allele frequency and 17% versus 32% for CG+GG frequency). Although, this difference was not statistically significant because of small number of patients, higher significance association was obtained when combining all patients from the 3 hospitals (OR=0.48, 95 CI=0.31-0.75, P=7×10−4, for dominant model of the C allele).
We investigated further whether the effect of OAS3-R381S was depending on type of immune response to the virus. There was no significant association of the polymorphism and DSS in patients with predominant IgM immune response. However, significant and nearly significant association was obtained in patients with predominant IgG response in each hospital and combined (Chit=19.41, OR=0.27 95 CI=0.14-0.53, P<10-5) (Table VII).
During 2004-2006, we obtained additional patients from previous 2 hospitals (RA and KK, Table IV). Association study of OAS3-R381S did not reveal a significant association with all hypotheses studied, however, significant association remained when these patients were added to the original case/control study (OR=0.48, 95 CI=0.31-0.75, P=7×10−4). This result prompted us to investigate the discordance of the results between the 2 periods.
One factor that could contribute to severity of dengue disease is a viral factor. We investigated prevalence of DV-4 serotypes during the 2001-2006 using data of patients from Siriraj Hospital and Khonkaen hospital of which we have data from 471 patients (Table VIII).
DV1 and DV2 serotypes were predominant during 2001-2003 while in 2004 DV1 decreased while epidemic of DV4 started in 2004 and was, then, predominant until 2006 (Table 5A). Severity of dengue disease was significantly different among different serotypes of the virus (P=0.012, Table 5B) with DV2 serotype having more severe cases than the other DV serotypes. Surprisingly, serotypes of the viruses were significantly different according to the type of immune response (Fisher exact P<10-4, Table 5C) with DV2 and DV4 showing with the predominant IgG. These results were similar to data from Ministry of Public Health. We hypothesized that the effect of OAS3-R381S could be specific to some viral serotypes. We found significant dominant protective effect of OAS3-R381S against DSS only with DV2 (OR=0.24, 95 CI=0.08-0.74, P=0.0045, Table IX).
Based on a systematic screening of OAS genes in DF/DHF/DSS patients and controls, we have shown association between polymorphisms of the OAS3 gene and the risk of shock (DSS), and the severity of the disease in patients with dengue virus infection. The variant (OAS3-R381S: rs2285933) is a G to C substitution 3rd nucleotide of codon 381 of OAS3, that changes amino acid from arginine to serine. The C variant is associated with dominant protection against DSS comparing with non-shock dengue disease, with frequency ratio of 0.13:0.32 (OR=0.48, 95 CI=0.31-0.75, P=7×104). Furthermore, significant association was found exclusively in patients with predominant IgG response (OR=0.46, 95 CI=0.28-0.74, P=8×104) and in patients infected by DV serotype 2 (OR=0.24, 95 CI=0.08-0.74, P=0.0045).
These results support a functional role of the non-synonymous polymorphism OAS3-R381S and demonstrate a role for OAS3 in the dengue pathogenesis, and interaction between a variant of OAS3 and serotype of the virus.
Our finding may have important consequences for the prediction of disease severity in DV infected patients, and for the development of OAS3-based prophylaxis and therapy against infections by Dengue virus and other positive-sense single-stranded RNA viruses of major medical importance.
| Number | Date | Country | Kind |
|---|---|---|---|
| 08290470.7 | May 2008 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB2009/005956 | 5/20/2009 | WO | 00 | 2/11/2011 |
