WO2016209805A1 - Compositions and methods for modulating viral infection - Google Patents
Compositions and methods for modulating viral infection Download PDFInfo
- Publication number
- WO2016209805A1 WO2016209805A1 PCT/US2016/038501 US2016038501W WO2016209805A1 WO 2016209805 A1 WO2016209805 A1 WO 2016209805A1 US 2016038501 W US2016038501 W US 2016038501W WO 2016209805 A1 WO2016209805 A1 WO 2016209805A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- amino acid
- virus
- seq
- mutant
- protein
- Prior art date
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5254—Virus avirulent or attenuated
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/24011—Flaviviridae
- C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
- C12N2770/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- Flavivirus is a genus of viruses that include West Nile virus, dengue virus, yellow fever virus, Zika virus, and several others. Most of the viruses are transmitted by the bite from an infected arthropod (e.g., mosquito, tick) and cause widespread morbidity and mortality throughout the world. Generally, no specific treatment is available for a flavivirus infection. Current treatments usually involve hospitalization, intravenous fluids, respiratory support, and prevention of secondary infections. Typically, no vaccines against a flavivirus infection exist.
- arthropod e.g., mosquito, tick
- Dengue virus infects approximately 390 million people annually, and 2.5 billion people live in areas at risk for dengue transmission, making DV the most prevalent arthropod-borne viral pathogen.
- DV infection can lead to a debilitating febrile disease known as dengue fever, or the more severe and potentially lethal dengue hemorrhagic fever/dengue shock syndrome (DFIF/DSS).
- DFIF/DSS dengue hemorrhagic fever/dengue shock syndrome
- serotypes of DV exist and infection by one serotype only confers long-lasting immunity to that particular serotype.
- Currently, there are no FDA-approved DV vaccines. Vaccination against only one serotype can lead to DFIF/DSS when an individual is subsequently infected with a different serotype due to antibody-dependent enhancement.
- a mutant flavivirus e.g., Dengue virus, West Nile virus, Zika virus
- a mutant flavivirus comprising a mutated NS3 protein.
- the mutated NS3 protein is deficient in 14-3-3 ⁇ binding.
- a virus comprising the mutated NS3 protein elicits an augmented innate immune response compared to a virus comprising a wild-type NS3 protein.
- a virus comprising the mutated NS3 protein produces a stronger
- a virus comprising the mutant NS3protein induces higher levels of interferon, interferon-stimulated genes, and/or proinflammatory cytokines compared to a virus comprising a wild-type NS3 protein.
- a mutated NS3 protein or mutant virus comprising the mutant protein has a reduced ability to inhibit the translocation of RIG-I to mitochondria/ mitochondrial -associated membranes, and RIG-I-dependent signaling compared to a wild-type NS3 protein or a wild-type virus.
- a virus comprising the mutated NS3protein elicits a stronger adaptive immune response in primary cells compared to a virus comprising a wild-type NS3 protein.
- the mutated NS3 protein comprises a mutation between amino acid 63 and amino acid 67 relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:3 .
- the mutation is an amino acid substitution, insertion, deletion, or combination thereof.
- the mutation comprises a substitution of at least one amino acid between amino acid 63 and amino acid 67 relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 with a different amino acid.
- the mutation comprises a substitution of at least one amino acid between amino acid 63 and amino acid 67 relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 , with lysine, e.g., substituting the amino acid at position 64 or 66, or both.
- the mutation comprises a substitution of the amino acids at positions 64 through 66 relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:3 with the amino acid sequence lysine-isoleucine-lysine.
- the mutant flavivirus is a dengue virus, a West Nile virus, or a Zika virus. In certain embodiments, the mutant flavivirus is a dengue virus serotype 1, a dengue virus serotype 2, a dengue virus serotype 3, or a dengue virus serotype 4, preferably a dengue virus serotype 2.
- a pharmaceutical composition comprising a mutant virus disclosed herein, and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition further comprises an adjuvant.
- a dengue virus vaccine comprising a mutant dengue virus disclosed herein.
- the dengue virus is a live virus.
- the vaccine further comprises an adjuvant.
- a mutated NS3 protein comprising a mutation between amino acid 63 and amino acid 67 relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:3.
- the mutation is an amino acid substitution, insertion, deletion, or combination thereof.
- the mutation is a substitution of at least one amino acid between amino acid 63 and amino acid 67 relative to the amino acid sequence of SEQ ID NO: 1, , SEQ ID NO: 2, or SEQ ID NO: 3 with a different amino acid.
- the mutation is a substitution of at least one amino acid between amino acid 63 and amino acid 67 relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 , with lysine, e.g., substituting the amino acid at position 64 or 66, or both.
- the mutation corresponds to substituting the amino acids at positions 64 through 66 relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:3 with the amino acid sequence lysine-isoleucine-lysine.
- the protein comprises an amino acid sequence of SEQ ID NO: 4, SEQ ID NO:5, or SEQ ID NO:6.
- a virus comprising a NS3 protein disclosed herein.
- nucleic acid encoding a protein described herein.
- the nucleic acid has a sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO 9.
- a virus comprising a nucleic acid disclosed herein.
- a vector or an expression vector comprising the nucleic acid.
- a cell e.g., a host cell
- a method for producing a protein is provided herein.
- the method includes culturing a cell comprising a nucleic acid disclosed herein under conditions suitable for expression of the protein. In some embodiments, the method includes isolating the protein from the cell(s) or from the medium in which the cell(s) is cultured. In some embodiments, the method further comprises isolating the protein.
- provided herein is a method for inducing in a subject an immune response against a flavivirus comprising administering to the subject a composition comprising a mutant viruses disclosed herein.
- provided herein is a method for protecting a subject from a flavivirus, comprising administering to the subject a mutant viruses disclosed herein.
- a method of treating a viral infection comprising administering a mutant virus disclosed herein, a mutant NS3 protein disclosed herein, a pharmaceutical composition disclosed herein, and/or a vaccine disclosed herein, to a subject (e.g., a subject in need thereof).
- a subject e.g., a subject in need thereof.
- the subject is human.
- the subject is exposed to dengue virus.
- the subject is exposed to a mosquito comprising the dengue virus.
- the subject was exposed to dengue virus or a mosquito, within the last 6 month, within the last month, within the last two weeks, within the last week, within the last 72 hours, within the last 48 hours, within the last 24 hours, within the last 12 hours, within the last 6 hours, within the last 4 hours, within the last 2 hours, or within the last hour.
- the subject does not have, but is at risk of developing a flavivirus infection.
- the subject is traveling to a region where a flavivirus is prevalent.
- the region is located in the United States, Argentina, Australia, Bangladesh, Barbados, Cambodia, Caribbean, Costa Sweden, Cuba, Dominican Republic, French Polynesia, Guadeloupe, El Salvador, Grenada, Guatemala, Guyana, Haiti, Honduras, India, Indonesia, Jamaica, Laos, Malaysia, Melanesia, Mexico, Micronesia, Portugal, Pakistan, Panama, Paraguay, The Philippines, Puerto Rico, Samoa, Western Saudi Arabia, Singapore, Sri Lanka, Suriname, Taiwan, Thailand, Trinidad and Tobago, Venezuela, Vietnam and/or China.
- Figure 1 includes 8 panels (Panels A-H), which show that the NS3 protein of DV interacts with 14-3-3 ⁇ .
- Panel A is the amino acid sequence of 14-3-3 ⁇ and specific peptides identified by MS upon affinity purification of FLAG-NS3-Pro (DV2, strain NGC) from HEK293T cells. Peptide coverage was -64.3%. Numbers indicate amino acids.
- Panel B is an image of an immunoblot showing HEK293T cells transfected with c-myc-tagged 14-3- 3 ⁇ and FLAG-tagged NS3-Pro or NS3-Hel.
- WCLs were subjected to FLAG- pulldown (FLAG-PD), followed by immunoblot (IB) with anti-c-myc and anti-FLAG.
- Panel C is an image of an immunoblot showing binding of FLAG- 14-3 -3 ⁇ and GST, GST- NS5, or GST-NS3, assessed in 293T cells by GST-PD and IB with anti-FLAG and anti- GST antibodies.
- Panel D is an image of an immunoblot showing HEK293T cells transfected with HA-tagged 14-3-3 ⁇ or 14-3-3 ⁇ together with GST or GST-NS3. WCLs were subjected to GST-PD, followed by IB with anti-HA and anti-GST.
- Panel E is an image of an immunoblot showing binding of endogenous 14-3 -3 ⁇ and GST, or GST-NS3 of DV2 (NGC), YFV (strain 17D), or HCV (strain Conl) in transfected 293T cells, assessed by GST-PD and IB with anti-14-3-38 antibody.
- Panel F is an image of an immunoblot showing Huh7 cells mock-infected or infected with DV2 NGC (MOI 1) for 28 h. WCLs subjected to immunoprecipitation (IP) with anti-14-3-38 antibody, followed by IB with anti- NS3 and anti-14-3-38.
- IP immunoprecipitation
- Panel G is an image showing Huh7 cells were transfected with FLAG- 14-3 -3 ⁇ and subsequently mock-infected, or infected with DV2 (NGC) at MOI 1 for 24 h. Cells were stained for FLAG (14-3-3 ⁇ ;), NS3 and NS4A and imaged by confocal microscopy. Nuclei were stained with DAPI.
- Panel H is an image of an immunoblot showing in vitro binding of recombinant 14-3-3 ⁇ and purified GST or GST-NS3 determined by IB with anti-14-3-38 antibody.
- Figure. 2 includes 3 panels (Panels A-C), which show that 14-3-3 ⁇ is critical for controlling DV replication.
- Panel B is a bar graph showing Huh7 cells were transfected with vector or c-myc-tagged 14-3-3 ⁇ and subsequently infected with the indicated DV serotypes (MOI 0.05), or HSV-1 (MOI 0.2).
- Figure 3 includes 5 panels (Panels A-E), which show that NS2B/3 inhibits RIG-I activation independent of proteolytic activity.
- Panel A is a bar graph depicting IFN- ⁇ luciferase activity in 293T cells transfected with GST or GST-RIG-I 2CARD together with vector, DV NS2B/3 WT or S135A, or HCV NS3/4A WT or S139A, normalized to constitutive pGK-P-gal.
- Panel C is a bar graph depicting 293T cells, that had been transfected with vector, DV NS2B/3 WT, NS2B/3 S135A, or NS3, subsequently infected with SeV (50 HAU/ml) for 18 h.
- Panel A Luciferase and ⁇ -gal activities were determined as in Panel A.
- Panel D is an image of an immunoblot showing GST or GST-NS3 was transfected into HEK293T cells. 48 h later, cells were infected with SeV (50 HAU/ml) for 16 h. WCLs were subjected to native PAGE, followed by immunoblot with anti-IRF3 antibody. WCLs were further used for SDS- PAGE, followed by IB with the indicated antibodies.
- Panel E is an image of an immunoblot showing GST or GST-NS3 transfected into HEK293T cells similar to Panel D, except WCLs were analyzed by IB 22 h after SeV infection with the indicated antibodies.
- Figure 4 includes 5 panels (Panels A-E), which shows that NS3 inhibits binding of RIG-I to 14-3-3 ⁇ , preventing the translocation of activated RIG-I to mitochondria/MAMs.
- Panel A is a an image of an immunoblot showing HEK293T cells were transfected with empty vector or RIG-I-FLAG together with GST or GST-NS3. 48 h later, cells were infected with SeV (50 HAU/ml) for 19 h. WCLs were subjected to FLAG-PD, followed by IB with anti-ubiquitin (Ub) and anti-FLAG antibodies.
- Panel B is an image of an immunoblot showing Huh7 cells were mock infected, or infected with DV2 NGC (MOI 1) or SeV (50 HAU/ml) for 18 h. WCLs were subjected to IP with anti-RIG-I, followed by IB with anti-Ub and anti-RIG-I.
- Panel C is an image of an immunoblot showing HEK293T cells were transfected with GST or GST-NS3. 48 h later, cells were infected with SeV (50 HAU/ml) for 23 h.
- WCLs were subjected to IP with anti-RIG-I (left), or anti-TREVI25 (right), followed by IB with anti-14-3-38, anti-TRIM25 and anti-RIG-I antibodies.
- Panel D is an immunoblot showing Huh7 cells were mock-infected, or infected with DV2 NGC (MOI 1) or SeV (50 HAU/ml) for 18 h.
- WCLs were subjected to IP with anti-RIG-I, followed by IB with anti-14-3-38, anti-TRIM25 and anti-RIG-I.
- Panel E is an immunoblot showing Huh7 cells were mock infected, or infected with DV2 NGC (MOI 1) or SeV (50 HAU/ml) for 22 h.
- WCLs were subjected to cytosol/mitochondria fractionation, followed by IB with anti-RIG-I, anti-MAVS and anti-GAPDH antibodies. Expression of RIG-I and NS3 was further determined in the WCL.
- Figure 5 includes 7 panels (Panels A-G), which show that NS3 binds to 14-3-38 using a phosphomimetic RxEP motif.
- Panel A is an image of an immunoblot showing 293T cells were transfected with the indicated GST-fused NS3 truncation constructs. 48 h later, WCLs were subjected to GST-PD, followed by IB with anti-14-3-38 and anti-GST antibodies.
- Panel B are sequence alignments of amino acid sequence of the NS3 region harboring the 14-3-3 binding motif (green) from DV (serotypes 1-4), and WNV, YFV and HCV.
- Panel C (left) is a ribbon representation of the crystal structure of DV4 NS3 protein.
- the protease domain is shown, as well as the linker and helicase domain.
- the RLEP motif (arrow) is illustrated.
- Panel C (right) is a close up view of the RLEP motif.
- Panel D is a bar graph showing bioinformatics analysis of 3280 known DV NS3 protein sequences. The most common residue for each position is shown as the consensus sequence (bottom) and is represented in blue in the bar graph. Polymorphisms for each position are represented by different colors. One polymorphism for E 66 or P 67 each was identified, as indicated in parenthesis.
- Panels E and F are immunoblots showing 293T cells transfected with GST, GST-NS3, or the indicated GST-NS3 mutants.
- Panel G is a bar graph showing HEK293T cells transfected with GST, GST-NS3 WT, or GST-NS3 KIKP .
- cells were infected with SeV (50 HAU/ml).
- cells were harvested for WCLs or subjected to mitochondria fractionation assay, followed by immunoblot analysis.
- Figure 6 includes 5 panels (Panels A-E), which show a recombinant DV virus encoding a NS3 KIKP mutant protein is attenuated in replication and elicits an enhanced antiviral immune response.
- Panel D is an image of an immunoblot showing Huh7 cells were mock infected, or infected with DV2 WT or DV2 KIKP at MOI 0.8. 20 h later, cells were harvested for WCLs, or subjected to mitochondria fractionation, followed by immunoblot analysis.
- Figure 7 includes 3 panels (Panels A-C), which show that DV KIKP is defective in antagonizing the innate immune response in primary human monocytes.
- Figure 8 includes 2 panels (Panels A and B).
- Panel A is an image of an immunoblot showing NS2B/3 does not cleave RIG-I, TRFM25 or 14-3-3 ⁇ .
- HEK293T cells transfected with TRIM25-FLAG, RIG-I-FLAG, FLAG-14-3-3 ⁇ or HA-STING together with empty vector or HA-tagged NS2B/3. 48 h later, WCLs were subjected to IB analysis with anti-HA and anti-FLAG antibodies.
- Panel A is an image of an immunoblot showing WT NS2B/3, but not the NS2B/3S135A mutant or NS3 expressed alone, is catalytically active.
- HEK293T cells were transfected with HA-tagged NS2B/3, NS2B/3S135A, or NS3, together with STING-HA.
- WCLs were analyzed by IB with anti-HA and anti-actin antibodies.
- Figure 9 includes 2 panels (Panels A and B).
- Panel A is an immunoblot showing RIG-I, but not TRIM25, directly binds to 14-3-3 ⁇ .
- In vitro binding assay was performed by incubating purified TRIM25-FLAG or RIG-I-FLAG with bacterially purified recombinant (r) human 14-3-3 ⁇ . Binding was determined by IB with anti-14-3-3e and anti-FLAG antibodies.
- Panel B is an immunoblot showing Ectopic expression of DV NS3 inhibits virus-induced relocalization of endogenous RIG-I from the cytosol to mitochondria.
- HEK293T cells were transfected with GST or GST-NS3. 48 h later, cells were infected with SeV (50 HAU/ml) for 20 h, followed by mitochondria fractionation assay and IB analysis.
- Figure 10 is a schematic representation of the domain structure of NS3 as well as GST-fused NS3 truncation mutants. 64RxEP67 motif and an overview of the results from the 14-3-3 ⁇ binding studies are also indicated. Numbers indicate amino acids.
- Figure 11 includes 3 panels (Panels A-C), which show the NS3KTKP mutant protein is impaired in inhibiting the RIG-I-14-3-3e interaction and RIG-I translocation to mitochondria.
- Panel A is an image of an immunoblot showing GST, GST-NS3 WT, or GST-NS3KIKP was transfected into HEK293T cells. 48 h later, cells were infected with SeV (50 HAU/ml) for 22 h. ISG (ISG54 or RIG-I) protein expression in the WCLs was determined by IB with the indicated antibodies.
- Panel B is an image of an immunoblot showing HEK293T cells were transfected with GST, GST-NS3 WT, or GST-NS3KIKP.
- Panel C is an image of an immunoblot showing HEK293T cells transfected with GST, GST-NS3 WT, or GST-NS3KIKP.
- SeV 50 HAU/ml
- cells were harvested for WCLs or subjected to mitochondria fractionation assay, followed by IB analysis.
- Figure 12 includes 5 panels (Panels A-E).
- Panel A is an image of an immunoblot showing the NS2B/3KIKP mutant protein is catalytically active.
- HEK293T cells were transfected with HA-tagged STING together with empty vector, or HA-tagged NS2B/3, NS2B/3S135A, or NS2B/3KIKP. 48 h later, WCLs were analyzed by IB with anti-HA and anti-actin antibodies.
- Panels B and C are bar graphs showing replication of DV2 WT and DV2 KIKP in Vero cells. Vero cells were infected with DV2 WT or DV2 KIKP at an MOI of 0.02.
- E DV2 K IKP, but not DVWT, strongly induces ISG protein expression.
- A549 cells were infected with DV2 WT or DV2KIKP (both MOI 0.2) for 24 h and subjected to immunofluorescence staining of endogenous ISG54 or RIG-I, NS3 and DAPI .
- Figure 13 includes two panels (Panels A and B).
- Panel B is a bar graph showing enhanced IFN- ⁇ secretion in T cells co-cultured with DV2 K iKP-infected moDCs.
- Figure 14 shows the amino acid sequence of the NS3 region harboring the 14-3-3- binding motif from DV (serotypes 1-4), West Nile Virus (WNV), Yellow Fever Virus (YFV) and HCV.
- DV serotypes 1-4
- WNV West Nile Virus
- YFV Yellow Fever Virus
- Figure 15 is an immunoblot showing binding of GST, GST NS3 of DV (DV2 NGC), YFV (17D), or WNV (NY99 or kunjin) in transfected HEK293T cells, assessed by GST-PD and IB with anti-14-3-3e antibody.
- Figure 16 shows IFN- luciferase activity in HEK293T cells transfected with GST- RIG-I 2 CARD together with GST, or GST-NS3 of DV (DV2 NGC) or WNV (NY99 or Kunjin), normalized to constitutive pGK-b-gal.
- Figure 17 is an immunoblot showing binding of GST, WNV GST-NS3 W T (NY99) or its mutant (GST-NS3 K IKP) and endogenous 14-3-3 ⁇ in transfected HEK293T cells by GST-PD an IB with an anti-14-3-3e antibody.
- kits for the treatment and/or prevention of flavivirus infection such as DV infection, WNV infection, or a Zika virus (ZV) infection.
- flavivirus infection such as DV infection, WNV infection, or a Zika virus (ZV) infection.
- proteins e.g., variant polypeptides and fragments thereof
- nucleic acids encoding the proteins
- methods for the production of proteins and methods for the use of viruses comprising such proteins in various applications, such as methods for treating and/or vaccinating against a number of conditions including, but not limited to, flavivirus infections such as dengue virus.
- flavivirus infections such as dengue virus.
- Flavivirus is a genus of viruses that includes, but is not limited to, Absettarov virus, Alkhurma virus (ALKV), Deer tick virus (DT), Gadgets Gully virus (GGYV), Kadam virus (KADV), Karshi virus, Kyasanur Forest disease virus (KFDV), Langat virus (LGTV), Louping ill virus (LIV), Mogiana tick virus (MGTV), Ngoye virus (NGOV), Omsk hemorrhagic fever virus (OFIFV), Powassan virus (POWV), Royal Farm virus (RFV), Sokuluk virus (SOKV), Tick-borne encephalitis virus (TBEV), Turkish sheep encephalitis virus (TSE), Kama virus (KAMV), Meaban virus (MEAV), Saumarez Reef virus (SREV), Tyuleniy virus (TYUV), Aedes flavivirus, Barkedji virus, Calbertado virus, Cell fusing agent virus, Chaoyang virus, Culex flavivirus,
- the NS3 protein of DV antagonizes the RIG-I (retinoic acid- inducible gene-I)-mediated IFN response through a proteolysis-independent mechanism. While the disclosure is not limited by any particular theory or mechanism of action, NS3 binds to the trafficking molecule 14-3 -3 ⁇ , blocking the translocation of RIG-I to mitochondria/MAMs and thereby inhibiting antiviral signal transduction. NS3 binds to 14- 3 -3 ⁇ using a highly conserved four-amino-acid sequence that mimics a canonical phospho- serine/threonine (pS/pT) motif found in cellular interaction partners of 14-3-3 proteins. Thus, a recombinant DV encoding a mutant NS3 protein deficient in 14-3 -3 ⁇ binding reduces it' s the ability to antagonize RIG-I and elicits an augmented innate immune response.
- RIG-I retinoic acid- inducible gene-I
- a mutant dengue virus comprising a mutated NS3 protein, wherein the mutated NS3 protein is deficient in 14-3-3 ⁇ binding.
- a mutated NS3 protein produces a stronger inflammatory response in a subject.
- a mutated NS3 protein or mutant virus comprising the mutant protein induces higher levels of interferon, interferon -stimulated genes, and proinflammatory cytokines.
- a mutated NS3 protein or mutant virus comprising the mutant protein has a reduced ability to inhibit the translocation of RIG-I to mitochondria/ mitochondrial-associated membranes, and RIG-I-dependent signaling.
- mutant flavivirus NS3 proteins are mutant flavivirus NS3 proteins.
- the protein is a variant of the NS3 protein expressed by a dengue virus.
- An exemplary amino acid sequence for a wild type NS3 protein from dengue virus serotype 2 is as follows (SEQ ID NO: 1):
- RVIDPRRCMK PVILTDGEER VILAGPMPVT HSSAAQRRGR IGRNPKNEND QYIYMGEPLE 481 NDEDCAHWKE AKMLLDNINT PEGIIPSMFE PEREKVDAID GEYRLRGEAR TTFVDLMRRG 541 DLPVWLAYRV AAEGINYADR RWCFDGVKNN QILEENVEVE IWTKEGERKK LKPRWLDARI 601 YSDPLALKEF KEFAAGRK
- the protein is a variant of the NS3 protein expressed by a West Nile virus.
- An exemplary amino acid sequence for a wild type NS3 protein for a West Nile Virus is as follows (SEQ ID NO: 2):
- the protein is a variant of the NS3 protein expressed by a Zika virus.
- An exemplary amino acid sequence for a wild type NS3 protein for a Zika Virus is as follows (SEQ ID NO: 3):
- the variant NS3 protein is deficient in 14-3-3 ⁇ binding.
- a virus comprising the variant NS3 protein produces a stronger inflammatory response in a subject than a wild-type virus.
- a virus comprising the variant NS3 protein induces higher levels of interferon, interferon - stimulated genes, and/or proinflammatory cytokines than a wild-type virus.
- a variant NS3 protein has a reduced ability to inhibit the translocation of RIG-I to mitochondria/ mitochondrial-associated membranes, and RIG-I-dependent signaling than wild-type NS3 protein.
- a virus comprising the variant NS3protein elicits a stronger adaptive immune response in primary cells than a wild-type virus.
- variant proteins described herein comprise one or more amino acid
- a variant protein comprises at least one (e.g., at least two, three, four, five, six, seven, eight, nine, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more than 100) amino acid substitutions, deletions, or insertions, relative to the wild-type, full-length NS3 protein from which it was derived.
- a variant protein comprises no more than 150 (e.g., no more than 145, 140, 135, 130, 125, 120, 1 15, 1 10, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 1 1, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitution(s), deletion(s), or insertion(s), relative to the wild-type, full- length NS3 protein from which it was derived.
- 150 e.g., no more than 145, 140, 135, 130, 125, 120, 1 15, 1 10, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 1 1, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s), deletion(s), or insertion(s), relative to the wild-type, full- length NS3 protein from which it
- Polypeptide “peptide,” and “protein” are used interchangeably and mean any peptide-linked chain of amino acids, regardless of length or post-translational modification.
- a variant protein described herein, or a fragment thereof includes an amino acid substitution between amino acid position 30 and amino acid position 90 relative to SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; an amino acid substitution between amino acid position 40 and amino acid position 80 relative to SEQ ID NO: l, SEQ ID NO:2, or SEQ ID NO:3; an amino acid substitution between amino acid position 50 and amino acid position 80 relative to SEQ ID NO: l, SEQ ID NO:2, or SEQ ID NO:3; an amino acid substitution between amino acid position 50 and amino acid position 75 relative to SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; an amino acid substitution between amino acid position 55 and amino acid position 75 relative to SEQ ID NO: l, SEQ ID NO:2, or SEQ ID NO:3; an amino acid substitution between amino acid position 60 and amino acid position 75 relative to SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; an amino acid substitution between amino acid position 60 and amino acid position 60 relative to SEQ
- a variant protein described herein includes a substitution of at least one amino acid between amino acid 63 and amino acid 67 relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3 with a different amino acid e.g., a substitution of an amino acid at position 64 or 66 with lysine.
- the amino acids at position 64 and 66, relative to SEQ ID NO: 1 are two of several amino acids (RxEP) highly conserved among dengue virus NS3 proteins ( Figure 6, Panel B). However, the exact position of these amino acid residues in a given polypeptide varies from species to species and with truncations or extension of the naturally-occurring sequence. In some
- a variant protein includes a substitution of the amino acids at position 64 through 66 with the sequence lysine-isoleucine-lysine.
- the variant protein described herein comprises a substitution at position 64 having the following amino acid sequence (SEQ ID NO: 4):
- the variant protein described herein comprises a substitution at position 66 having the following amino acid sequence (SEQ ID NO: 5):
- the variant protein described herein comprises a substitution at position 64and position 66 having the following amino acid sequence (SEQ ID NO: 6):
- the term "conservative substitution” refers to the replacement of an amino acid present in the native sequence in a given polypeptide with a naturally or non- naturally occurring amino acid having similar steric properties. Where the side-chain of the native amino acid to be replaced is either polar or hydrophobic, the conservative substitution should be with a naturally occurring amino acid, a non-naturally occurring amino acid that is also polar or hydrophobic, and, optionally, with the same or similar steric properties as the side-chain of the replaced amino acid.
- Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; lysine, histidine and arginine; and phenylalanine and tyrosine.
- A alanine
- R arginine
- N asparagine
- D aspartic acid
- C cysteine
- G glycine
- Q glutamic acid
- E glutamic acid
- H histidine
- I isoleucine
- L leucine
- K methionine
- M phenylalanine
- F proline
- S serine
- non-conservative substitutions refers to replacement of the amino acid as present in the parent sequence by another naturally or non-naturally occurring amino acid, having different electrochemical and/or steric properties.
- the side chain of the substituting amino acid can be significantly larger (or smaller) than the side chain of the native amino acid being substituted and/or can have functional groups with significantly different electronic properties than the amino acid being substituted.
- a variant protein described herein, or a fragment thereof has an amino acid sequence that is at least 80 (e.g., at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99) % identical to: (i) the amino acid of SEQ ID NO:2; (ii) the amino acid of SEQ ID NO:3; or (iii) the amino acid of SEQ ID NO:4with the proviso that the variant protein or fragment thereof comprises an amino acid substitution at position 64, an amino acid substitution at position 66, or combinations thereof.
- a variant protein described herein, or a fragment thereof has an amino acid sequence that is at least 80 (e.g., at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99) % identical to: (i) the amino acid of SEQ ID NO:4; (ii) the amino acid of SEQ ID NO:5; or (iii) the amino acid of SEQ ID NO:6.
- Percent amino acid sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical to the amino acids in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software, such as BLAST software or ClustalW2. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.
- the proteins disclosed herein can be produced using any appropriate technique in the art.
- a nucleic acid encoding a fusion protein can be inserted into an expression vector that contains transcriptional and translational regulatory sequences, which include, e.g., promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, transcription terminator signals, polyadenylation signals, and enhancer or activator sequences.
- the regulatory sequences include a promoter and transcriptional start and stop sequences.
- the expression vector can include more than one replication system such that it can be maintained in two different organisms, for example in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification.
- telomere sequences can be selected by simultaneously introducing drug resistance genes such as E. coli gpt (Mulligan and Berg (1981) Proc Natl Acad Sci USA 78:2072) or Tn5 neo (Southern and Berg (1982) Mol Appl Genet 1:327).
- the selectable marker gene can be either linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection (Wigler et al. (1979) Cell 16:77).
- a second class of vectors utilizes DNA elements which confer autonomously replicating capabilities to an extrachromosomal plasmid.
- These vectors can be derived from animal viruses, such as bovine papillomavirus (Sarver et al. (1982) Proc Natl Acad Sci USA, 79:7147), cytomegalovirus, polyoma virus (Deans et al. (1984) Proc Natl Acad Sci USA 81: 1292), or SV40 virus (Lusky and Botchan ( ⁇ 9 ) Nature 293 :79).
- the expression vectors can be introduced into cells in a manner suitable for subsequent expression of the nucleic acid.
- the method of introduction is largely dictated by the targeted cell type, discussed below.
- Exemplary methods include CaP0 4 precipitation, liposome fusion, cationic liposomes, electroporation, viral infection, dextran-mediated transfection, polybrene-mediated transfection, protoplast fusion, and direct microinjection.
- Appropriate host cells for the expression of recombinant proteins include yeast, bacteria, insect, plant, and mammalian cells (e.g., rodent cell lines, such as Chinese Hamster Ovary (CHO) cells).
- mammalian cells e.g., rodent cell lines, such as Chinese Hamster Ovary (CHO) cells.
- bacteria such as E. coli, fungi such as Saccharomyces cerevisiae and Pichia pastoris, insect cells such as SF9, mammalian cell lines (e.g., human cell lines), as well as primary cell lines.
- a protein can be produced from the cells by culturing a host cell transformed with the expression vector containing nucleic acid encoding the polypeptide, under conditions, and for an amount of time, sufficient to allow expression of the proteins.
- Such conditions for protein expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation.
- proteins expressed in E. coli can be refolded from inclusion bodies (see, e.g., Hou et al. (1998) Cytokine 10:319-30).
- a fusion protein described herein can be expressed in mammalian cells or in other expression systems including but not limited to yeast, baculovirus, and in vitro expression systems (see, e.g., Kaszubska et al. (2000) Protein Expression and Purification 18:213-220).
- the recombinant proteins can be isolated.
- the term "purified” or “isolated” as applied to any of the proteins described herein refers to a polypeptide that has been separated or purified from components ⁇ e.g., proteins or other naturally-occurring biological or organic molecules) which naturally accompany it, e.g., other proteins, lipids, and nucleic acid in a prokaryotic or eukaryotic cell expressing the proteins.
- a polypeptide is purified when it constitutes at least 60 ⁇ e.g., at least 65, 70, 75, 80, 85, 90, 92, 95, 97, or 99) %, by weight, of the total protein in a sample.
- the recombinant proteins can be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample.
- Standard purification methods include electrophoretic, molecular, immunological, and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse- phase HPLC chromatography.
- an antibody can be purified using a standard anti-antibody column ⁇ e.g., a protein-A or protein-G column).
- Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. See, e.g., Scopes (1994) "Protein Purification, 3 rd edition," Springer- Verlag, New York City, New York. The degree of purification necessary will vary depending on the desired use. In some instances, no purification of the expressed proteins will be necessary.
- Methods for determining the yield or purity of a purified protein include, e.g., Bradford assay, UV spectroscopy, Biuret protein assay, Lowry protein assay, amido black protein assay, high pressure liquid chromatography (EfPLC), mass spectrometry (MS), and gel electrophoretic methods ⁇ e.g., using a protein stain such as Coomassie Blue or colloidal silver stain).
- the expression of a protein ⁇ e.g., a mutant NS3 protein disclosed herein can also be determined by detecting and/or measuring expression of a protein.
- Methods of determining protein expression generally involve the use of antibodies specific for the target protein of interest.
- methods of determining protein expression include, but are not limited to, western blot or dot blot analysis, immunohistochemistry ⁇ e.g., quantitative immunohistochemistry), immunocytochemistry, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbent spot (ELISPOT; Coligan et al., eds. (1995) Current Protocols in Immunology. Wiley, New York), or antibody array analysis (see, e.g., U.S. Patent Application Publication Nos.
- the presence or amount of protein expression can be determined using a western blotting technique.
- a lysate can be prepared from a biological sample, or the biological sample itself, can be contacted with Laemmli buffer and subjected to sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE- resolved proteins, separated by size, can then be transferred to a filter membrane (e.g., nitrocellulose) and subjected to immunoblotting techniques using a detectably-labeled antibody specific to the protein of interest. The presence or amount of bound detectably- labeled antibody indicates the presence or amount of protein in the biological sample.
- a filter membrane e.g., nitrocellulose
- an immunoassay can be used for detecting and/or measuring the protein expression of a protein.
- an immunoassay can be performed with an antibody that bears a detection moiety (e.g., a fluorescent agent or enzyme).
- a detection moiety e.g., a fluorescent agent or enzyme.
- Proteins from a biological sample can be conjugated directly to a solid-phase matrix (e.g., a multi-well assay plate, nitrocellulose, agarose, sepharose, encoded particles, or magnetic beads) or it can be conjugated to a first member of a specific binding pair (e.g., biotin or streptavidin) that attaches to a solid-phase matrix upon binding to a second member of the specific binding pair (e.g., streptavidin or biotin).
- a specific binding pair e.g., biotin or streptavidin
- Such attachment to a solid-phase matrix allows the proteins to be purified away from other interfering or irrelevant components of the biological sample prior to contact with the detection antibody and also allows for subsequent washing of unbound antibody.
- the presence or amount of bound detectably-labeled antibody indicates the presence or amount of protein in the biological sample.
- Methods for generating antibodies or antibody fragments specific for a protein can be generated by immunization, e.g., using an animal, or by in vitro methods such as phage display.
- a polypeptide that includes all or part of a target protein can be used to generate an antibody or antibody fragment.
- the antibody can be a monoclonal antibody or a preparation of polyclonal antibodies.
- Methods for detecting or measuring gene expression can optionally be performed in formats that allow for rapid preparation, processing, and analysis of multiple samples. This can be, for example, in multi -welled assay plates (e.g., 96 wells or 386 wells) or arrays (e.g., nucleic acid chips or protein chips).
- Stock solutions for various reagents can be provided manually or robotically, and subsequent sample preparation (e.g., RT-PCR, labeling, or cell fixation), pipetting, diluting, mixing, distribution, washing, incubating (e.g., hybridization), sample readout, data collection (optical data) and/or analysis (computer aided image analysis) can be done robotically using commercially available analysis software, robotics, and detection instrumentation capable of detecting the signal generated from the assay. Examples of such detectors include, but are not limited to,
- spectrophotometers e.g., detecting the presence or level of a target protein in a cell
- exemplary high-throughput cell-based assays can utilize ArrayScan® VTI HCS Reader or KineticScan® HCS Reader technology (Cellomics Inc., Pittsburg, PA).
- a recombinant virus comprising a mutant NS3 protein described herein.
- cDNA of a dengue virus comprising a nucleic acid sequence mutations that encode the mutant NS3 protein.
- Suitable cell lines for propagating a recombinant dengue vims include mammalian cells, such as Vero cells, AGMK cells, BHK-21 cells, COS-I or COS-7 cells, MDCK cells, CV-I cells, LLC-MK2 cells, primary cell lines such as fetal Rhesus lung (FRhL-2) cel ls, BSC-I ceils, and MRC-5 cells, or human diploid fibroblasts, as well as avian ceils, chicken or duck embryo derived cell lines, e.g., AGE1 cells, and primary, chicken embryo fibroblasts, and mosquito cell lines, such as C6/36.
- mammalian cells such as Vero cells, AGMK cells, BHK-21 cells, COS-I or COS-7 cells, MDCK cells, CV-I cells, LLC-MK2 cells, primary cell lines such as fetal Rhesus lung (FRhL-2) cel ls, BSC-I ceils, and MRC-5 cells, or human dip
- a recombinant dengue virus is used to infect the host cell (for example, selected from among the suitable cell types listed above). After virus adsorption, the cultures are fed with medium capable of supporting growth of the ceils. The host cells are maintained in culture until the desired vims titer is achieved.
- the vims is harvested by common methods known in the art including slow-speed centrifugation, or by filtration through a filter of pore size of 0.45 ⁇ .
- Methods for concentrating recovered virus are within the scope of a person with ordinary skill in the art and include, for example, ultrafiltration (e.g., with a membrane of no greater than 300 kDa pore size), or precipitation with polyethylene glycol (PEG) 8000.
- Methods for purifying viruses are known to a person with ordinary skill in the art and include continuous or multi-step sucrose gradients, purification by column chromatography using size exclusion, ion exchange, adsorption, or affinity columns, or purification by partitioning in polymer two-phase or multi-phase systems, and any combination thereof.
- Methods for assaying for virus positive fractions include plaque assay, hemagglutination (HA) assay, and/or antigen assays such as immunoassays.
- nucleic acid molecules that encode the mutant NS3 protein described herein.
- the nucleic acids may be present, for example, in whole cells, in a cell lysate, or in a partially purified or substantially pure form.
- the nucleic acid has a sequence of SEQ ID NO: 7:
- Nucleic acid molecules provided herein can be obtained using standard molecular biology techniques. For example, nucleic acid molecules described herein can be cloned using standard PCR techniques or chemically synthesized. For nucleic acids encoding antibodies expressed by hybridomas, cDNAs encoding the light and/or heavy chains of the antibody made by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques.
- vectors that contain the isolated nucleic acid molecules described herein.
- the term "vector,” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
- plasmid which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
- viral vector Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
- Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
- vectors e.g., non-episomal mammalian vectors
- vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby be replicated along with the host genome.
- certain vectors are capable of directing the expression of genes. Such vectors are referred to herein as "recombinant expression vectors" (or simply, “expression vectors”).
- cells that contain a nucleic acid described herein (e.g., a nucleic acid encoding an NS3 protein described herein).
- the cell can be, for example, prokaryotic, eukaryotic, mammalian, avian, murine and/or human.
- the cell is a hybridoma.
- the nucleic acid provided herein is operably linked to a transcription control element such as a promoter.
- the cell transcribes the nucleic acid provided herein and thereby expresses a protein described herein.
- the nucleic acid molecule can be integrated into the genome of the cell or it can be extrachromosomal.
- compositions and/or vaccines comprising a mutant dengue virus described herein.
- the pharmaceutical compositions and/or vaccines described herein include a virus comprising a mutant NS3 protein together with one or more excipients and/or adjuvants.
- the pharmaceutical composition and/or vaccine described herein comprises a mutant flavivirus (e.g., DV, WNV, or ZV) viral genome and/or mutant gene encoding mutant NS3.
- the pharmaceutical composition and/or vaccine can contain genetic material, such as a heterologous gene insert expressing the mutant protein.
- the mutant NS3 can be expressed in cells of a susceptible species immunized with the vaccine containing mutant DV , WNV, or ZV and/or mutant NS3. Immunity against wild type DV, WNV, or ZV can thereby be conferred in a species and/or tissue normally susceptible to a DV, WNV, or ZV infection.
- the mutant NS3 virus has reduced ability to bind to the trafficking molecule 14-3 -3 ⁇ and reduced ability to block of the translocation of RIG-I to mitochondria/MAMs.
- a mutant flavivirus e.g., DV, WNV, or ZV
- encoding a mutant NS3 protein described herein deficient in 14-3-3 ⁇ binding has reduced ability to antagonize RIG-I and elicits an augmented innate immune response.
- the present disclosure affords a pharmaceutical composition and/or vaccine to treat and/or prevent flavivirus infections or other disease states related to or caused by flavivirus infections, e.g., dengue fever, yellow fever, Zika fever, microcephaly.
- the mutant flavivirus is able to induce an immune response in a subject, which results in the treated subject's immune system to fight a wild type flavivirus.
- a pharmaceutical composition and/or vaccine having the mutant flavivirus and/or mutant NS3 is taken by subjects who have been infected by flavivirus to improve an immune response to a wild type flavivirus .
- the pharmaceutical composition and/or vaccine may further comprise an adjuvant that can augment the immune response by increasing delivery of antigen, stimulating cytokine production, and/or stimulating antigen presenting cells.
- the adjuvant can be administered concurrently with the pharmaceutical composition and/or vaccine composition disclosed herein, e.g., in the same composition or in separate compositions.
- an adjuvant can be administered prior or subsequent to the pharmaceutical composition and/or vaccine composition disclosed herein.
- adjuvants include, but are not limited to: aluminum salts, non-toxic bacterial fragments, cholera toxin (and detoxified fractions thereof), chitosan, homologous heat-labile of E.
- coli and detoxified fractions thereof
- lactide/glycolide homo and copolymers PHA/GA
- polyanhydride e.g. trimellitylimido-L-tyrosine, DEAE-dextran
- saponins complexed to membrane protein antigens Immune stimulating complexes— ISCOMS
- bacterial products such as lipopolysaccharide (LPS) and muramyl dipeptide, (MDP), liposomes, cochelates, proteinoids, cytokines (interleukins, interferons), genetically engineered live microbial vectors, non-infectious pertussis mutant toxin, neurimidase/galactose oxidase, and attenuated bacterial and viral toxins derived from mutant strains.
- ISCOMS immune stimulating complexes
- LPS lipopolysaccharide
- MDP muramyl dipeptide
- liposomes cochelates, proteinoids, cytok
- the mutant DV is able to induce an immune response in a subject against one, two, three or all four serotypes of the dengue virus ⁇ e.g., dengue virus serotype 1, dengue virus serotype 2, dengue virus serotype 3, or dengue virus serotype 4) .
- the pharmaceutical composition and/or vaccine may comprise a combination of mutant proteins from two, three or all four serotypes of the dengue virus.
- the mutant DV is a dengue virus serotype 2.
- the pharmaceutical composition , vaccine and/or adjuvant can be administered to a subject, e.g., a human subject, using a variety of methods that depend, in part, on the route of administration.
- the route can be, e.g., intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneal (IP) injection, or
- HVI intramuscular injection
- provided herein is a method for inducing an immune response against a flavivirus in a subject comprising administering to the subject a composition ⁇ e.g., a vaccine composition) disclosed herein.
- a method for protecting a subject from a flavivirus comprising administering to the a composition disclosed herein.
- a method of treating a subject for flavivirus infection comprising administering to the subject a composition disclosed herein.
- a "subject,” as used herein, can be any mammal.
- a subject can be a human, a non-human primate (e.g., monkey, baboon, or chimpanzee), a horse, a cow, a pig, a sheep, a goat, a dog, a cat, a rabbit, a guinea pig, a gerbil, a hamster, a rat, or a mouse.
- the subject is an infant (e.g., a human infant).
- the subject is exposed to a flavivirus due to the subject's exposure to a mosquito comprising the flavivirus.
- the subject may be exposed to a Aedes mosquitoes, particularly A. aegypti which live between the latitudes of 35° North and 35° South below an elevation of 1,000 metres (3,300 ft).
- Aedes mosquitoes particularly A. aegypti which live between the latitudes of 35° North and 35° South below an elevation of 1,000 metres (3,300 ft).
- Such a subject may be at risk of developing a flavivirus infection and disease states related to or caused by such an infection.
- the subject does not have, but is at risk of developing a dengue virus infection.
- a subject "at risk” may or may not have detectable disease, and may or may not have displayed detectable disease prior to the treatment methods described herein.
- At risk denotes that an individual who is determined to be more likely to develop a symptom based on conventional risk assessment methods or has one or more risk factors that correlate with development of a particular condition. An individual having one or more of these risk factors has a higher probability of developing a condition than an individual without these risk factors.
- Examples (i.e., categories) of risk groups are well known in the art and discussed herein, such as those subjects who are traveling to a region of the world where the dengue virus is prevalent. For example, in some embodiments the region is in the United States, Argentina, Australia, Bangladesh, Barbados, Cambodia, Caribbean, Costa Rica, Cuba, Dominican Republic, French Polynesia,
- HEK293T, Huh7, Huh7.5, Vero and A549 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES and 1% penicillin-streptomycin (Gibco).
- DMEM Dulbecco's Modified Eagle's Medium
- FBS fetal bovine serum
- HEPES fetal bovine serum
- penicillin-streptomycin Gibco
- BHK-21 cells were propagated in Minimum Essential Medium Alpha (MEM-oc) supplemented with 10% FBS, 10 mM HEPES and 1% penicillin-streptomycin.
- C6/36 cells were cultured in Eagle's Minimum Essential Medium (EMEM) supplemented with 10% FBS and 1% penicillin-streptomycin, and grown at 28°C.
- EMEM Eagle's Minimum Essential Medium
- K562 cells and primary CD 14 + monocytes were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS, 1% non-essential amino acid solution (Gibco) and 1% penicillin-streptomycin.
- DV2 NGC, DV1 276 RK1, DV2 16681, DV3 BC 188/97 and DV4 814699 were propagated in C6/36 cells.
- SeV (Cantell) was purchased from Charles River Laboratories.
- HSV-1 was a kind gift from David Knipe (Harvard).
- Plasmids and Transfections pQCXIP-NS2B/3-HA and pQCXIP-NS3-HA were generated by subcloning NS2B/3 (containing NS2B and NS3) or NS3 of DV2 (strain NGC) into pQCXIP vector using Notl and BamHI sites.
- GST-NS3 and GST-NS5 were generated by subcloning NS3 or NS5 of DV2 (strain NGC) into pEBG vector between BamHI and Clal.
- NS3 of YF V kindly provided by Richard Kuhn, Purdue University
- NS3 of HCV were subcloned into the pEBG vector.
- pEF-BOS-FLAG-NS3-Pro (aa 1-179), pEF-BOS-FLAG-NS3-Hel (aa 169- 618), pEF-BOS-FLAG-NS5-MTase (aa 1-319) and pEF-BOS-FLAG-NS5-Pol (aa 297-901) were generated by subcloning into pEF-BOS-FLAG vector using Notl and Sail sites.
- 14-3- 3 ⁇ (Uniprot: P62258-1) was purchased as a cDNA clone and subcloned into pEF-BOS and pCAGGS vectors with an N-terminal FLAG and c-myc tag, respectively.
- HA-tagged 14-3- 3 ⁇ was provided by Satoshi Inoue (University of Tokyo) and has been described (Urano et al., Nature 417, 871-875 (2002)).
- pQCXIP-STING-HA was generated by subcloning STING (clone ID 5762441, Thermo Scientific) into pQCXIP vector using Notl and BamHI sites.
- STING clone ID 5762441, Thermo Scientific
- the plasmids encoding the HCV NS3/4A protease complex (pcDNA3-FLAG- NS3/4A) and its S139A catalytically-inactive mutant were a kind gift of Zhijian Chen (Li et al., PNAS 102, 17717-17722 (2005)).
- Plasmids encoding GST-RIG-I(2CARD), RIG-I- FLAG and TRIM25-FLAG have been described previously (Gack et al., Nature 446, 916- 920 (2007); Wies et al., Immunity 38, 437-449 (2013)).
- the DV NS3 truncation mutants GST-NS3Q-92), GST-NS3(93-168), GST-NS3(43-92), GST-NS3(82-168), GST-NS3(63- 168), and GST-NS3(43-168) were generated by PCR using GST-NS3 full-length as template. All constructs were sequenced to verify 100% agreement with the original sequence. Transfections were performed using the calcium phosphate method, or with TurboFectin 8.0 (Origene), Lipofectamine and Plus reagent, or Lipofectamine 2000 (all Life Technologies) according to the manufacturer's instructions.
- SMARTpool M-017302-03 -0005) as well as a non-targeting control siRNA were purchased from Dharmacon.
- K562 cells were seeded into 12-well plates and transfected with 300 nM siRNA using Lipofectamine RNAiMAX (Life Technologies) according to the
- Knockdown of endogenous 14-3-3 ⁇ was determined by western blot analysis.
- Antibodies and Reagents were used: anti-FLAG (M2, Sigma), anti-HA (HA-7, Sigma), anti-GST (Sigma), anti-c-myc (9E10), anti- -actin (Abeam), anti-RIG-I (Alme-1, Adipogen), anti-TRFM25 (BD
- anti-prM (2H2, Merck Millipore) was conjugated to DyLight 633 using a commercial kit (Thermo Scientific) and used to detect DV-infected cells.
- Anti-CD 14-FITC (M5E2, BD Biosciences) was used to determine purity of CD14 + monocytes. Isotype control antibodies were purchased from BD Biosciences.
- Luciferase Reporter Assay FEK293T cells were seeded into 12-well plates. The following day, cells were transfected with 200 ng IFN- ⁇ luciferase construct, 300 ng ⁇ -gal- expressing pGK- -gal, and 100 ng - 1 ⁇ g of plasmid encoding effector protein. To stimulate IFN- ⁇ promoter activity, 2 ng of GST-RIG-I-2CARD was co-transfected, or cells were infected with SeV (50 HAU/ml) 48 hours after transfection. Cells were harvested and assayed for luciferase activity (Promega). Luciferase values were normalized to ⁇ - galactosidase activity to control for transfection efficiency.
- HEK239T cells were transfected with pEF-BOS-FLAG-NS3-Pro, pEF-BOS-FLAG-NS3-Hel, pEF-BOS-FLAG- NS5-MTase or pEF-BOS-FLAG-NS5-Pol. Two days later, cells were lysed with NP-40 buffer supplemented with protease inhibitor cocktail (Sigma). Clarified lysates were mixed with a -50% slurry of anti-FLAG-conjugated sepharose beads (Sigma) and incubated for 4 h at 4°C.
- Huh7 cells were grown on chamber slides or on cover slips in 24-well plates, and then infected with DV2 or SeV at indicated titers, or mock infected. Cells were harvested at indicated time points and fixed with 4% (w/v) paraformaldehyde for 20 min, permeabilized with 0.2% (v/v) Triton-X-100 in PBS, and blocked with 10% (v/v) goat serum or FBS in PBS for 1 h.
- anti-14-3-3e Proteintech
- anti-NS3 GT2811 or GTX 124252, Genetex
- anti-NS4A GTX 124249, Genetex
- anti- ISG54 12604-1-AP, Proteintech
- anti-RIG-I Alme-1, Adipogen
- anti-FLAG Anti-FLAG
- Cells were mounted in DAPI-containing Vectashield (Vector Labs) to co-stain nuclei. All laser scanning images were acquired on an Olympus IX8I confocal microscope.
- NP 006752.1 Bacterially-purified recombinant human 14-3-3 ⁇ protein (NP 006752.1) was purchased from Sino Biological. GST or GST-NS3 (DV2, strain NGC) expressed in HEK293T cells was immobilized on glutathione-conjugated sepharose beads in NP-40 buffer and incubated with recombinant 14-3-3 ⁇ protein (final concentration of 10 ⁇ g/ml) for 2 h at 4°C. After extensive washing with NP-40 buffer, bound proteins were eluted from the beads with 2x Laemmli buffer and heated at 95°C for 5 min, followed by SDS-PAGE and western blot analysis.
- TRFM25-FLAG and RIG-I-FLAG were purified from transfected HEK293T cells using anti-FLAG-conjugated sepharose beads and tested for binding to recombinant 14-3-3 ⁇ .
- Mitochondria Fractionation Assay HEK293T or Huh7 cells were infected with DV or SeV at indicated titers, or mock infected. 20 -24 hours later, a portion of cells was harvested for WCLs, and another portion for fractionation assay using a commercial mitochondria/cytosol fractionation kit (MIT 1000, Merck Millipore) according to the manufacturer's instructions. Briefly, cells were disrupted in Isotonic Mitochondrial Buffer using a Dounce homogenizer.
- Ly sates were subjected to low-speed centrifugation to pellet nuclei and unbroken cells. Supernatant was subsequently centrifuged at 10,000 x g for 30 min at 4°C. The supernatant containing the cytosol and microsome fraction ('cytosolic fraction') as well as the pellet containing the enriched mitochondrial fraction were subjected to a bicinchoninic acid (BCA) assay. Equal amounts of protein were loaded for SDS-PAGE and analyzed by western blot. Anti-GAPDH and anti-MAVS western blot analyses served as controls.
- BCA bicinchoninic acid
- cells were washed once in PBS, fixed in 1% (w/v) paraformaldehyde, permeabilized with 0.1% saponin (Sigma), and then stained with anti-prM-DyLight 633 in permeabilization buffer for -40 min at 4°C.
- NS3 protein sequences from full genome DV sequences were analyzed with NIAID Virus Pathogen Database and Analysis Resource (ViPR) online through the website at http://www.viprbrc.org.
- ViPR NIAID Virus Pathogen Database and Analysis Resource
- RNA Quantitative Real Time-PCR
- RNA extraction kit OEGA Bio-Tek
- Equal amounts of RNA typically 10 - 100 ng
- RNA were used in an one-step qRT-PCR reaction (Superscript III Platinum One-Step qRT-PCR kit with ROX, Life Technologies) with commercially available primers with FAM reporter dye for the indicated target genes (IDT).
- IDTT FAM reporter dye for the indicated target genes
- Expression level for each target gene was calculated by normalizing against GAPDH using the AACT method and expressed as fold levels compared to mock-infected cells. All qRT-PCR reactions were run on a 7300 RT- PCR System or 7500 FAST RT-PCR System (both ABI).
- DV2 K i K p was generated based on an infectious clone of DV2 16681, pD2/IC-30P, kindly provided by Claire Huang (CDC) and described previously (Butrapet et al., J. Virology 74, 3011-3019 (2000); Kinney et al., Virology 230, 300-308.(1997)). PCR was used to generate mutant pD2/IC-30P harboring R64K and E66K mutations in the NS3 gene.
- the wild-type and mutant infectious clone plasmids were linearized by Xbal digestion and in vitro transcribed using the T7 promoter (RiboMAX Large Scale RNA Production System, Promega) with the addition of a m 7 G(5')ppp(5')A RNA cap structure analog (New England Biolabs).
- the in vitro transcribed RNA was purified using Micro Bio-Spin columns (Bio Rad) and transfected into Vero cells using Lipofectamine 2000. Viral supernatants were harvested and used to propagate the wild-type and mutant virus in Vero cells. Vero cells were further used to titer the recombinant viruses using a FACS-based assay (Lambeth et al., J. Clinical
- PBMCs peripheral blood mononuclear cells
- PBMCs peripheral blood mononuclear cells
- CD 14 + monocytes were positively selected from PBMCs using anti-CD14 magnetic microbeads according to the manufacturer's instructions (Miltenyi Biotec). CD14 + monocytes were rested overnight in growth media before use, or cryopreserved for use in future experiments. The purity of CD14 + cells was routinely -90%, as determined by anti-CD 14-FITC staining (BD
- NS3 protease amino acids (aa) 1-179) and helicase (aa 169-619) domains (FLAG-NS3-Pro and FLAG-NS3-Hel), as well as the NS5 methyltransferase (aa 1-319) and polymerase (aa 297-901) domains (FLAG-NS5-MTase and FLAG-NS5-Pol) was utilized.
- Co-IP co-immunoprecipitation
- 14-3-3 ⁇ expression suppressed DV2 replication and 14-3-3 ⁇ overexpression inhibited the replication of four other DV strains representing all four serotypes (DV1-4), but had no effect on herpes simplex virus- 1 (HSV-1), an unrelated DNA virus.
- HSV-1 herpes simplex virus- 1
- 14-3 -3 ⁇ expression in K562 cells was silenced using short interfering RNAs (siRNAs). Knockdown of 14-3-3 ⁇ in K562 cells significantly enhanced DV replication as compared to non-targeting control siRNA, supporting a role for 14-3 -3 ⁇ in controlling DV replication.
- Example 3 - NS2B/3 inhibits RIG-I activation independent of proteolytic activity
- Example 5 - NS3 binds to 14-3 -3 ⁇ using a phosphomimetic RxEP motif
- GST-fused NS3-Pro truncation fragments were constructed and tested for their abilities to bind endogenous 14-3-3 ⁇ by Co-IP.
- Full-length GST-NS3 served as a positive control ( Figures 5, Panel A and Figure 10).
- Full-length GST-NS3, GST-NS3 i -92 and GST-NS3 43 -92 efficiently interacted with endogenous 14-3-3 ⁇ , while other NS3 fragments did not bind 14- 3-3 ⁇ under the same conditions.
- Rxx(pS/pT)xP canonical high-affinity binding motif
- x denotes any residue
- pS/pT indicates a phosphorylated serine/threonine residue
- Phosphorylation of S/T in Rxx(pS/pT)xP has been shown to be essential for 14-3-3 binding, as dephosphorylation of this residue abrogates 14-3-3 interaction (Yaffe et al., Cell 91, 961-971 (1997)).
- E 66 was replaced with Lys (K 66 ), a positively charged amino acid (NS3 RIKP ).
- NS3 RIKP exhibited profoundly diminished binding to 14-3-3 ⁇ , indicating that the
- Example 6 The 14-3-3 ⁇ - ⁇ 3 ⁇ 4 deficient NS3KTKP mutant protein is impaired in suppression of RIG-I translocation and IFN- ⁇ induction
- the NS3 KIKP mutant protein that exhibited a near-complete loss of 14-3 -3 ⁇ binding was characterized functionally.
- the inhibitory effect of WT NS3 and the NS3 KIKP mutant on the complex formation of endogenous RIG-I and 14-3 -3 ⁇ triggered by SeV infection were compared. While WT NS3 potently inhibited SeV-induced RIG-I- 14-3 -3 ⁇ binding, NS3 KIKP did not affect their interaction ( Figure 11, Panel A). In line with this, while WT NS3 potently blocked the translocation of endogenous RIG-I to MAVS-containing mitochondrial fractions, NS3 KIKP expression did not inhibit RIG-I translocation (Figure 5, Panel G).
- Example 7 A recombinant DV encoding a NS3KTKP mutant protein is attenuated in replication and elicits enhanced levels of IFNs, ISGs and proinflammatory cytokines
- a recombinant DV encoding the NS3 KIKP mutant protein that is impaired in 14-3-3 ⁇ binding and RIG-I antagonism was constructed. Since NS3, as part of the NS2B/3 protease complex, processes the viral polyprotein and is therefore essential for DV replication, whether a NS2B/3 KIKP mutant protein retains proteolytic activity was determined.
- Huh7 cells were infected with DV2 WT or DV2 KIKP using MOIs (MOI 0.3 and 1, respectively) that resulted in comparable infectivity (-75% of cells infected at 2 d postinfection as determined by flow cytometry [data not shown]).
- DV2 KIKP elicited markedly higher levels of IFNB1, ISGs (ISG15, IFIH1 and ⁇ 7), and proinflammatory cytokines (TNF, IL6 and CCL5) than DV2 WT ( Figure 6, Panel B).
- DV2 KIKP infection of A549 cells robustly induced ISG protein expression (ISG54 and RIG-I) in neighboring non-infected cells as determined by confocal immunofluorescence microscopy.
- ISG protein induction was low in response to DV2 WT infection ( Figure 12, Panel E).
- CD14 + monocytes infected with DV KIKP exhibited enhanced IL-6 protein secretion as compared to cells infected with DV2 WT ( Figure 7, Panel B).
- Example 8 The Dengue DV2KTKP mutant virus elicits an augmented T cell response
- T cells co-cultured with DV2 K iKP-infected moDCs secreted significantly higher levels of IFN-at 96 hours after infection as compared to T cells co- cultured with DV2WT-infected moDCs, demonstrating greater T cell activation by
- Example 9 West Nile Virus (WNV) NS3 protein antagonizes RIG-I activation
- dengue virus NS3 harbors a RxEP motif to usurp 14-3-3 ⁇ binding.
- a sequence alignment of multiple flavivirus NS3 proteins shows that West Nile virus harbors a RLDP motif (Fig. 14).
- D is also a phosphomimetic residue like E, it was determined whether WNV NS3 binds 14-3-3 ⁇ to antagonize RIG-I activation and type-I interferon (IFN) induction.
- NS3 of both NY99 and Kunjin strains of WNV were able to bind endogenous 14-3-3 ⁇ ( Figure 15).
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201680048414.9A CN108472353A (en) | 2015-06-22 | 2016-06-21 | Composition and method for adjusting virus infection |
US15/739,428 US20180185467A1 (en) | 2015-06-22 | 2016-06-21 | Compositions and methods for modulating viral infection |
BR112017027770A BR112017027770A2 (en) | 2015-06-22 | 2016-06-21 | compositions and methods for modulating viral infection |
EP16815135.5A EP3310382A4 (en) | 2015-06-22 | 2016-06-21 | Compositions and methods for modulating viral infection |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201562183018P | 2015-06-22 | 2015-06-22 | |
US62/183,018 | 2015-06-22 | ||
US201662295635P | 2016-02-16 | 2016-02-16 | |
US62/295,635 | 2016-02-16 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2016209805A1 true WO2016209805A1 (en) | 2016-12-29 |
Family
ID=57586686
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2016/038501 WO2016209805A1 (en) | 2015-06-22 | 2016-06-21 | Compositions and methods for modulating viral infection |
Country Status (5)
Country | Link |
---|---|
US (1) | US20180185467A1 (en) |
EP (1) | EP3310382A4 (en) |
CN (1) | CN108472353A (en) |
BR (1) | BR112017027770A2 (en) |
WO (1) | WO2016209805A1 (en) |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107828744A (en) * | 2017-11-10 | 2018-03-23 | 山东农业大学 | Utilize the method for mosquito source C6/36 cell line proliferation culture duck tembusu viruses |
WO2018132658A1 (en) * | 2017-01-13 | 2018-07-19 | Sestan Nenad | Oncolytic viruses and methods of use |
JP2021502115A (en) * | 2017-11-09 | 2021-01-28 | アンスティテュ・パストゥール | Zika virus chimeric polyepitope containing non-structural proteins, and its use in immunogenic compositions |
US11491217B2 (en) | 2017-12-06 | 2022-11-08 | Emory University | Chimeric viruses encoding mutant zika virus envelope glycoproteins |
US11648304B2 (en) | 2017-11-03 | 2023-05-16 | Takeda Vaccines, Inc. | Zika vaccines and immunogenic compositions, and methods of using the same |
US11975062B2 (en) | 2017-11-30 | 2024-05-07 | Takeda Vaccines, Inc. | Zika vaccines and immunogenic compositions, and methods of using the same |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20080152633A1 (en) * | 2005-02-16 | 2008-06-26 | The University Of Queensland | Flavivirus Replicon Constructs for Tumor Therapy |
WO2012065105A2 (en) * | 2010-11-12 | 2012-05-18 | Thomas Monath | Chimeric flavivirus vaccines |
-
2016
- 2016-06-21 EP EP16815135.5A patent/EP3310382A4/en not_active Withdrawn
- 2016-06-21 CN CN201680048414.9A patent/CN108472353A/en active Pending
- 2016-06-21 BR BR112017027770A patent/BR112017027770A2/en not_active Application Discontinuation
- 2016-06-21 US US15/739,428 patent/US20180185467A1/en not_active Abandoned
- 2016-06-21 WO PCT/US2016/038501 patent/WO2016209805A1/en unknown
Non-Patent Citations (4)
Title |
---|
DATABASE Uniprot 1 April 2015 (2015-04-01), XP055505146, Database accession no. P29990 * |
DENG ET AL.: "NS3 protein of Hepatitis C virus associates with the tumour suppressor p53 and inhibits its function in an NS3 sequence-dependent manner", JOURNAL OF GENERAL VIROLOGY, vol. 87, 2006, pages 1703 - 1713, XP055341072 * |
See also references of EP3310382A4 * |
YANG ET AL.: "A Novel Dengue Virus Inhibitor, BP13944, Discovered by High-Throughput Screening with Dengue Virus Replicon Cells Selects for Resistance in the Viral NS2B/NS3 Protease", ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, vol. 58, no. 1, January 2014 (2014-01-01), pages 110 - 119, XP055341069 * |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2018132658A1 (en) * | 2017-01-13 | 2018-07-19 | Sestan Nenad | Oncolytic viruses and methods of use |
US11648304B2 (en) | 2017-11-03 | 2023-05-16 | Takeda Vaccines, Inc. | Zika vaccines and immunogenic compositions, and methods of using the same |
US11730802B2 (en) | 2017-11-03 | 2023-08-22 | Takeda Vaccines, Inc. | Zika vaccines and immunogenic compositions, and methods of using the same |
US11964008B2 (en) | 2017-11-03 | 2024-04-23 | Takeda Vaccines, Inc. | Method for inactivating zika virus and for determining the completeness of inactivation |
JP2021502115A (en) * | 2017-11-09 | 2021-01-28 | アンスティテュ・パストゥール | Zika virus chimeric polyepitope containing non-structural proteins, and its use in immunogenic compositions |
JP7225254B2 (en) | 2017-11-09 | 2023-02-20 | アンスティテュ・パストゥール | Zika virus chimeric polyepitopes comprising nonstructural proteins and their use in immunogenic compositions |
CN107828744A (en) * | 2017-11-10 | 2018-03-23 | 山东农业大学 | Utilize the method for mosquito source C6/36 cell line proliferation culture duck tembusu viruses |
US11975062B2 (en) | 2017-11-30 | 2024-05-07 | Takeda Vaccines, Inc. | Zika vaccines and immunogenic compositions, and methods of using the same |
US11491217B2 (en) | 2017-12-06 | 2022-11-08 | Emory University | Chimeric viruses encoding mutant zika virus envelope glycoproteins |
Also Published As
Publication number | Publication date |
---|---|
CN108472353A (en) | 2018-08-31 |
BR112017027770A2 (en) | 2018-09-11 |
US20180185467A1 (en) | 2018-07-05 |
EP3310382A4 (en) | 2019-01-02 |
EP3310382A1 (en) | 2018-04-25 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20180185467A1 (en) | Compositions and methods for modulating viral infection | |
Wang et al. | Zika virus infection induces host inflammatory responses by facilitating NLRP3 inflammasome assembly and interleukin-1β secretion | |
Shomuradova et al. | SARS-CoV-2 epitopes are recognized by a public and diverse repertoire of human T cell receptors | |
Muller et al. | The flavivirus NS1 protein: molecular and structural biology, immunology, role in pathogenesis and application as a diagnostic biomarker | |
Kubinski et al. | Tick-borne encephalitis virus: A quest for better vaccines against a virus on the rise | |
Lee et al. | Probing molecular insights into Zika virus–host interactions | |
Teoh et al. | The SARS coronavirus E protein interacts with PALS1 and alters tight junction formation and epithelial morphogenesis | |
Laurent-Rolle et al. | The NS5 protein of the virulent West Nile virus NY99 strain is a potent antagonist of type I interferon-mediated JAK-STAT signaling | |
Zhang et al. | Identification of host proteins involved in Japanese encephalitis virus infection by quantitative proteomics analysis | |
Lundberg et al. | Zika virus non-structural protein NS5 inhibits the RIG-I pathway and interferon lambda 1 promoter activation by targeting IKK epsilon | |
Testa et al. | Conserved MHC class I–presented dengue virus epitopes identified by immunoproteomics analysis are targets for cross-serotype reactive T-Cell response | |
EP3119801B1 (en) | Distinguishing flavivirus infection using a recombinant mutant envelope protein | |
Eyre et al. | Genome-wide mutagenesis of dengue virus reveals plasticity of the NS1 protein and enables generation of infectious tagged reporter viruses | |
Sinigaglia et al. | Immature particles and capsid-free viral RNA produced by Yellow fever virus-infected cells stimulate plasmacytoid dendritic cells to secrete interferons | |
Kant Upadhyay | Biomarkers in Japanese encephalitis: a review | |
Yamanaka et al. | Evaluation of single-round infectious, chimeric dengue type 1 virus as an antigen for dengue functional antibody assays | |
Yoshii et al. | Single point mutation in tick-borne encephalitis virus prM protein induces a reduction of virus particle secretion | |
Diwaker et al. | Microtubule-dependent trafficking of alphaherpesviruses in the nervous system: the ins and outs | |
Tan et al. | Rapid purification of recombinant dengue and West Nile virus envelope Domain III proteins by metal affinity membrane chromatography | |
Khanam et al. | Immune-mediated pathogenesis in dengue virus infection | |
Dechtawewat et al. | Mass spectrometric analysis of host cell proteins interacting with dengue virus nonstructural protein 1 in dengue virus-infected HepG2 cells | |
Haslwanter et al. | Genotype-specific features reduce the susceptibility of South American yellow fever virus strains to vaccine-induced antibodies | |
Ho et al. | Development of SARS-CoV-2 variant protein microarray for profiling humoral immunity in vaccinated subjects | |
Fricke et al. | Antibodies targeting KSHV gH/gL reveal distinct neutralization mechanisms | |
Jouan et al. | Distinct antiviral signaling pathways in primary human hepatocytes and their differential disruption by HCV NS3 protease |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 16815135 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
REG | Reference to national code |
Ref country code: BR Ref legal event code: B01A Ref document number: 112017027770 Country of ref document: BR |
|
ENP | Entry into the national phase |
Ref document number: 112017027770 Country of ref document: BR Kind code of ref document: A2 Effective date: 20171221 |