# US 11,111,276 B2 · Epitope-substituted vaccine for use in improving safety and immunogenicity against dengue viruses > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,111,276 B2 - Document ID: US11111276B2 - Status: Granted - Kind: B2 - Application number: 16/597,850 - Filed: 2019-10-10 - Published: 2020-01-30 - Granted: 2021-09-07 - Earliest claimed: 2015-02-09 - Assignees: Academia Sinica - Applicants: Academia Sinica - Inventors: Han-Chung Wu; Chung-Tao Tang - Web page: https://patentgrep.com/patent/US11111276B2 ## CPC - C07K 14/005: from viruses - A61K 39/12: Viral antigens - A61K 39/39: characterised by the immunostimulating additives, e.g. chemical adjuvants - A61P 31/14: for RNA viruses - A61P 37/04: Immunostimulants - C07K 16/116: Togaviridae (F); Matonaviridae (F); Flaviviridae (F) - C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) - G01N 33/56983: {Viruses} - A61K 2039/505: {comprising antibodies} - A61K 2039/53: {DNA (RNA) vaccination} - A61K 2039/55505: {Inorganic adjuvants} - A61K 2039/575: {humoral response} - C07K 2317/34: Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues - C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding - C12N 2770/24122: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes - C12N 2770/24123: Virus like particles [VLP] - C12N 2770/24134: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein - C12N 2770/24151: Methods of production or purification of viral material - C12N 2770/24171: Demonstrated in vivo effect - G01N 2333/185: Flaviviruses or Group B arboviruses, e.g. yellow fever virus, japanese encephalitis, tick-borne encephalitis, dengue - G01N 2469/20: Detection of antibodies in sample from host which are directed against antigens from microorganisms - Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE ## Abstract Isolated mutant dengue virus E protein variants are disclosed. The variant comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 1 and has one or more amino acid residue substitutions at position corresponding to Asn8 (N8), Arg9 (R9), Val12 (V 12) and/or Glu13 (E13). The variant may comprise an amino acid sequence that is at least 90% identical to the SEQ ID NO: 1 and lack an infection-enhancing antibody-binding motif comprising the amino acid sequence of SEQ ID NO: 28 at domain I. An isolated nucleic acid sequence encoding the variant, a plasmid expressing the variant, a plasmid expressing a virus-like particle comprising the variant, a DNA vaccine, and a method of detecting the presence of a dengue virus in a biological sample are also disclosed. ## Claims ### Claim 1 A method for neutralizing and protecting against one or more dengue virus serotypes infection, and/or for neutralizing a dengue virus, reducing, alleviating antibody dependent enhancement of dengue virus infection, and/or increasing survival rate in a subject in need thereof, comprising: administering to the subject in need thereof a therapeutically effective amount of a plasmid expressing a mutant dengue virus E protein comprising an amino acid sequence that is at least 80% identical to SEQ ID NO; 1, wherein the mutant dengue virus E protein has one or more amino acid residue substitutions at position corresponding to Asn8, Arg9, Val12, and/or Glu13 of SEQ ID NO: 1. ### Claim 2 (depends on 1) The method of claim 1, wherein the mutant dengue virus E protein has one amino acid residue substitution at the Asn8 with Arg. ### Claim 3 (depends on 1) The method of claim 1, further comprising steps of preparing immunized sera as follows: (a) collecting serum samples from the subject in need thereof after the administering step; and (b) preparing the immunized sera from the collected serum samples. ### Claim 4 (depends on 1) The method of claim 1, wherein the mutant dengue virus E protein has one amino acid residue substitution at the Asn8 with Ala, Glu, or Arg. ### Claim 5 (depends on 1) The method of claim 1, wherein the plasmid is administered in a form of gold-coated plasmid DNA-complex. ### Claim 6 (depends on 1) The method of claim 1, wherein prior to the administering step a serum sample from the subject in need thereof exhibits a reactivity with an isolated peptide comprising the amino acid sequence of SEQ ID NO: 28. ### Claim 7 (depends on 6) The method of claim 6, wherein the isolated peptide is less than 15 amino acid residues in length and is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-12, and 14-26. ### Claim 8 (depends on 1) The method of claim 1, prior to the administering step further comprising detection of the dengue virus infection in the subject in need thereof, wherein the detection comprises the steps of: (a) contacting a blood sample from the subject in need thereof with an isolated mutant dengue virus E protein having one amino acid residue substitution at position corresponding to Asn8 of SEQ ID NO: 1 with Arg, or with an isolated peptide comprising the amino acid sequence of SEQ ID NO: 28; (b) allowing dengue virus antibodies in the blood sample and the isolated mutant dengue virus E protein or the isolated peptide to form a complex; and (c) detecting the presence of the dengue virus antibodies in the complex and thereby detecting the dengue virus infection. ### Claim 9 (depends on 1) The method of claim 1, wherein the plasmid expressing a mutant dengue virus E protein comprises one amino acid residue substitution at position corresponding to Asn8, Arg9, or Glu13 of SEQ ID NO: 1. ### Claim 10 A method for eliciting neutralizing antibodies protecting against one or more dengue virus serotypes infection, and/or reducing, alleviating antibody dependent enhancement of dengue virus infection, comprising: immunizing a subject in need thereof a therapeutically effective amount of a DNA vaccine comprising a plasmid expressing a mutant dengue virus E protein, the mutant dengue virus E protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, wherein the mutant dengue virus E protein has one or more amino acid residue substitutions at position corresponding to Asn8, Arg9, Val12, and/or Glu13 of SEQ ID NO: 1. ### Claim 11 (depends on 10) The method of claim 10, wherein the subject in need thereof is immunized with vaccine at least 3 times at a 3-week interval. ### Claim 12 (depends on 11) The method of claim 11, further comprising: (i) collecting a serum sample from the subject in need thereof 3 weeks after the third immunization; and (ii) preparing immunized sera comprising the neutralizing antibodies. ### Claim 13 (depends on 10) The method of claim 10, wherein the mutant dengue virus E protein has one amino acid residue substitution at the Asn8 with Ala, Glu, or Arg. ### Claim 14 (depends on 13) The method of claim 13, wherein the subject in need thereof is immunized with the DNA vaccine at least 3 times at a 3-week interval. ### Claim 15 (depends on 14) The method of claim 14, further comprising: (i) collecting a serum sample from the subject in need thereof 3 weeks after the third immunization; and (ii) preparing, immunized sera comprising the neutralizing antibodies. ### Claim 16 A method for neutralizing and protecting against one or more dengue virus serotypes infection, and/or for neutralizing a dengue virus, and/or reducing, alleviating antibody dependent enhancement of dengue virus infection in a subject in need thereof, comprising: immunizing the subject in need thereof a therapeutically effective amount of a DNA vaccine comprising: (a) a recombinant DNA comprising a nucleotide sequence encoding a mutant dengue virus E protein and a promoter operably linked to the nucleotide sequence, wherein the mutant dengue virus E protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 1, and has one or more amino acid residue substitutions at position corresponding to Asn8, Arg9, Val12, and/or Glu13 of SEQ ID NO: 1; and (b) gold or tungsten, wherein the recombinant DNA and gold or tungsten forms a complex. ### Claim 17 (depends on 16) The method of claim 16, wherein the mutant dengue virus E protein has one amino acid residue substitution at position corresponding to Asn8, Arg9, or Glu3 of SEQ ID NO: 1. ### Claim 18 (depends on 16) The method of claim 16, wherein the mutant dengue virus E protein has one amino acid residue substitution at the Asn8 with Ala, Glu, or Arg. ### Claim 19 (depends on 18) The method of claim 18, wherein the mutant dengue virus E protein has one amino acid residue substitution at the Asn8 with Arg.