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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • 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
  • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
  • C12N7/04—Inactivation or attenuation; Producing viral sub-units
  • C12N7/045—Pseudoviral particles; Non infectious pseudovirions, e.g. genetically engineered
• • 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
  • A61K2039/5258—Virus-like particles
• • 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
  • C12N2770/24123—Virus like particles [VLP]
• • 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
• • 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
  • C12N2770/24151—Methods of production or purification of viral material
• • 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

• the present invention relates to highly immunogenic virus like particles (VLPs) comprising recombinant envelop (E) protein of the Dengue virus (DENV). More particularly, the invention relates to the development of recombinant DENV envelope E based Dengue vaccine candidate and a method for producing it using Pichia pastoris expression.
• VLPs highly immunogenic virus like particles
• DENV envelope E recombinant envelop protein of the Dengue virus
• Dengue viruses members of flavi virus genus of the Flaviviridae family, impose one of the largest social and economic burdens of any mosquito-borne viral pathogens.
• DENV infection may be asymptomatic or result in a range of manifestations, from mild dengue fever (DF) to more severe and potentially fatal dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS).
• DF mild dengue fever
• DHF dengue hemorrhagic fever
• DSS dengue shock syndrome
• Over 2.5 billion people representing a staggering 40% of world population, in more than 100 countries, are at risk from DENV infection, with at least 50-100 million infections each year.
• E protein being the major structural protein present on the surface of mature Dengue virions
• the E protein binds to host cell surface receptor and contains type specific and dominant neutralization determinants [Lindenbach BD, Thiel HJ, Rice CM. (2007). Flavivindae: The viruses and their replication. In Fields of Virology, 5th edition (Eds.-in chief nipe, D.M., Howley, P. M.), pp. 1101-1 152; Wolters Kluwer and Lippincott Williams & Wilkins, Philadelphia.] making it highly efficacious.
• the E protein contains multiple conformational neutralizing epitopes and can more importantly elicit virus-neutralizing antibodies.
• Vaccine 28 2705- 2715]. While the insect cell-expressed recombinant E protein is in its native conformation, it does not form VLPs.
• the E protein expressed in insect cells using a baculovirus vector tends to form large unorganized aggregates [Kelly, E. P., Greene, J; J. King, A. D., Innis, B. L. (2000). Purified dengue 2 virus envelope glycoprotein aggregates produced by baculovirus are immunogenic in mice. Vaccine 18: 2549- 2559]. Therefore, the design of an effective dengue recombinant antigen with an easy and inexpensive expression system leading to the development of a safe, efficacious, and cost effective dengue vaccine is of utmost importance and urgently needed.
• Another object of the invention is to provide an expression system for the expression of recombinant E protein in higher level and with proper confirmation.
• Still another object of the invention is to provide a safe, efficacious and inexpensive dengue vaccine.
• the present invention provides highly immunogenic virus like particles (VLPs) comprising recombinant envelop (herein after E) protein of Dengue virus (DENV).
• VLPs virus like particles
• DEV Dengue virus
• the envelop protein E comprises 393-395 amino acid residues of the ecto-domain of Dengue virus serotypes selected from DENV-1, DENV- 2, DENV-3 or DENV- 4.
• the ecto-domain has amino acid sequence selected from the sequences having SEQ IDs 1, 2, 3 and 4.
• virus like particles have particle size between 20nm-
• Another embodiment of the invention provides a polynucleotide sequence encoding a recombinant envelop protein E comprising a pre-membrane (prM) carboxy terminal sequence; a sequence encoding ecto-domain of envelop protein E; and a sequence encoding 6x histidine tag.
• prM pre-membrane
• Yet another embodiment of the invention provides a recombinant envelop protein E encoded by a polynucleotide sequence comprising a pre-membrane carboxy terminal sequence; a sequence encoding ecto-domain of envelop protein E; and a sequence encoding 6x histidine tag.
• said ecto-domain nucleotide sequence is linked to said histidine tag nucleotide sequence through a nucleotide sequence encoding penta- glycine linker.
• ecto-domain nucleotide sequence is selected from sequences having SEQ IDs 5, 6, 7, and 8.
• pre-membrane carboxy terminal sequence is selected from sequences having SEQ IDs 9, 10, I I and 12.
• Yet another embodiment of the invention provides a method for producing virus like particles comprising recombinant envelop protein E, said method comprising: transforming host cells with an expression vector comprising a recombinant polynucleotide, wherein the polynucleotide comprises a nucleotide sequence encoding 36 carboxy terminal amino acid residues of pre-membrane domain, followed by a sequence encoding ecto-domain of the Envelop protein and 6x histidine tag; and culturing the transformed host cell under conditions whereby envelop (E) proteins are expressed and assembled into VLPs.
• the host cell is Pichia pastoris.
• the transformed cultures are grown in an environment of methanol at a temperature of 30°C.
• Still another embodiment of the invention provides a recombinant envelop protein E having 393-395 amino acid residues.
• VLPs virus like particles
• Yet another embodiment of the invention provides an expression vector comprising a polynucleotide sequence for the expression of recombinant envelop protein E.
• the vector used is pPICZ A and is a shuttle vector.
• polynucleotide sequence is integrated at the EcoRI and Notl sites of the vector.
• Figure 1 Vector construct for expression of recombinant DENV E proteins in P. pastoris.
• DENV. E gene is cloned in pPICZ A vector (Invitrogen) at EcoRI and Notl sites.
• the box at N terminus denotes 3' prM sequences and the box at C terminus denotes the 6x histidine tag for purification.
• Figure 2 Western blot analyses of DENV E protein expression in P. pastoris.
• FIG. 3 Purification profile of DENV- 1 (A), DENV-2 (B), DENV-3 (C) and
• DENV-4 E proteins from total cell lysate by affinity-chromatography.
• the solid curve indicates UV absorbance at 280nm.
• the dashed curve indicates the imidazole step gradient. Purified peak fractions were analysed by SDS-PAGE gel (inset, iane 2) and confirmed by Western blot analysis (inset, second lane 3). The dashed line in the insets demarcates the SDS-PAGE and Western blot lanes.
• FIG. 4 The recombinant E protein preparations were negatively stained with uranyl acetate and observed by electron microscopy, DENV E proteins were found to assemble into VLPs, with particle size varying from 20nm to 80nm.
• FIG. 5 Evaluation of the immunogenic ity of DENV-3 VLPs in Balb/c mice.
• Anti-DENV-3 antibodies in sera from DENV-3 E VLP-immunized mice collected after the 1st (curve 'b') and second (curve 'c') boosts were analyzed using DENV-3 VLPs coated on the microtiter wells. Captured anti-DENV-3 antibodies were revealed using anti-mouse IgG-HRPO conjugate and TMB substrate.
• the ELISA reactivity of sera from PBS-immunized control mice is represented by curve 'a'.
• the y-axis represents ELISA reactivity (absorbance at 450 nm) and x-axis represents sera dilution in thousands.
• FIG. 6 Evaluation of the immunogenicity of DENV-2 VLPs in Balb/c mice.
• Anti-DENV-2 antisera from DENV-2 E VLP-immunized mice, collected after the 1st (curve 'b') and second (curve V) boosts were analyzed by ELISA using DENV-2 VLPs coated on the microtiter wells. Captured anti-DENV-2 antibodies were revealed using anti-mouse IgG-HRPO conjugate (1 : 10,000 dilution) and TMB substrate.
• the ELISA reactivity of sera from PBS-immunized control mice is represented by curve 'a'.
• the y-axis represents ELISA reactivity (absorbance at 450 nm) and x-axis represents sera dilution in thousands (lk-64k).
• FIG. 7 Evaluation of the immunogenicity of DENV E VLPs through ELISA: (A) anti-DENV-1 E response (curve a), (B) anti-DENV-4 E response (curve b).
• the ELISA reactivity of Mock sera (PBS-immunized mice) is represented by curve "c" in both graphs.
• the y-axis represents ELISA reactivity (absorbance at 450 nm) and x-axis represents sera dilution in thousands.
• Figure 8 Determination of serum virus-neutralizing antibody titres in Balb/C mice immunized with recombinant DENV-2 E VLPs. Serial dilutions of immune sera were separately pre-incubated with each of the four DENV serotypes, followed by determination of inhibition of virus infectivity using a FACS-based assay. The serum dilution causing 50% inhibition is expressed as FNT50 value on the y-axis.
• FIG. 9 DENV-2 enhancing antibody titers in antisera raised using recombinant DENV-2 and DENV-3 E VLPs.
• DENV-2 was pre-incubated with serial dilutions of anti-DENV-2 E antiserum (curve 'b'), anti-DENV-3 E (curve 'c') antiserum or PBS-immunized antiserum (curve 'a') and used to infect 562 cells.
• a control DENV-2 infection (spot'd') in which antibody pre-incubation was omitted (No Ab) was done in parallel.
• FACS analysis was done 24 hours later to determine the percentage of infected cells.
• FIG. 10 DENV-2 enhancing antibody titers in antisera raised using recombinant DENV-3 E VLPs and recombinant DENV-3 EDIII.
• DENV-2 was pre- incubated with serial dilutions of anti-DENV-3 E antiserum (curve 'c'), anti-DENV-3 EDIII antiserum (curve 'b') or PBS-immunized antiserum (curve 'a') and used to infect K562 cells.
• a control DENV-2 infection (spot d) in which antibody pre-incubation was omitted (No Ab) was done in parallel.
• FACS analysis was done 24 hours later to determine the percentage of infected cells.
• Figure 1 1 AG 129 mice that were immunized with PBS (diamonds) and recombinant DENV-2 E VLPs (circles) were challenged with a lethal dose of virulent DENV-2, followed by monitoring of survival rates over a period of 18 days post- challenge.
• the upper-most curve shows the corrected survival curve (taking into account only the 6 mice that displayed anti-DENV-2 antibodies in ELISA).
• Figure 12 Nucleotide sequence of the synthetic DENV-1 E gene, codon- optimized for expression in P.pastoris.
• the prM carboxy terminal sequence, the penta- glycine linker and 6x His tag are highlighted in grey, green and yellow, respectively.
• the start and stop codons are underlined. Restriction sites for cloning are shown in italics.
• Figure 13 Nucleotide sequence of the synthetic DENV-2 E gene, codon- optimized for expression in P.pastoris.
• the prM carboxy terminal sequence, the penta- glycine linker and 6x His tag are highlighted in grey, green and yellow, respectively.
• the start and stop codons are underlined. Restriction sites for cloning are shown in italics.
• Figure 14 Nucleotide sequence of the synthetic DENV-3 E gene, codon- optimized for expression in P.pastoris.
• the prM carboxy terminal sequence, the penta- glycine linker and 6x His tag are highlighted in grey, green and yellow, respectively.
• the start and stop codons are underlined. Restriction sites for cloning are shown in italics.
• Figure 15 Nucleotide sequence of the synthetic DENV-4 E gene, codon- optimized for expression in P.pastoris.
• the prM carboxy terminal sequence, the penta- glycine linker and 6x His tag are highlighted in grey, green and yellow, respectively.
• the start and stop codons are underlined. Restriction sites for cloning are shown in italics.
• Figure 16 Amino acid sequences of recombinant DENV-1 E (A), DENV-2 E (B), DENV-3 E (C) and DENV-4 E (D) proteins predicted to be encoded by the P. ⁇ astora-optimized DENV-1 E, DENV-2 E, DENV-3 E and DENV-4 E synthetic genes, respectively, shown in the preceding pages.
• the C-terminal prM sequences preceding the E sequences are highlighted in grey.
• the penta-glycine linker and 6x-His tag engineered into the C-terminus are highlighted in green and yellow, respectively.
• the present invention provides highly immunogenic virus like particles (VLPs) comprising recombinant envelop (E) protein of Dengue virus (DENV).
• VLPs recombinant envelop
• DEV Dengue virus
• the recombinant protein comprises ecto-domain of the envelop protein E of Dengue virus of serotypes 1, 2, 3 and 4.
• the envelope E protein being the major structural protein present on the surface of mature Dengue virions.
• the invention provides gene constructs capable of expressing a polypeptide that can elicit type specific immune response in a host against dengue virus.
• the gene constructs comprise sequences encoding carboxy terminal amino acid residues for pre-membrane (prM) sequence linked to the encoding sequence corresponding to ecto-domain of the dengue envelope E proteins and a 6x histidine tag encoding sequence.
• the ecto-domain of said envelope E protein is linked to said histidine tag (SEQ ID 26) through a penta-glycine linker (SEQ ID 25).
• nucleotide sequences encoding penta-rglycine linker are given in sequence listing under SEQ IDs 17, 18, 19 and 20.
• nucleotide sequences encoding histidine tag are given in sequence listing under SEQ IDs 21, 22, 23, and 24.
• the polynucleotide encoding the ecto-domain of the envelop/surface protein of either serotype of the Dengue virus is selected from DENV-1, DENV-2, DENV-3 or DENV- 4 has sequence of SEQ IDs 5, 6, 7 and 8, respectively.
• the corresponding amino acid residues have sequences with SEQ IDs 1, 2, 3 and 4.
• polypeptide expressed by the gene constructs of present invention possesses
• pre-membrane which is the signal sequence for the envelop protein. This sequence helps in the translocation of protein from cytosol to ER lumen. The signal sequence gets cleaved at trans golgi network during process.
• the signal sequences used in the gene constructs are different and are specific for the ectodomain sequence taken from serotype of the Dengue virus.
• nucleotide sequences and amino acid sequences of the prM carboxy terminal sequence used for different type of DENV serotypes are given below:
• the invention provides encoding nucleotide sequences that express recombinant
• sequences of the four synthetic DENV E genes are given in Figures 12-15.
• amino acid sequences encoded by synthetic DENV E genes are given in Figure 16.
• Another aspect of the invention provides vector constructs containing the
• DENV E genes The gene constructs are codon optimized for expression in P.pastoris and cloned into pPICZ A at EcoRl and Notl site.
• the plasmid vectors are electroporated into P.pastoris and analyzed for expression. Clones of P.pastoris harbouring the E genes of each subtype are identified.
• Plasmid pPICZ A is a shuttle vector.
• the recombinant envelope E protein . specific for a dengue virus subtype expressed by the gene constructs form yet another aspect of the invention.
• P.pastoris clones harbouring the E gene of each subtype are cultured and analyzed for expression of the DENV E proteins by immunoassay using monoclonal antibody (mAb) specific for each of the E protein for DENV-1, DENV-2, DENV-3 and DENV-4.
• mAb monoclonal antibody
• the expressed DENV E proteins are purified by affinity chromatography and fractions analyzed by Western blot method. The amino acid sequences of the proteins encoded by these .genes are given in Figure 16.
• the purified DENV recombinant E proteins further assembles into highly immunogenic virus like particles (VLPs).
• VLPs virus like particles
• the formation of VLP without the co-expression of prM is unexpected.
• VLPs are highly immunogenic and have the potential to elicit DENV neutralizing antibodies and confer protection against lethal DENV challenge in AG 129 model.
• the VLPs consist of 393-395 amino acid residues of ecto-domain of Dengue virus serotypes viz DENV-1, DENV-2, DENV-3 or DENV- 4.
• VLP-based tetravalent dengue vaccine can also be developed by a person skilled in the art from the teachings of the present invention.
• Another aspect of the invention provides methods of producing virus neutralizing antibodies against specific subtype of dengue virus comprising immunization of a host with the DENV E VLP of the invention.
• Immunization can comprise the use of one or more adjuvants well known in the art. immune sera of mice immunized with recombinant proteins could neutralize the infectivity of DENV in Plaque Reduction Neutralization Tests (PRNT).
• PRNT Plaque Reduction Neutralization Tests
• the type-specific antibodies produced against the recombinant E proteins of this invention can also be used for the passive immunization as well as for other therapeutic use.
• the use of recombinant E proteins of the present invention as diagnostic antigens in immunoassays forms another aspect of this invention.
• the recombinant E proteins of the invention may also be used directly in vaccine formulations by a person skilled in the art.
• Compositions containing DENV rE proteins of the invention in conventional forms well known in the art are also encompassed in this invention.
• the vaccines of the invention contain an immunogenically effective amount of the recombinant E protein as an active ingredient.
• the vaccine may be introduced into a subject optionally with adjuvant.
• the E genes of DENVs codon-optimized for P.pastoris expression, were synthesized by Genscript and Biobasic Inc.
• the genes contain sequences encoding 36 carboxy terminal amino acid (aa) residues of prM followed by the first 395 aa residues of E (393 aa in the case of DENV-3 E), representing the ectodomain, and a 6x histidine tag.
• a penta-glycine linker was inserted between the ectodomain and the 6x histidine tag.
• the sequences of the four synthetic DENV E genes are presented in Figures 12-15.
• the amino acid sequences of the proteins encoded by these genes are given in Figure 16.
• the DENV E genes were cloned into pPICZ A (shuttle vector for P.pastoris) at EcoRl and Notl sites ( Figure 1).
• the plasmids were electroporated into P.pastoris (KM71H cells) and analyzed for gene integration by colony PCR and for expression of the recombinant DENV E proteins using DENV E-protein specific monoclonal antibodies (mAbs). Based on this four P.pastoris clones, each harbouring the E gene of one DENV serotype have been identified.
• Example 2 Expression of DENV E proteins Shake-flask cultures of the four P.pastoris clones were set up to reach log phase. Expression of DENV E protein was carried out at 30°C, 250 rpm by adding 1% methanol every 12 hrs. The cultures were induced for 72 hours and 1 ml of each induced culture was collected every 24 hrs. Expression of DENV E ( ⁇ 48kDa) proteins was analysed in induced biomass and confirmed by Western blot analysis using an in- house mAb specific for the carboxy-terminal part of the E protein for DENV-1, -2 and - 3 and a commercial mAb for DENV-4 ( Figure 2).
• P.pastoris these were used to immunize Balb/c mice on days 0, 30 and 60.
• the antigens were formulated in alum. Immune sera were collected 1 week after the 1st and
• FIG. 5 shows the results of ELISA performed with sera from DENV-3 E VLP-immunized mice.
• the microtiter wells were coated with DENV-3 VLPs to capture polyclonal DENV-3 antibodies from the immune sera and revealed with anti-mouse IgG-HRPO in conjunction with TMB substrate. This showed that the DENV VLPs are highly immunogenic.
• a comparison of the ELISA titers of sera obtained from the first and second booster immunizations show that there is a clear boosting effect. This was evident with DENV-2 VLP immunization as well, as shown in Figure 6.
• the FACS based Neutralization titers required for 50% inhibition is designated as the FNT50 titer.
• mice were used (Johnson, A. J., Roehrig, J. T. (1999). New mouse model for dengue virus vaccine testing. J. Virol. 73: 783-786). These mice were immunized with recombinant DENV-2 E antigen formulated in alum on days 0, 30 and 90 and challenged on day 102 with a virulent strain of DENV-2 (1.4xl0 8 PFU/mouse) obtained by alternate passaging of DENV-2 strain NGC between C6/36 mosquito cells and AG 129 mice as described previously (Shresta, S., Sharar, K.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Genetics & Genomics (AREA)
• Virology (AREA)
• Medicinal Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Microbiology (AREA)
• Engineering & Computer Science (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Biochemistry (AREA)
• Immunology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Public Health (AREA)
• Gastroenterology & Hepatology (AREA)
• Epidemiology (AREA)
• Animal Behavior & Ethology (AREA)
• Biophysics (AREA)
• Veterinary Medicine (AREA)
• Mycology (AREA)
• Pharmacology & Pharmacy (AREA)
• Biomedical Technology (AREA)
• Biotechnology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Molecular Biology (AREA)
• General Engineering & Computer Science (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Peptides Or Proteins (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)

Priority Applications (1)

Applications Claiming Priority (1)

Publications (1)

Family

ID=47428791

Family Applications (1)

Country Status (1)

Citations (3)

• 2012
  • 2012-10-26 WO PCT/IN2012/000707 patent/WO2014064707A1/en active Application Filing

Patent Citations (3)

Non-Patent Citations (10)

Similar Documents

Legal Events