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
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
• • 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
• • 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
  • A61K2039/5256—Virus expressing foreign proteins
• • 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
  • A61K2039/70—Multivalent vaccine
• • 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
  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/10011—Arenaviridae
  • C12N2760/10034—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
  • C12N2770/24141—Use of virus, viral particle or viral elements as a vector
  • C12N2770/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
• • 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 invention relates to chimeric Flavivirus based vaccines.
• the invention further relates to vaccines against viruses such as Lassa Virus.
• VSV-LASV-GPC VSV based LASV
• MOPV Mopeia virus
• clone ML29 a DNA vaccine called INO-4500
• pLASV-GPC a DNA vaccine encoding the LASV-GPC gene from Josiah strain f and it is from Inovio company (pLASV-GPC).
• yellow fever virus 17D has been used as vector for Lassa virus glycoprotein (GPC) or its subunits GP1 and GP2 (Bredenbeek et ai. (2006) Virology 345, 299-304 and Jiang et at. (2011) Vaccine 29, 1248-1257)).
• GPC Lassa virus glycoprotein
• SSP signal peptide
• INO-4500 pLASV- GPC
• This vaccine requires multiple high doses delivered via dermal electroporation in order to achieve full protection and enhance the vaccine immune response.
• This multi-dose administration regimen will be very challenging to implement in the rural areas of West Africa where LASV is endemic and the main outbreaks have occurred.
• ML29 is classified in risk group 2 by the EU and risk group 3 by US CDC what is an obstacle for further development of this vaccine.
• VSV-LASV-GPC still requires a cold chain to preserve it which involves high cost and still there are no studies concerning its safety.
• the approach involving YF17D as vector to express Lassa glycoprotein precursor was not successful in NHP studies (0% survival, marmosets).
• this vaccine candidate showed issues of genetic instability that did not allow to scale-up the technology as required for vaccine production. Summary of the invention
• the resulting PLLAV-YFV17D-LASV- GPC launches viable live-attenuated viruses expressing functional LASV-GPC and YFV-17D proteins.
• the PLLAV-YFV17D-LASV-GPC construct can be used directly as vaccine what involves that this vaccine is thermostable.
• the vaccine induces immune responses against both LASV and YFV after one-single shot.
• a second similar construct has been generated in which the cleavage site has been restored (R246A mutation was restored to R246R). (Additional information in the attached data)
• PLLAV-YFV17D-LASV-GPC is a dual vaccine inducing YFV and Lassa virus specific immunity.
• PLLAV-YFV17D-LASV-GPC can also be used as stable seed for the production of tissue culture-derived live-attenuated vaccine not only in the PLLAV modality, but also unexpectedly the recombinant YFV17D-LASV-GPC virus appears to be genetically more than that disclosed in prior art by Bredenbeek et al. and Jiang et al. (cited above).
• a polynucleotide comprising a sequence of a live, infectious, attenuated
• Flavivirus wherein a nucleotide sequence encoding at least a part of a arenavirus glycoprotein protein is located at the intergenic region between the E and NS1 gene of said Flavivirus, such that a chimeric virus is expressed, characterised in that the encoded sequence C terminally of the E protein of said Flavivirus and N terminally of the signal peptide of the NS1 protein of said Flavivirus comprises in the following order :
• polynucleotide according to claim 1 wherein the sequence of the live, infectious, attenuated Flavivirus is Yellow Fever virus, typically the YF17D strain.
• glycoprotein comprises the R207C, G360C and E329P stabilizing mutations.
• polynucleotide according to any one of claims 1 to 13, wherein the sequence of the chimeric virus at the junction of the NS1 signal sequence and the GP1 domain comprises the sequence of SEQ ID NO: 11.
• junctions connecting the flavirus NS1 signal sequence, the Lassavirus G protein, the TM2 protein and the second NS1 signal sequence provide a fingerprint for the encoded proteins.
• encoded sequences can be defined by sequences having the sequence of SEQ ID NO:2 or SEQ ID NO: 4, comprising the sequences with SEQ ID NO: 11, SEQ ID: NO 12 and SEQ ID N013; and wherein outside SEQ ID NO: 11, SEQ ID: NO 12 and SEQ ID N013 , a number of amino acids may differ from SEQ ID NO:2 or SEQ ID NO:4, e.g. differing up to 20, up to 10, or up to 5 compared to SEQ ID NO:2 or SEQ ID NO: 4, or e.g. having a sequence identity of at least 95 %, 96 %, 97 %, 98% or 99 % with SEQ ID NO: 2 or SEQ ID NO: 4.
• a polynucleotide in accordance to any one of claims 1 to 17, for use as a medicament for use as a medicament.
• a chimeric live, infectious, attenuated Flavivirus wherein at least a part of an arenavirus Glycoprotein is located between the E and NS1 protein of said Flavivirus, such that C terminally of the E protein and N terminally of the signal peptide of the NS1 protein the virus comprises in the following order :
• 26. A chimeric virus encoded by a nucleotide in accordance to any one of claims 21 to 23, for use in the prevention of an Arenaviral infection and in the prevention of the Flavivirus.
• a method of preparing a vaccine against an arenaviral infection comprising the steps of: providing a BAC which comprises: an inducible bacterial ori sequence for amplification of said BAC to more than 10 copies per bacterial cell, and a viral expression cassette comprising a cDNA of a arenaviral-flaviviral chimeric virus according to any one of claims 1 to 16, and comprising cis-regulatory elements for transcription of said viral cDNA in mammalian cells and for processing of the transcribed RNA into infectious RNA virus,
• step a) transfecting mammalian cells with the BAC of step a) and passaging the infected cells
• Figure 1 Schematic representation of 1) PLLAV-YFV17D-LASV-GPC and 2) PLLAV-YFV17D-LASV-GPCCS.
• Figure 2 A) Plaque phenotype of YFV17D-LASV-GPC compared to YFV17D.
• Figure 3 Schematic vaccination schedule. AG129 mice were vaccinated with PLLAV-YFV17D-LASV-GPC (25 ug, i.p.) or YFV17D-LASV-GPC (375 PFU).
• Figure 4 Analysis of cellular immunity in vaccinated AG129 mice.
• Figure 5 A) Plaque phenotype of YFV17D-LASV-GPCcs compared to YFV17D.
• FIG. 6 A) Schematic vaccination schedule. AG129 mice were vaccinated subcutaneous (SC) with YFV17D-LASV-GPCcs (250 PFU). B) Analysis of cellular immunity in vaccinated AG129 mice. Representative IFN-gamma ELISPOT wells after 48 hours splenocyte stimulation with the indicated antigen. Spots per six hundred thousand splenocytes in IFN-gamma ELISPOT after 48 hours of stimulation with the indicated antigen. For each mouse, samples were analyzed in duplicates and values are normalized by subtracting the number of spots in control wells (ovalbumin peptide stimulated).
• the present invention is exemplified for Yellow Fever virus, but is also applicable using other viral backbones of Flavivirus species such, but not limited to, Japanese Encephalitis, Dengue, Murray Valley Encephalitis (MVE), St. Louis Encephalitis (SLE), West Nile (WN), Tick-borne Encephalitis (TBE), Russian Spring-Summer Encephalitis (RSSE), Kunjin virus, Powassan virus, Kyasanur Forest Disease virus, Zika virus, Usutu virus, Wesselsbron and Omsk Hemorrhagic Fever virus.
• MVE Murray Valley Encephalitis
• SLE St. Louis Encephalitis
• WN West Nile
• TBE Tick-borne Encephalitis
• RSSE Russian Spring-Summer Encephalitis
• Kunjin virus Powassan virus, Kyasanur Forest Disease virus, Zika virus, Usutu virus, Wesselsbron and Omsk Hemorrhagic Fever virus.
• Flaviviridae which comprises the genus Flavivirus but also the genera, Pegivirus, Hepacivirus and Pestivirus.
• Hepacivirus C hepatitis C virus
• Hepacivirus B GB virus B
• the genus Pegivirus comprises eg Pegivirus A (GB virus A), Pegivirus C (GB virus C), and Pegivirus B (GB virus D).
• the genus Pestivirus comprises e.g. Bovine virus diarrhea virus 1 and Classical swine fever virus (previously hog cholera virus).
• the Flavivirus which is used as backbone can itself by a chimeric virus composed of parts of different Flavivirus.
• C and NS1-5 region are from Yellow Fever and the prME region is of Japanese encephalitis or of Zika virus.
• the present invention is exemplified for the G protein of Lassa virus but is also applicable to G proteins of other arenaviruses.
• the present invention relates to nucleotide sequence and encoded proteins wherein within the RNA or copy DNA (cDNA) of a flavivirus a glycoprotein of an arenavirus is inserted
• Arenaviruses are comprised of two RNA genome segments and four proteins, the polymerase L, the envelope glycoprotein GP (also referred to in the present invention as G protein or GPC), the matrix protein Z, and the nucleoprotein NP.
• Mammarenavirus arenaviruses are divided into two major subgroups: the Old World (OW) and the New World (NW) complex.
• the Old World lineage consists of the prototypic LCMV and other viruses endemic to the African continent, including Lassa (LASV), Mopeia (MOPV), Ippy, and Mobala (MOBV) viruses.
• LASV Lassa
• MOPV Mopeia
• Ippy Ippy
• MOBV Mobala
• Clade B is the most important in term of human disease, since it contains the major viruses causing hemorrhagic fevers (HF) in South America, i.e. Junin (JUNV), Machupo (MACV), Guanarito (GTOV) and Sabia (SABV) viruses but also other non-pathogenic viruses, like Tacaribe (TCRV) and Amapari virus (AMPV).
• the present invention envisages chimeric constructs based on G proteins of any of the above groups, subgroups or species are used.
• Preferred embodiment are constructs based on G proteins of the LASV inserted within a flavivirus RNA or cDNA.
• the present invention envisages chimeric constructs based on G proteins of Reptarenavirusse or Hartmanivirusses are used.
• the present invention is exemplified with G protein of Lassa virus strain Josiah. This sequence of this protein is accessible for example as UniProtKB P08669 database entry or as NCBI NP_694870.1 database entry.
• the arenavirus envisaged is a virus wherein the protein sequence of the G protein has a sequence identity of at least 70, at least 80, at least 90, at least 95, or least 99 % identity with the G protein of Lassa virus strain Josiah, as disclosed in the above cited database entries.
• the constructs of the present invention allow a proper presentation of the encoded insert into the ER lumen and proteolytic processing.
• the encoded protein by the insert lacks the N terminal signal sequence and a GP2 transmembrane domain.
• two transmembrane domains of e.g. WNV are fused c terminally to the glycoprotein sequence .
• any immunogenic protein can be presented via the vector of the present invention that the protein lacks an N terminal membrane targeted domain and contains at the C terminus a targeting membrane followed by a cytoplasmic sequence to allow the connection with the transmembrane membrane preceding the NS1 protein.
• Flavivirus is used as backbone and a G protein of Lassa virus as insert.
• Flaviviruses have a positive single-strand RNA genome of approximately 11,000 nucleotides in length.
• the genome contains a 5' untranslated region (UTR), a long open-reading frame (ORF), and a 3' UTR.
• the ORF encodes three structural (capsid [C], precursor membrane [prM], and envelope [E]) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins.
• C capsid [C]
• prM precursor membrane
• E envelope
• NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 proteins seven nonstructural proteins.
• the structural proteins form viral particles.
• the nonstructural proteins participate in viral polyprotein processing, replication, virion assembly, and evasion of host immune response.
• C-signal peptide regulates Flavivirus packaging through coordination of sequential cleavages at the N terminus (by viral NS2B/NS3 protease in the cytoplasm) and C terminus (by host signalase in the endoplasmic reticulum [ER] lumen) of the signal peptide sequence.
• the positive-sense single-stranded genome is translated into a single polyprotein that is co- and post translationally cleaved by viral and host proteins into three structural [Capsid (C), premembrane (prM), envelope (E)], and seven non- structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) proteins.
• the structural proteins are responsible for forming the (spherical) structure of the virion, initiating virion adhesion, internalization and viral RNA release into cells, thereby initiating the virus life cycle.
• the non-structural proteins on the other hand are responsible for viral replication, modulation and evasion of immune responses in infected cells, and the transmission of viruses to mosquitoes.
• the intra- and inter- molecular interactions between the structural and non-structural proteins play key roles in the virus infection and pathogenesis.
• the E protein comprises at its C terminal end two transmembrane sequences, indicated as TM1 and TM2.
• NS1 is translocated into the lumen of the ER via a signal sequence corresponding to the final 24 amino acids of E and is released from E at its amino terminus via cleavage by the ER resident host signal peptidase (Nowak et al. (1989) Virology 169, 365-376).
• the NS1 comprises at its C terminal a 8-9 amino acids signal sequence which contains a recognition site for a protease (Muller & Young (2013) Antiviral Res. 98, 192-208)
• constructs of the present invention are chimeric viruses wherein a Lassa G protein is inserted at the boundary between the E and NS1 protein. However additional sequence elements are provided N terminally and C terminally of the G protein insert.
• the invention relates to polynucleotide comprising a sequence of a live, infectious, attenuated Flavivirus wherein a nucleotide sequence encoding at least a part of a arenavirus G protein is inserted at the intergenic region between the E and NS1 gene of said Flavivirus, such that a chimeric virus is expressed, characterised in that the encoded sequence C terminally of the E protein of said Flavivirus and N terminal the NS1 protein of said Flavivirus comprises in the following order :
• sequence elements are provided which are substrates for a signal peptidase. These can vary in length and in sequence, and can be as short as one amino acid as shown in Jang et al. cited above. A discussion on suitable recognition sites for signalling proteases is found in Nielsen et a/. (1997) Protein Eng. 10, 1-6.
• the signal peptide at the N terminus of the NS1 protein will be used (or a fragment which allows proteolytic processing).
• the same signal peptide (or fragment) of the NS1 protein of the Flavivirus backbone is introduced.
• the invention equally relates to polynucleotides comprising a sequence of a live, infectious, attenuated Flavivirus.
• a nucleotide sequence encoding at least a part of an arenavirus G protein is inserted at the intergenic region between the E and NS1 gene of said Flavivirus.
• Additional sequences are provided such that when the chimeric virus is expressed such that the encoded sequence from the C terminally of the E protein to the N terminus of the signal peptide of the NS1 protein comprises in the following order: a further signal peptide (or cleavable fragment thereof) of a Flavivirus NS1 gene, C terminal to the E protein and N terminal to the NS1 protein, a arenavirus G protein lacking a functional signal peptide and a transmembrane sequence of the GP2 domain.
• This G protein is C terminally positioned from a NS1 signal peptide.
• C terminally of the G protein is the sequence of a Flavivirus TM1 and TM2 transmembrane domain of a Flavivirus.
• TM sequence follows the NS1 protein, including its native signal peptide sequence.
• G protein and the TM domains are flanked at N terminus and C terminus by an NS1 sequence.
• NS1 sequence In the embodiments disclosed in the examples the protein and DNA sequence of both NS1 are identical.
• both NS1 signal sequences have the sequence DQGCAINFG [SEQ ID NO: 10].
• constructs of the present invention did not show recombination due to the presence of this repetitive sequence. Sequence modifications can be introduced or NS1 sequences from different Flavivirus can be used to avoid presence of identical sequences, as long as the encoded peptide remains a target from the protease which processes these NS1 Nterminal signal sequences.
• the G protein is of Lassa virus, preferably of the Josiah strain of Lassa virus.
• the nucleotide sequence of the G protein is codon optimized.
• constructs of the present invention typically contain a defective G protein signal by partial or complete removal of this sequence or by the introduction of mutations which render the signal protein non-functional.
• the TM domains which are located C terminally of the G protein and N terminally of the NS1 is generally of a Flavivirus, typically from the E protein, and more typical a TM domains of an E protein. In preferred embodiments these TM domains of an E protein are from a different Flavivirus than the virus forming the backbone.
• the examples of present invention describe the TM1 and TM2 domain of the E protein of the West Nile virus. These domain have the sequence GGMSWITQGLLGALLLWMGINARD [SEQ ID NO: 14] and
• sequence elements form a continuous sequence without any intervening sequence elements. It is submitted that in between these sequence elements, additional amino acids may be present as long as the localisation of the protein at either the ER lumen or cytosol is not disturbed and proteolytic processing is maintained.
• nucleotide sequence can be that of the virus itself or can refer to a sequence in a vector.
• a suitable vector for cloning Flavivirus and chimeric version are, amongst other technologies, Bacterial Artificial Chromosomes, as described in more detail below.
• the methods and compounds of the present invention have medicinal application, whereby the virus or a vector encoding the virus can be used to vaccinate against the arenavirus which contains the G protein that was cloned in the Flavivirus.
• the proteins from the Flavivirus equally provide protection such that the compounds of the present invention can be used to vaccinate against a Flavivirus and an arenavirus using a single virus or DNA vaccine.
• Bacterial Artificial Chromosomes and especially the use of inducible BACS as disclosed by the present inventors in WO2014174078, is particularly suitable for high yield, high quality amplification of cDNA of RNA viruses such as chimeric constructs of the present invention.
• a BAC as described in this publication BAC comprises:
• RNA virus genome a viral expression cassette comprising a cDNA of an the RNA virus genome and comprising cis-regulatory elements for transcription of said viral cDNA in mammalian cells and for processing of the transcribed RNA into infectious RNA virus.
• RNA virus genome is a chimeric viral cDNA construct of an RNA virus genome and an arenavirus G protein .
• the viral expression cassette comprises a cDNA of a positive-strand RNA virus genome, an typically a RNA polymerase driven promoter preceding the 5' end of said cDNA for initiating the transcription of said cDNA, and an element for RNA self-cleaving following the 3' end of said cDNA for cleaving the RNA transcript of said viral cDNA at a set position.
• the BAC may further comprise a yeast autonomously replicating sequence for shuttling to and maintaining said bacterial artificial chromosome in yeast.
• a yeast ori sequence is the 2m plasmid origin or the ARS1 (autonomously replicating sequence 1) or functionally homologous derivatives thereof.
• RNA polymerase driven promoter of this first aspect of the invention can be an RNA polymerase II promoter, such as Cytomegalovirus Immediate Early (CMV- IE) promoter, or the Simian virus 40 promoter or functionally homologous derivatives thereof.
• CMV- IE Cytomegalovirus Immediate Early
• the RNA polymerase driven promoter can equally be an RNA polymerase I or III promoter.
• the BAC may also comprise an element for RNA self-cleaving such as the cDNA of the genomic ribozyme of hepatitis delta virus or functionally homologous RNA elements.
• “Acceptable carrier, diluent or excipient” refers to an additional substance that is acceptable for use in human and/or veterinary medicine, with particular regard to immunotherapy.
• an acceptable carrier, diluent or excipient may be a solid or liquid filler, diluent or encapsulating substance that may be safely used in systemic or topic administration.
• a variety of carriers, well known in the art may be used.
• These carriers may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulphate and carbonates, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulphates, organic acids such as acetates, propionates and malonates and pyrogen-free water.
• any safe route of administration may be employed for providing a patient with the DNA vaccine.
• oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intra-muscular, intra-dermal, subcutaneous, inhalational, intraocular, intraperitoneal, intracerebroventricular, transdermal and the like may be employed.
• Intra-muscular and subcutaneous injection may be appropriate, for example, for administration of immunotherapeutic compositions, proteinaceous vaccines and nucleic acid vaccines. It is also contemplated that microparticle bombardment or electroporation may be particularly useful for delivery of nucleic acid vaccines.
• Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, suppositories, aerosols, transdermal patches and the like. These dosage forms may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release of the therapeutic agent may be effected by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose. In addition, the controlled release may be effected by using other polymer matrices, liposomes and/or microspheres.
• DNA vaccines suitable for oral or parenteral administration may be presented as discrete units such as capsules, sachets or tablets each containing a pre determined amount of plasmid DNA, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion.
• Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more agents as described above with the carrier which constitutes one or more necessary ingredients.
• the compositions are prepared by uniformly and intimately admixing the DNA plasmids with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.
• compositions may be administered in a manner compatible with the dosage formulation, and in such amount as is effective.
• the dose administered to a patient should be sufficient to effect a beneficial response in a patient over an appropriate period of time.
• the quantity of agent (s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof, factors that will depend on the judgement of the practitioner.
• DNA vaccine may be delivered by bacterial transduction as using live- attenuated strain of Salmonella transformed with said DNA plasmids as exemplified by Darji et a/. (2000) FEMS Immunol Med Microbiol 27, 341-349 and Cicin-Sain et al. (2003) J Virol 77, 8249-8255 given as reference.
• the DNA vaccines are used for prophylactic or therapeutic immunisation of humans, but can for certain viruses also be applied on vertebrate animals (typically mammals, birds and fish) including domestic animals such as livestock and companion animals.
• the vaccination is envisaged of animals which are a live reservoir of viruses (zoonosis) such as monkeys, dogs, mice, rats, birds and bats.
• vaccines may include an adjuvant, i.e. one or more substances that enhances the immunogenicity and/or efficacy of a vaccine composition
• life vaccines may eventually be harmed by adjuvants that may stimulate innate immune response independent of viral replication.
• Non limiting examples of suitable adjuvants include squalane and squalene (or other oils of animal origin); block copolymers; detergents such as Tween-80; Quill A, mineral oils such as Drakeol or Marcol, vegetable oils such as peanut oil; Corynebacterium-derived adjuvants such as Corynebacterium parvum; Propionibacterium-derived adjuvants such as Propionibacterium acne; Mycobacterium bovis (Bacille Calmette and Guerin or BCG); interleukins such as interleukin 2 and interleukin 12; monokines such as interleukin 1; tumour necrosis factor; interferons such as gamma interferon; combinations such as saponin- aluminium hydroxide or Quil-A aluminium hydroxide; liposomes; ISCOMt) and ISCOMATRIX (B) adjuvant; mycobacterial cell wall extract; synthetic glycopeptides such as muramyl dipeptides or other derivatives; A
• PLLAV-YFV17D-LASV-GPC Lassa glycoprotein with the N-terminal signal peptide sequence (SSP) and the GP2 transmembrane domain (TM) deleted.
• the LASV glycoprotein cleavage site was mutated (R246A) to keep the precursor GPC (GP1 and GP2 linked).
• R207C and G360C bind GP1 and GP2 covalently
• E329P described in Hastie et al (2017) Science 356, 923- 928, were introduce to improve stability.
• PLLAV-YFV17D-LASV-GPC was transfected into BHK21J cells and typical CPE was observed as well as the virus supernatant harvested from them formed markedly smaller plaques compared to the plaque phenotype of YFV17D ( Figure 2A). Therefore, the resulting transgenic virus (YFV17D-LASV-GPC) is further attenuated, and virus yields were at least 10-fold less compared to YFV17D.
• the stability of PLLAV-YFV17D-LASV-GPC was determined by performing RT-PCR to detect the transgene insert in virus samples that were harvested during serial passage of the YFV17D-LASV-GPC ( Figure 2B). Sequencing of the RT-PCR products showed that LASV-GPC insert with no mutations can be detected at least until passage 5 in BHK21J cells.
• mice were monitored daily for morbidity/mortality and blood was sampled for serological analysis at baseline and with two-week intervals.
• the vaccine was safe as no adverse effects were observed in any of the vaccinated mice.
• Some animals (4 of the 9 mice) were boosted two weeks after first inoculation with the PLLAV or LAV YFV 17 D- LASV-GPC using same dose and route than in the first vaccination ( Figure 3).
• the immunogenicity analysis for YFV17D-LASV-GPC revealed that at 14 days post vaccination there were specific antibodies against LASV in 3 and 1 mice vaccinated with PLLAV or LAV respectively.
• T-cell response is the main determinant responsible for providing protection against LASV infection. Therefore, the T cells responses were analyzed in both groups at 4 months post vaccination. This analysis showed that there was T cells responses against LASV and YFV in all the mice vaccinated with YFV17D-LASV-GPC (LAV) and in 7 out of 9 after vaccination with the PLLAV version ( Figure 4). These T cell responses can hence be considered to confer immunity and protection against LASV infection.
• construct#2 PLLAV-YFV17D-LASV-GP CCS signal peptide deleted, transmembrane domain gp2 deleted,cleavage site restored(R246R)aND MutatloNS R207C,E329P aND G360C) -End YFE (amino acids 1-40)
• Lassa Josiah strain G protein sequence SEQ ID NO: 9 (amino acids 1-58: signal sequence (amino acids 59-259: GP1 domain)
• amino acids 438-481 transmembrane domain and cytoplasmic tail
• HLSIPNFNQY 150 EAMSCDFNGG KISVQYNLSH SYAGDAANHC GTVANGVLQT FMRMAWGGSY 200 IALDSGRGNW DCIMTSYQYL IIQNTTWEDH CQFSRPSPIG YLGLLSQRTR 250 DIYISRRLLG TFTWTLSDSE GKDTPGGYCL TRWMLIEAEL KCFGNTAVAK 300 259

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• 2020
  • 2020-09-11 BR BR112022004378A patent/BR112022004378A2/pt unknown
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  • 2020-09-11 WO PCT/EP2020/075541 patent/WO2021048402A1/en not_active Ceased
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• 2022
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