WO2021255221A1 - Vaccin contre le virus de la grippe porcine comprenant une construction d'acide nucléique codant pour des antigènes de lignées de virus spécifiques - Google Patents

Vaccin contre le virus de la grippe porcine comprenant une construction d'acide nucléique codant pour des antigènes de lignées de virus spécifiques Download PDF

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WO2021255221A1
WO2021255221A1 PCT/EP2021/066548 EP2021066548W WO2021255221A1 WO 2021255221 A1 WO2021255221 A1 WO 2021255221A1 EP 2021066548 W EP2021066548 W EP 2021066548W WO 2021255221 A1 WO2021255221 A1 WO 2021255221A1
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nucleic acid
antigen
swine
acid sequence
iav
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PCT/EP2021/066548
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English (en)
Inventor
Mark A MOGLER
Basav Hangalapura NAGARAJ
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Intervet International B.V.
Intervet Inc.
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Priority to EP21733126.3A priority Critical patent/EP4168428A1/fr
Priority to CN202180043379.2A priority patent/CN115836082A/zh
Priority to JP2022577345A priority patent/JP2023530135A/ja
Publication of WO2021255221A1 publication Critical patent/WO2021255221A1/fr

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    • 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
    • C07K14/08—RNA viruses
    • C07K14/11—Orthomyxoviridae, e.g. influenza virus
    • 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
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses

Definitions

  • the nucleic acid construct typically comprises, in the order from 5’ to 3’, the backbone virus nonstructural protein open reading frame, a subgenomic promoter followed by the first HA antigen gene sequence, interstitial sequence, a second subgenomic promoter sequence followed by a second HA antigen gene, and finally the backbone virus 3’ untranslated region.
  • the present invention further provides nucleic acid constructs in which the IAV-S HA antigens are arranged in the specific order as defined in the first aspect and in which the IAV-S antigens are from the specific strains as defined in the second aspect.
  • the present invention provides an immunogenic composition, comprising the RNA replicon particle as described herein.
  • the present invention provides an immunogenic composition comprising a combination of RNA replicon particles, the combination comprising a first RNA replicon particle comprising the nucleic acid construct according to the first embodiment and a second RNA replicon particle comprising the nucleic acid construct according to the second embodiment.
  • the vaccine of the present invention may be used in the prevention or treatment of a disease caused by a Swine influenza A virus in a subject.
  • the present invention provides a method of immunizing a porcine against a swine influenza A virus, the method comprising administering to the porcine an immunologically effective amount of the vaccine of the present invention.
  • the present invention further provides replicon particles as described in the third aspect, wherein the nucleic acid constructs encode IAV-S HA antigens, which are arranged in the specific order as defined in the first aspect and/or in which the IAV-S antigens are from the specific strains as defined in the second aspect in combination with replicon particles as described in the fourth aspect.
  • Figure 4 HI antibody titers induced by single-gene RNA particle encoding one HA antigen of Gent 1984 lineage IAV-S.
  • Figure 6 HI antibody titers induced by dual-gene RNA particle encoding one HA antigen of pandemic (EUHAl-11) and another of Gentl984 (EUHA3-4) or one HA antigen of Scotl994 (EUH1- 15, EUHAl-17) and another of EurAsianAvian (EUHAl-3 & EUHAl-5) lineage IAS strains in two different positions.
  • Figure 7 Neuraminidase inhibition (NI) antibody titers induced by single-gene RNA particle encoding one NA antigen of EurAsianAvian (EA) lineage IAV-S.
  • NI Neuraminidase inhibition
  • EA EurAsianAvian
  • Figure 9 (NI) antibody titers induced by single-gene RNA particle encoding one NA antigen of Scot/94 lineage IAV-S.
  • Figure 12 NI antibody titers induced by dual-gene RNA particles encoding one NA antigen of EurAsianAvian (EUNA1-2) and another NA of Gentl984 (EUNA2-7) or triple-gene RNA particles encoding one NA antigen each of EurAsianAvian (EUNA1-2), Gentl984 (EUNA2-7) and Scotl994 (EUHNA2-6) or Pdm09 (EUNA1-4) lineage IAV-S strains in different positions. Definition of terms:
  • a nucleic acid construct is an artificially constructed segment of nucleic acid (e.g. DNA, RNA, mRNA), typically for transplantation into a target cell.
  • nucleic acid e.g. DNA, RNA, mRNA
  • pig or “swine” or “porcine” are used interchangeably and include all domesticated porcine species, unless otherwise indicated.
  • replicon refers to a modified RNA viral genome that lacks one or more elements (e.g., coding sequences for structural proteins) that if they were present, would enable the successful propagation of the parental virus in cell cultures or animal hosts. In suitable cellular contexts, the replicon will amplify itself and may produce one or more sub-genomic RNA species.
  • RNA replicon particle is an RNA replicon packaged in structural proteins, e.g., the capsid and glycoproteins, which may be derived from an alphavirus, e.g., is an alphavirus RNA replicon particle as described by Pushko et al, [Virology 239(2):389-401 (1997)], but may also be a Sindbis virus [Bredenbeek et al., 1993, J. of Virol., vol. 67, p. 6439-6446], and Semliki Forest virus [Liljestrom & Garoff, 1991, Biotechnology (NY), vol. 9, p.
  • RNA RP of the present invention is an alphavirus RNA RP.
  • non-IAV-S is used to modify terms such as pathogen, and/or antigen (or immunogen) to signify that the respective pathogen, and/or antigen (or immunogen) is neither an IAV-S pathogen nor an IAV-S antigen (or immunogen) and that a non- IAV-S protein antigen (or immunogen) does not originate from an IAV-S.
  • adjuvant and “immune stimulant” are used interchangeably herein, and are defined as one or more substances that cause stimulation of the immune system.
  • an adjuvant is used to enhance an immune response to one or more vaccine antigens/isolates.
  • adjuvants are agents that nonspecifically increase an immune response to a particular antigen, thus reducing the quantity of antigen necessary in any given vaccine, and/or the frequency of injection necessary in order to generate an adequate immune response to the antigen of interest.
  • an adjuvant is used to enhance an immune response to one or more vaccine antigens/isolates.
  • a “nonadjuvanted vaccine” is a vaccine or a multivalent vaccine that does not contain an adjuvant.
  • pharmaceutically acceptable is used adjectivally to mean that the modified noun is appropriate for use in a pharmaceutical product.
  • an excipient in a pharmaceutical vaccine it characterizes the excipient as being compatible with the other ingredients of the composition and not disadvantageously deleterious to the intended recipient animal, e.g., porcine.
  • the nucleic acid constructs according to the first and/or second embodiment of the first and/or second aspect may be included in an expression cassette incorporating the nucleic acid sequences encoding hemagglutinin (HA) antigens as described above as heterologous genes together with transcription- and/or expression controlling nucleic acid sequences, such as alphavirus sub-genomic promoter sequences etc, and which are suitable for expression of the HA antigens.
  • Such expression cassettes can be generated using well known techniques by incorporating the heterologous nucleic acid sequences encoding the HA antigens in a vector, such as DNA vectors or RNA vectors.
  • the vector can be a viral replicon backbone, such as an RNA replicon particle (RP), and preferably is an alphavirus RNA replicon particle.
  • RP RNA replicon particle
  • alphavirus RNA replicon particle is well known as “non-transmissible”, “single-cycle”, or “propagation-incompetent” virus like particle vector.
  • the genome can encode one or more heterologous genes from its 26S subgenomic promoter.
  • the RP can replicate within the target cell without producing a progeny, and in this way deliver and express heterologous antigen(s) to the immune system of a target animal.
  • the alphavirus RNA RP may be based on a human Venezuelan equine encephalitis vaccine (VEEV) TC-83 strain.
  • the first RNA replicon particle preferably an alphavirus RNA replicon particle, comprising a first nucleic acid construct comprising, in the order from 5 ’ to 3 ’ of the nucleic acid sequence: a first nucleic acid sequence encoding a first HA antigen of IAV-S of the Scot/94 lineage, and a second nucleic acid sequence encoding a second HA antigen of IAV-S of the EA lineage, and the immunogenic composition further comprises:
  • the second RNA replicon particle preferably an alphavirus RNA replicon particle, comprising a nucleic acid construct comprising third and fourth nucleic acid sequences encoding, in the order from 5’ to 3’ of the nucleic acid sequence, third and fourth HA antigens of IAV-S, wherein the third HA antigen encoded by the third nucleic acid sequence is of the Scot/94 lineage from strain A/swine/Italy/3033-1/2015 (H1N2), preferably of SEQ ID NO.
  • the present invention provides an immunogenic composition, such as a vaccine, comprising at least first, second and third RNA replicon particles, the first RNA replicon particle comprising a nucleic acid construct, comprising, in the order from 5 ’ to 3 ’ of the nucleic acid sequence, first and second nucleic acid sequences encoding first and second HA antigens of IAV-S, wherein the first HA antigen is of the Scot/94 lineage, and the second HA antigen is of the EA lineage, the second RNA replicon particle comprising a nucleic acid construct comprising, in the order from 5 ’ to 3 ’ of the nucleic acid sequence, third and fourth nucleic acid sequences encoding third and fourth HA antigens of IAV-S, wherein the third HA antigen is of the Gent/84 lineage, and the fourth HA antigen is of the pdm09 lineage, and the third RNA replicon particle comprises a nucleic acid construct, comprising
  • one or more RNA RPs that encode one or more HAs and/or NAs originating from IAV-S can be added together with one or more other vectors encoding one or more porcine antigen and/or one or more killed and/or modified (attenuated) live virus isolates such as one or more killed or modified live IAS-V strain, one or more killed and/or modified live PRRS virus, one or more killed and/or modified live PCV, one or more killed, and/or modified live TGE, one or more killed and/or modified live PPRV, one or more killed and/or modified live PPV, one or more killed and/or modified live PRV and one or more killed and/or modified live PED.
  • one or more killed or modified live IAS-V strain such as one or more killed and/or modified live PRRS virus, one or more killed and/or modified live PCV, one or more killed, and/or modified live TGE, one or more killed and/or modified live PPRV, one or more killed and/or modified live PPV, one
  • one or more alphavirus RNA replicon particles (RPs) that encode one or more HAs or NAs originating from IAV-S can be added together with one or more other vectors encoding one or more porcine antigen and/or added together with one or more killed and/or modified (attenuated) live bacteria that can infect swine too, including one or more killed and/or modified live Pasteurella multocida (of one or more multiple serotypes), Salmonella ssp., Escherichia coli (of one or more multiple serotypes), Haemophilus parasuis, Lawsonia intracellularis, Mycoplasma ssp.
  • RPs alphavirus RNA replicon particles
  • the immunogenic composition of the present invention can be used as a vaccine, which may be a non- adjuvanted vaccine or an adjuvanted vaccine.
  • the present invention further comprises vaccines (multivalent) vaccines comprising the immunogenic compositions of the present invention.
  • the vaccines are a nonadjuvanted vaccine.
  • the vaccines comprise an adjuvant.
  • the vaccines and multivalent vaccines of the present invention can be administered as a primer vaccine and/or as a booster vaccine.
  • a vaccine of the present invention is administered as a one-shot vaccine (one dose), without requiring subsequent administrations.
  • the primer vaccine and the booster vaccine can be administered by the identical route.
  • the primer vaccine and the booster vaccine are both administered by intradermal injection. In other embodiments of this type, the primer vaccine and the booster vaccine are both administered by intramuscular injection. In alternative embodiments, in the case of the administration of both a primer vaccine and a booster vaccine, the administration of the primer vaccine can be performed by one route and the booster vaccine by another route. In certain embodiments of this type, the primer vaccine can be administered by intradermal injection and the booster vaccine can be administered orally. In related embodiments of this type, the primer vaccine can be administered by intramuscular injection and the booster vaccine can be administered orally. In other embodiments of this type, the primer vaccine can be administered by intramuscular injection and the booster vaccine can be administered by intradermal injection. In still other embodiments of this type, the primer vaccine can be administered by intradermal injection and the booster vaccine can be administered by intramuscular injection. The skilled artisan will appreciate that the vaccine composition is preferably formulated appropriately for each type of recipient animal and route of administration.
  • the vaccines of the present invention are administered in 0.05 mL to 3 mL doses.
  • the dose administered is 0.1 mL to 2 mL.
  • the dose administered is 0.2 mL to 1.5 mL.
  • the dose administered is 0.3 to 1.0 mL.
  • the dose administered is 0.4 mL to 0.8 mL.
  • a nucleic acid construct for use in the prevention of a disease caused by a Swine influenza A virus (IAV-S) in a subject comprising, in the order from 5’ to 3’ of the nucleic acid sequence: a first nucleic acid sequence encoding a first hemagglutinin (HA) antigen of IAV-S of the A/swine/Scotland/410440/1994-like Hl hU N2 (Scot/94) lineage, and a second nucleic acid sequence encoding a second HA antigen of IAV-S of the Eurasian avian-like Hl av Nl (EA) lineage.
  • HA hemagglutinin
  • EA Eurasian avian-like Hl av Nl
  • nucleic acid construct for use as defined in [1] or [2], wherein the first HA antigen encoded by the first nucleic acid sequence comprises an amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having at least 85% sequence identity thereof.
  • nucleic acid construct for use as defined in any one of [1] to [4], wherein the second HA antigen encoded by the second nucleic acid sequence comprises an amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least 90% sequence identity thereof.
  • RNA replicon particle comprising the nucleic acid construct as defined in any one of [1] to
  • RNA replicon particle comprising the nucleic acid construct as defined in any one of [6] to [10] ⁇
  • RNA replicon particle as defined in [13], which is a Venezuelan Equine Encephalitis (VEE) alphavirus RNA replicon particle
  • a method of immunizing a porcine against a swine influenza A virus comprising administering to the porcine an immunologically effective amount of the vaccine of any one of [18] to [20]
  • a nucleic acid construct comprising, in the order from 5’ to 3’ of the nucleic acid sequence: a first nucleic acid sequence encoding a first HA antigen of IAV-S of the
  • a nucleic acid construct for use in the prevention of a disease caused by a Swine influenza A virus in a subject comprising first and second nucleic acid sequences: a first nucleic acid sequence encoding a first HA antigen of IAV-S of the A/swine/Scotland/410440/1994-like Hl hU N2 (Scot/94) lineage from strain A/swine/Italy/3033-1/2015 (H1N2), and a second nucleic acid sequence encoding a second HA antigen of IAV-S of the Eurasian avian-like Hl av Nl (EA) lineage from strain A/swine/Italy/28762-3/2013 (H1N1).
  • a first nucleic acid sequence encoding a first HA antigen of IAV-S of the A/swine/Scotland/410440/1994-like Hl hU N2 (Scot/94) lineage from strain A/
  • RNA replicon particle comprising the nucleotide construct as defined in any one of [4] to
  • the present invention provides the following embodiments:
  • RNA replicon particle is an alphavirus RNA replicon particle.
  • a vaccine comprising the immunogenic composition as defined in any one of the preceding [1] to [13]
  • nucleic acid construct for use as defined in any one of [1] to [5], wherein the third NA antigen is from strain A/swine/England/373/2010 (H1N1) or A/swine/Italy/179057/2015 (H1N1).
  • RNA replicon particle as defined in [9] which is an alphavirus RNA replicon particle.
  • a vaccine comprising the immunogenic composition as defined in [12] or [13]
  • VEE replicon vectors designed to express haemagglutinin (HA) or neuraminidase (NA) genes were constructed as previously described [see, U.S. 9,441 ,247 B2; the contents of which are hereby incorporated herein by reference], with the following modifications.
  • the TC-83-derived replicon vector “pVEK” [disclosed and described in U.S. 9,441 ,247 B2] was digested with restriction enzymes Ascl and Pad.
  • the synthetic gene cassette was then ligated into the digested pVEK vector, and the resulting clones were re-named “pVHV-respective RP code.
  • the “pVHV” vector nomenclature was chosen to refer to pVEK-derived replicon vectors containing transgene cassettes cloned via the Ascl and Pad sites in the multiple cloning site of pVEK.
  • RNA for the replicon and helper components were combined and mixed with a suspension of Vero cells, electroporated in 4 mm cuvettes, and returned to OptiPro SFM cell culture media (Thermo Fisher, Waltham, MA). Following overnight incubation, alphavirus RNA replicon particles were purified, formulated in phosphate buffered saline with 5 % sucrose (w/v) and 1 % swine serum, passed through a 0.22 micron membrane filter, and dispensed into aliquots for storage. Titer of functional RP was determined by immunofluorescence assay on infected Vero cell monolayers. Batches of RP were identified according to the gene encoded by the packaged replicon (Tables la&b).
  • the new synthetic construct also contained 50 nucleotides of heterologous non-coding sequence, and a third copy of the native TC- 83 sgRNA promoter and 5’ untranslated sgRNA region sequence to the 5’ of the third NA gene sequence.
  • the 3’ region from the third NA gene sequence consisted of the 3’ untranslated region of TC-83, until the corresponding Sphl site of the parental pVDG vector.
  • the triple-gene vectors were termed “pVTG” to differentiate them from related vectors pVEK, pVHV, and pVDG.
  • the cells and media were centrifuged in the presence of prepared Cellufme Sulfate® resin, washed with phosphate buffered saline containing 5% sucrose (w/v), and eluted with 200 mM Na 2 SC> 4 + 5% sucrose (w/v) buffer.
  • Eluted RP were passed through a 0.22 micron membrane filter, and dispensed into aliquots for storage. Titer of functional RP was determined by immunofluorescence assay on infected Vero cell monolayers.
  • RNA particles, NA source strains, lineages and GenBank accession number (Accession#) are listed in Table lb. RNA particles, NA source strains, lineages and GenBank accession number (Accession#)
  • RNA particle vaccines encoding either single or multiple HA or NA genes with Xsolve50 adjuvant per pig per time. The respective vaccination was repeated at approximately 8 weeks of age and the blood samples were collected approximately 9 weeks of age and were used for either Hemagglutination inhibition (HI) assay or Neuraminidase inhibition (NI) assays to quantify the levels of antigen specific antibody levels.
  • HI Hemagglutination inhibition
  • NI Neuraminidase inhibition
  • Hemagglutination inhibition (HI) assay All serum samples were heat inactivated at 56°C for 30 min, subsequently treated with 0.25% periodate, followed by 0.75% glycerol and adsorbed with 2.6% chicken red blood cells to remove non specific agglutinins.
  • serial dilutions of pre-treated serum were incubated for 1 hour with 8 hemagglutination units of SIV strains listed in table lc or Id as HA antigens. Subsequently, the mixture was incubated with 0.2% chicken red blood cells for 1 hour at room temperature and plates were read for inhibition of agglutination. The reciprocal of the highest serum dilution that completely inhibited erythrocyte agglutination was assigned as the HI titer and expressed in log base 2 values.
  • Test antigens were titrated to determine the dilution that is able to yield 70% of the maximum signal. Equal volumes of NA antigen were added to serial dilutions of serum in fetuin-coated wells during the overnight 37 °C incubation. Optical density (OD) values were normalized to the values from positive control wells containing no serum. Neuraminidase inhibition titers were defined as the reciprocal of the interpolated serum dilution having an extinction value equal to 50% inhibition in comparison with the control and were expressed in log base 2 values.
  • the adjuvanted vaccine was administered to 5 pigs in two-intramuscular (IM) vaccinations at 5 and 8 weeks of age (2 mL per dose; 3 x 5 x 10 6 RP/dose, Vaccinates).

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Abstract

La présente invention concerne une construction d'acide nucléique codant pour un premier antigène d'hémagglutinine (HA) de la grippe porcine A (IAV-S) de la lignée Scot/94 issue de la souche A/porcine/Italie/3033-1/2015 (H1N2) ou une séquence d'acides aminés ayant au moins 85 % d'identité de séquence et pour un second antigène d'hémagglutinine (HA) de la grippe porcine A (IAV-S) de la lignée de type aviaire eurasien (EA) issue de la souche A/porcine/Italie/28762-3/2013 (H1N1) ou une séquence d'acides aminés ayant au moins 90 % d'identité de séquence, et une construction d'acide nucléique qui code pour un premier antigène d'HA d'IAV-S de la lignée Gent/84 issue de la souche A/porcine/Italie/240849/2015 (H3N2) ou une séquence d'acides aminés ayant au moins 95 % d'identité de séquence et pour un second antigène d'HA d'IAV-S de la lignée pandémique 09 (pdm09) issue de la souche A/porcine/Angleterre/373/2010 (H1N1) ou une séquence d'acides aminés ayant au moins 95 % d'identité de séquence. Selon d'autres modes de réalisation, la présente invention concerne des particules de réplicon d'ARN comprenant une ou les deux constructions d'acide nucléique, une composition immunogène, telle qu'un vaccin, qui peut être utilisée contre une infection par le virus de la grippe A, et comprenant les particules de réplicon. L'invention concerne en outre des méthodes de fabrication et d'utilisation du vaccin.
PCT/EP2021/066548 2020-06-19 2021-06-18 Vaccin contre le virus de la grippe porcine comprenant une construction d'acide nucléique codant pour des antigènes de lignées de virus spécifiques WO2021255221A1 (fr)

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EP21733126.3A EP4168428A1 (fr) 2020-06-19 2021-06-18 Vaccin contre le virus de la grippe porcine comprenant une construction d'acide nucléique codant pour des antigènes de lignées de virus spécifiques
CN202180043379.2A CN115836082A (zh) 2020-06-19 2021-06-18 包含编码特定病毒谱系抗原的核酸构建体的甲型猪流感病毒疫苗
JP2022577345A JP2023530135A (ja) 2020-06-19 2021-06-18 特定のウイルス系統の抗原をエンコードする核酸構築物を含むブタインフルエンザａウイルスワクチン

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