WO2016127262A1 - Nucléoprotéine d'orthomyxovirus multimérisée et ses utilisations - Google Patents

Nucléoprotéine d'orthomyxovirus multimérisée et ses utilisations Download PDF

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WO2016127262A1
WO2016127262A1 PCT/CA2016/050133 CA2016050133W WO2016127262A1 WO 2016127262 A1 WO2016127262 A1 WO 2016127262A1 CA 2016050133 W CA2016050133 W CA 2016050133W WO 2016127262 A1 WO2016127262 A1 WO 2016127262A1
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nlp
influenza
nlps
recombinant
ssrna
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Denis Leclerc
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Folia Biotech Inc.
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16123—Virus like particles [VLP]
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    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
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    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16151—Methods of production or purification of viral material

Definitions

  • the present invention relates to the field of influenza vaccines and, in particular, to multimerized orthomyxovirus nucleoprotein and influenza vaccines comprising same.
  • TIV Trivalent inactivated vaccine
  • HA variable surface proteins
  • NA neuraminidase
  • influenza viruses are subjected to minor (drift) and major (shift) antigenic change which means that new vaccines have to be made each year to match the dominating strains (Ferguson NM, and Anderson RM., 2002, Nat Med, 8:562-3). Furthermore, these vaccines will be ineffective in case of a pandemic caused by the emergence of a novel influenza A strain that expresses variant HA and NA proteins (Luke CJ, and Subbarao K., 2006, Emerg Infect Dis , 12:66-72). [0003] Immunization with conserved influenza proteins, such as nucleoprotein (NP) or matrix protein (M1/M2), is known to induce a broad, heterotypic response against a multitude of strains.
  • NP nucleoprotein
  • M1/M2 matrix protein
  • Influenza virus NP is an internal protein, the major role of which is to encapsidate the viral genome to form a ribonucleoprotein. NP has been shown to be capable of binding single-stranded RNA with high affinity and little or no sequence specificity (Kingsbury D, et al, 1987, Virology, 156:396-403; Portela A, and Digard P., 2002, J Gen Virol, 83:723-34).
  • NP is extremely well conserved among influenza A strains (Heiny AT, et al, 2007, PLoS One, l l :el l90; Shu LL, et al, 1993, J Virol, 67:2723-9) and contains dominant CTL epitopes for most human HLA-types (Stanekova Z, and Vareckova E., 2010, Virol J, 7:351; Tan PT, et al, 2011, Hum Vaccin, 7:402-9).
  • NP immunization leads to a protection mainly mediated by CD8+ T lymphocytes that recognize CTL epitopes presented by infected cells (Wang M, et al, 2007, Vaccine, 25:2823-31; Yewdell JW, et al, 1985, Proc Natl Acad Sci USA, 82: 1785-9; Taylor PM, and Askonas BA., 1986, Immunology, 58:417-20) and that this protection is heterosubtypic (Epstein SL, et al, 2005, Vaccine, 23:5404-10; Guo L, et al, 2010, Arch Virol, 155: 1765-75; Wang W, et al, 2012, PLoS One, 7:e52488).
  • Canadian Patent No. 1,270,438 describes a T-cell inducing material comprising influenza virus NP, which is obtained by fragmentation of influenza virus and is capable of protection against heterologous strains of influenza.
  • International (PCT) Patent Application Publication No. WO 2010/021289 describes methods of oral administration of influenza HA or NP with a mucosal adjuvant, such as a CpG oligonucleotide.
  • International (PCT) Patent Application Publication No. WO 2010/144797 describes vaccine compositions comprising a pharmaceutically acceptable carrier and an antigen preparation, the antigen preparation comprising influenza NP and optionally influenza M protein.
  • International (PCT) Patent Application Publication No. WO 2014/085580 describes vaccine compositions comprising a dendritic cell targeting agent and an influenza antigen such as HA or NP. The antigen is attached, fused, coupled or conjugated to the targeting agent.
  • Chinese Patent Application No. CN101899461 describes a fusion gene encoding NP and M2e optimized using E. coli codons. Immunization of mice with the fusion protein provided protection against heterologous influenza virus.
  • VLPs viral- like-particles
  • J Viral structural proteins expressed in an ordered and repetitive fashion to form viral- like-particles (VLPs) are considerably more immunogenic than in their soluble form (Justewicz DM, et al, 1995, J Virol, 69:5414-21 ; Jennings G, and Bachmann M., 2008, Biol Chem, 389:521-36).
  • VLPs are composed of multiple copies of one or more recombinant viral structural proteins that can assemble spontaneously upon expression (Jennings G, and Bachmann M., 2008, ibid).
  • VLPs can activate both arms of the adaptive response by stimulating B-cell-mediated immunity, CD4 proliferative responses and cytotoxic T lymphocyte (CTL) responses (Noad R, and Roy P., 2003, Trends Microbiol, 11:438-44; Plummer EM, and Manchester M., 2011, Wiley Interdiscip Rev Nanomed Nanohiotechnol, 3: 174-96).
  • CTL cytotoxic T lymphocyte
  • NLPs using the recombinant DENV-2 capsid protein and oligonucleotides as a scaffold have been reported.
  • the NLPs were more immunogenic than the capsid protein alone and induced protective CD4+ and CD8+ cells in a viral encephalitis murine model (Gil
  • a VLP comprised of hepatitis B virus core protein, a recombinant M2 protein with three copies of M2e and NP epitopes has been described (Gao et al, 2013, Antiviral Res, 98:4-11). Administration of this 3M2e-NP-HBc VLP together with the SPOl oil-in-water adjuvant was able to protect mice against challenge with a heterologous influenza virus strain.
  • Chinese Patent No. CN101643721 describes a recombinant virus-like particle containing influenza matrix protein Ml, NA, HA and an M2eNP fusion protein.
  • the present invention relates to multimerized orthomyxovirus nucleoprotein and uses thereof.
  • One aspect of the invention relates to a nucleocapsid-like particle (NLP) having a rod-like shape and comprising a plurality of recombinant orthomyxoviral nucleoprotein (NP) polypeptides assembled with a single-stranded RNA (ssRNA) scaffold, the ssRNA being between about 120 and about 5000 nucleotides in length.
  • NLP nucleocapsid-like particle
  • NP orthomyxoviral nucleoprotein
  • ssRNA single-stranded RNA
  • NLPs of the present disclosure can comprise recombinant NP polypeptides having a sequence derived from an orthomyxovirus.
  • the NLPs can comprise recombinant NP polypeptides having a sequence derived from influenza virus.
  • the NLPs can comprise recombinant NP polypeptides having a sequence derived from an influenza virus type B or type A NP.
  • Another aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an NLP as described above and a pharmaceutically acceptable carrier or diluent.
  • compositions of the present disclosure can further comprise an adjuvant.
  • the adjuvant comprises Papaya mosaic virus (PapMV) virus-like particles (VLPs).
  • the PapMV VLPs comprise one or more influenza antigens fused to the PapMV coat protein.
  • the PapMV VLPs can be used as a vaccine platform that presents the one or more influenza antigens to immune cells.
  • the one or more influenza antigens are derived from the influenza M2 protein, for example, the M2e peptide or a fragment thereof.
  • Another aspect of the invention relates to a vaccine comprising the pharmaceutical composition.
  • NLP nucleocapsid-like particle
  • NP recombinant influenza nucleoprotein
  • ssRNA single-stranded RNA
  • Another aspect of the invention relates to a method of inducing an immune response against orthomyxoviruses in a subject comprising administering to the subject an effective amount of an NLP, a pharmaceutical composition, or a vaccine as described above.
  • the immune response is against influenza virus infection.
  • Another aspect of the invention relates to a method of vaccinating a subject against orthomyxovirus infection, more specifically an influenza virus infection, comprising administering to the subject an effective amount of an NLP, a pharmaceutical composition, or a vaccine as described above.
  • Another aspect of the invention relates to a fusion protein comprising an influenza nucleoprotein (NP) polypeptide and an M2e peptide, the M2e peptide fused to the C-terminus of the NP polypeptide and having the sequence EVETPIRNE [SEQ ID NO:9].
  • NP influenza nucleoprotein
  • M2e peptide fused to the C-terminus of the NP polypeptide and having the sequence EVETPIRNE [SEQ ID NO:9].
  • nucleocapsid-like particle having a rod-like shape and comprising a plurality of the fusion proteins described above assembled with a single-stranded RNA (ssRNA) scaffold, the ssRNA being between about 120 and about 5000 nucleotides in length.
  • NLP nucleocapsid-like particle
  • ssRNA single-stranded RNA
  • Another aspect of the invention relates to a method of inducing an immune response against influenza virus in a subject comprising administering to the subject an effective amount of an NLP having a rod-like shape and comprising a plurality of the fusion proteins described above assembled with a single-stranded RNA (ssRNA) scaffold, the ssRNA being between about 120 and about 5000 nucleotides in length.
  • Another aspect of the invention relates to a method of vaccinating a subject against influenza virus infection comprising administering to the subject an effective amount of an NLP having a rod-like shape and comprising a plurality of the fusion proteins described above assembled with a single-stranded RNA (ssRNA) scaffold, the ssRNA being between about 120 and about 5000 nucleotides in length.
  • an NLP having a rod-like shape and comprising a plurality of the fusion proteins described above assembled with a single-stranded RNA (ssRNA) scaffold, the ssRNA being between about 120 and about 5000 nucleotides in length.
  • ssRNA single-stranded RNA
  • Figure 1 shows results from the production and purification of recombinant nucleoprotein (NP):
  • A shows the partial amino acid sequence of the NP protein produced (based on GenBank: ACP41106.1), which includes a C-terminal 6xHis tag;
  • B shows SDS- PAGE evaluation of the induction and purification of NP;
  • C shows Western-Blot analysis using anti-NP antibody (B, C)
  • Lane 1 bacterial lysate before induction.
  • Lane 2 bacterial lysate after 16h induction at 22°C.
  • Lane 3 shows the elution profile of the purified rNP on a Superdex 200 Size-exclusion chromatography (SEC) column.
  • SEC Size-exclusion chromatography
  • Figure 2 shows the biochemical characterization of NLPs formed with poly-U and poly-C ssRNA (NLP-Poly-U and NLP-Poly-C):
  • A shows the elution profile of both NLPs on a Superdex 200 SEC column;
  • B Left panel: Size distribution of the NLPs as measured by dynamic light scattering (DLS), and
  • Right panel Size of the NLPs as measured under a temperature gradient. Dotted line indicates 37°C, and
  • C Transmission electron microscope images at magnification 49 OOOx of rNP, NLP -Poly-U and NLP-Poly-C.
  • FIG. 3 shows that immunization of mice with NLP-Poly-C or NLP-Poly-U leads to similar humoral responses: BALB/c mice (5/group) were vaccinated twice at a 14-day interval with rNP, NLP-Poly-C or NLP-Poly-U by intramuscular (i.m) route. Serum was obtained at day 14 and 28 and ELISA assays were conducted to evaluate the levels of total IgG (A) and IgG2a (B) titers. * P ⁇ 0.05, ** P ⁇ 0.01.
  • Figure 4 shows the stability of the NLPs: elution profiles of both NLPs on a Superdex 200 SEC column after being kept for 3 months at 2-8°C. Monomeric NP elutes near 18 mL and absorb more at 280 than 254 nm. Various lengths of NLP elute between 8-12 mL and absorb more at 254 nm than 280 nm because of the presence of RNA.
  • Figure 5 shows the biochemical characterization of NLP-Poly-U produced by an alternative process: A. Elution profile of NLP-Poly-U on a Superdex 200 SEC column; B. Left panel: Size distribution of NLP-Poly-U as measured by dynamic light scattering (DLS).
  • DLS dynamic light scattering
  • FIG. 6 shows that multimerization of the monomeric rNP increases the humoral and cellular immune response to NP: BALB/c mice (10/group) were immunized once intramuscularly with formulation buffer or 0.5 ⁇ g of either rNP or NLP-Poly-U. Serum was obtained 14 days post-immunization and ELISAs were conducted to evaluate the levels of total IgG (A: left panel) and IgG2a (A: right panel). 14 days post-immunization, spleens were extracted and the ELISPOT assay was performed.
  • the number of IFN- ⁇ secreting cells was determined by subtracting the spots in media alone from the number of spots counted in wells reactivated with either the H-2K D peptide (B: left panel) or NLP-Poly-U (B: right panel).
  • a boost immunization using the same antigen quantity was performed using 5 mice/group. 7 days after the boost, an ELISPOT assay (C) was performed. ** P ⁇ 0.01, *** P ⁇ 0.001.
  • Figure 7 shows the effect of adjuvant on immunization with monomeric or multimerized NP: BALB/c mice (10/group) were immunized once intramuscularly with formulation buffer or 0.5 ⁇ g of either rNP or NLP-Poly-U adjuv anted with 5, 10, 20 or 40 ⁇ g of PapMV VLPs (PAL). Serum was obtained 14 days post-immunization and ELISAs were conducted to evaluate the levels of total IgG (A: top panels) and IgG2a (A: bottom panels). 14 days post-immunization, spleens were extracted and the ELISPOT assay was performed.
  • BALB/c mice (10/group) were immunized once intramuscularly with formulation buffer or 0.5 ⁇ g of either rNP or NLP-Poly-U adjuv anted with 5, 10, 20 or 40 ⁇ g of PapMV VLPs (PAL). Serum was obtained 14 days post-immunization and ELISAs were conducted to evaluate the levels of total
  • the number of IFN- ⁇ secreting cells was determined by subtracting the spots in media alone from the number of spots counted in wells reactivated with either the H-2K D peptide (B: left panel) or NLP-Poly-U (B: right panel).
  • a boost immunization using the same antigen quantity was performed using 5 mice/groups. 7 days after the boost, ELISPOT assay (C) was performed. ** P ⁇ 0.01, *** P ⁇ 0.001.
  • Figure 8 shows that boost-immunization increases both the humoral and cellular responses to NP: BALB/c mice (5/group) were immunized once or twice intramuscularly at a 14-day interval with 10 ⁇ g of NLP alone or combined with 40 ⁇ g of PAL. Blood was collected on day 13 and on day 21. ELISAs were conducted to evaluate the levels of total IgG (A: left panel) and IgG2a (A: right panel). Splenocytes of mice immunized once were harvested 14 days after the immunization and reactivated with the H-2K D peptide (B: left panel) or NLP-Poly-U (B: right panel) to evaluate the IFN- ⁇ secretion by the T cells.
  • FIG. 9 shows the humoral response in vaccinated mice prior to influenza challenge: BALB/c mice (10/group) were immunized once intramuscularly with various vaccine formulations. At day 14 post-immunization, blood was collected and IgG2a titers to NP were measured. *** P ⁇ 0.001.
  • Figure 10 shows the effect of a single dose immunization in protection from infection with a lethal dose of influenza H1N1 : BALB/c mice (10/group) were immunized once intramuscularly with various vaccine formulations. 14 days post-immunization, the mice were challenged with either 120 pfu (1 LD 50 ) or 240 pfu (2 LD 50 ) of A/WSN/33 (H1N1) influenza virus and monitored for weight loss (A), survival (B) and clinical symptoms (C) for 14 days.
  • A weight loss
  • B survival
  • C clinical symptoms
  • Clinical symptom levels were noted as follows: 1 - lightly spiked fur; 2 - spiked fur, curved back; 3 - spiked fur, curved back, difficulty to move, light dehydration; 4 - spiked fur, curved back, difficulty to move, severe dehydration, closed eyes, ocular secretion. Statistical analysis is applied between each group. *P ⁇ 0.05, **P ⁇ 0.01 and ***P ⁇ 0.001.
  • Figure 11 shows the humoral and cellular responses of mice surviving the influenza virus challenge described in Figure 10: (A) IgG2a titers to NP were assessed in mice serum collected either just before infection or 14 days post-infection, and (B) the splenocytes of 5 mice from the Buffer group and from the 10 ⁇ g NLP + 40 ⁇ g PAL group were harvested 7 days post-infection and re-stimulated with the H-2K D peptide to evaluate IFN- ⁇ secretion by T cells. ND: non-detected. *** P ⁇ 0.001.
  • Figure 12 presents graphs summarizing the in vivo effect of NLP with and without PAL adjuvant on IgG2a titers: (A) anti-NP immune response elicited by NP vs. NLP; (B) anti-NP immune response elicited by NLP vs. NLP + PAL (amount of PAL: 20 meg), and (C) anti-NP immune response elicited by NP + PAL vs. NLP + PAL (amount of NP: 0.5 meg).
  • GTT Geometric Mean Titer
  • Figure 13 presents the amino acid sequences of: (A) influenza A/California/04/2009 [H1N1] NP (GenBank: ACP41106.1) [SEQ ID NO:4]; (B) influenza A/California/04/2009 [H1N1] NP comprising a 6xHis tag and additional C-terminal amino acids (underlined) that result from inclusion of Spel and Mlul restriction sites spaced by GCA in the corresponding DNA sequence [SEQ ID NO:5], and (C) recombinant NP as shown in (B) fused at the C- terminus to a M2e derived peptide (underlined) [SEQ ID NO: 6].
  • Figure 14 presents a flow-chart summarizing the process for the production and purification of NLP.
  • Figure 15 is a SDS-PAGE gel showing the purification of the His-tagged recombinant rNP protein from E. coli.
  • Figure 16 presents the DNA sequences encoding the ssRNA scaffolds (A) SRT500 [SEQ ID NO:7], and (B) SRT1517 [SEQ ID NO:8]. In the corresponding RNA sequences, the T nucleotides are replaced with U's.
  • Figure 17 shows the multimerization of rNP into NLPs using the SRT1517 ssRNA scaffold: (A) dynamic light scattering analysis showing that the particles have an average length of 50 nm, and (B) electron micrograph showing the shape of the multimerized NP NLPs that look like elongated structures with irregular edges (magnification 49 OOOx).
  • Figure 18 shows the multimerization of NP into NLPs using the SRT500 ssRNA scaffold: (A) dynamic light scattering analysis showing an average particle length of 30 nm and a width of 13-18 nm, and (B) electron micrograph showing the shape of the multimerized NP NLPs that look like elongated structures with irregular edges (magnification 49 OOOx).
  • Figure 19 presents a flow-chart summarizing the process for the production of NPM2e NLPs using NP C-terminally fused to a M2e peptide ("NPM2ec").
  • Figure 20 shows (A) a SDS-PAGE analysis of in process samples taken from the assembly reaction and following polishing steps during the production of NLP-rNPM2e; (B) dynamic light scattering analysis of NLPs formed by multimerization of NPM2ec with a ssRNA scaffold showing an average particle length of 30 nm and width of 13-18nm, and (C) electron micrograph showing the shape of the multimerized NPM2e NLPs that look like elongated structures with irregular edges (magnification 49 OOOx).
  • Figure 21 presents the amino acid sequences of representative NPs: (A) influenza type B NP (GenBank: NP_056661.1) [SEQ ID NO: 10], (B) influenza B/Wisconsin/01/2010 (GenBank: AFH57958) [SEQ ID NO:31], (C) influenza A/New York/78/2002 [H1N2] NP (GenBank: AAY78943.1) [SEQ ID NO: 11], and (D) influenza A/Switzerland/9243/99 [H3N2] NP (GenBank: CAD30200.1) [SEQ ID NO: 12].
  • Figure 22 presents (A) the RNA sequence encoding the SRT1517 ssRNA scaffold [SEQ ID NO:32]; and (B) the 76nt long ssDNA used for assembly of the closed ring structure [SEQ ID NO: 33].
  • Figure 23 presents a flow-chart summarizing the process for the production of rodlike NLPs using the SRT1517 ssRNA scaffold of Fig. 22A.
  • Figure 24 shows the structural characterization of the rod-like NLPs prepared according to the process of Fig. 23: (A) electron micrograph of the nanoparticles; (B) dynamic light scattering of the nanoparticles; and (C) elution profile on Superdex 200 SEC column with a peak at 9.07mL.
  • Figure 25 shows the structural characterization of the closed ring form of NP prepared according to the process of Fig. 23 with the 76nt ssDNA used as the scaffold: (A) electron micrograph of the closed ring nanoparticles; (B) dynamic light scattering of the rings; and (C) elution profile on Superdex 200 SEC column with a peak at 10.32mL.
  • Figure 26 shows that multimerization of the influenza NP into a rod-like form improves the immune response directed to the NP antigen: ELISAs were made with the serum of the immunized animals (5 per group) against the GST-NP antigen devoid of a 6xH tag: (A) Total IgG; and (B) IgG2a.
  • Figure 27 shows the effect of different vaccine formulations. BALB/c mice (10/group) were immunized twice i.m.
  • Figure 29 shows (A) dynamic light scattering analysis of influenza B NLPs; and (B) electron micrograph showing the shape of the multimerized influenza B NLPs (magnification 173 OOOx).
  • Figure 30 presents the RNA sequence encoding the optimized influenza B amino acid sequence of influenza B [SEQ ID NO:34] .
  • Figure 31 presents a flow-chart summarizing the process for the production of influenza B NP.
  • the present invention relates generally to nucleocapsid-like particles (NLPs) formed by multimerization of recombinant orthomyxoviral nucleoprotein (NP) with a single-stranded RNA (ssRNA) scaffold.
  • NLPs are formed by multimerization of recombinant influenza nucleoprotein with a single-stranded RNA scaffold.
  • NP has been previously reported to multimerize with ssRNA, but into a closed ring structure rather than an NLP (Ye et al, 2006, Nature, 444: 1078-1082; Chenavas et al, 2013, PLoS Pathogen, 9(3):el003275; Tarus et al, 2012, Biochimie, 94:776-785).
  • the NLPs described herein are capable of eliciting an improved antibody response against NP when compared to monomeric recombinant NP and, are further capable of an unexpected improved antibody response when compared to the closed ring structure.
  • the NLPs described herein have potential use in vaccines for protection against influenza virus infections.
  • influenza NP is conserved across various strains of influenza virus
  • certain embodiments of the invention contemplate the use of the NLPs as a vaccine to provide protection against infection with heterologous influenza strains (that is, strains other than the strain from which the NP comprised by the NLPs is derived).
  • the NLPs are also capable of improving the CTL response to NP when compared to the monomeric recombinant NP.
  • fusion of the NP at the C-terminus to a heterologous peptide does not interfere with the ability of the NP to form NLPs.
  • the invention relates to NLPs formed from an NP -peptide fusion protein in which the peptide comprises one or more epitopes from another influenza protein. NLPs comprising such fusion proteins can be used to induce an immune response against both NP and the protein from which the one or more epitopes are derived.
  • the disclosed NLPs are capable of improving the immune response to NP when used alone, certain embodiments as demonstrated herein contemplate the use of the NLPs in combination with an adjuvant in order to further enhance the immune response. According to certain embodiments, combination of the disclosed NLPs with an adjuvant that further presents one or more epitopes from another influenza protein, for example, can be used to induce an immune response against both NP and the protein from which the one or more epitopes are derived.
  • NLPs of the present disclosure can be combined with adjuvant, such as PapMV VLPs which according to certain embodiments can comprise one or more influenza antigens fused to the PapMV coat protein, to induce an immune response against the NP and the one or more influenza antigens fused to the PapMV VLP.
  • adjuvant such as PapMV VLPs which according to certain embodiments can comprise one or more influenza antigens fused to the PapMV coat protein, to induce an immune response against the NP and the one or more influenza antigens fused to the PapMV VLP.
  • the term “about” refers to an approximately +/-10% variation from a given value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.
  • the use of the word “a” or “an” when used herein in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of "one or more,” “at least one” and “one or more than one.”
  • the terms “comprising,” “having,” “including” and “containing,” and grammatical variations thereof, are inclusive or open-ended and do not exclude additional, unrecited elements and/or method steps.
  • the term “consisting essentially of when used herein in connection with a composition, use or method, denotes that additional elements and/or method steps may be present, but that these additions do not materially affect the manner in which the recited composition, method or use functions.
  • the term “consisting of when used herein in connection with a composition, use or method excludes the presence of additional elements and/or method steps.
  • composition, use or method described herein as comprising certain elements and/or steps may also, in certain embodiments consist essentially of those elements and/or steps, and in other embodiments consist of those elements and/or steps, whether or not these embodiments are specifically referred to.
  • subject and patient refer to an animal in need of treatment.
  • animal refers to both human and non-human animals, including, but not limited to, mammals, birds and fish, and encompasses domestic, farm, zoo, laboratory and wild animals, such as, for example, cows, pigs, horses, goats, sheep and other hoofed animals; dogs; cats; chickens; ducks; non-human primates; guinea pigs; rabbits; ferrets; rats; hamsters and mice.
  • Administration of the disclosed NLPs "in combination with" one or more additional agents is intended to include simultaneous (concurrent) administration and consecutive administration.
  • Consecutive administration encompasses various orders of administration of the agent(s) and the NLPs to the subject with administration of the agent(s) and the NLPs being separated by a defined time period that may be short (for example in the order of minutes) or extended (for example in the order of days or weeks).
  • Immunization and “vaccination” are used interchangeably herein to refer to the administration of a vaccine to a subject for the purposes of raising a protective immune response. Immunization can be accomplished using various methods depending on the subject to be treated including, but not limited to, intraperitoneal injection (i.p.), intravenous injection (i.v.), intramuscular injection (i.m.), oral administration, intranasal administration, spray administration and immersion.
  • intraperitoneal injection i.p.
  • intravenous injection i.v.
  • intramuscular injection i.m.
  • oral administration intranasal administration
  • spray administration and immersion.
  • nucleic acid or amino acid sequence indicates that, when optimally aligned, for example using the methods described below, the nucleic acid or amino acid sequence shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with a defined second nucleic acid or amino acid sequence (the "reference sequence”).
  • reference sequence the “reference sequence”
  • Substantial identity may be used to refer to various types and lengths of sequence, such as full-length sequence, functional domains, coding and/or regulatory sequences, promoters, and genomic sequences.
  • Percent identity between two amino acid or nucleic acid sequences can be determined in various ways that are within the skill of a worker in the art, for example, using publicly available computer software such as Smith Waterman Alignment (Smith, T. F. and M. S. Waterman (1981) J Mol Biol 147: 195-7); "BestFit” (Smith and Waterman, Advances in Applied Mathematics, 482-489 10 (1981)) as incorporated into GeneMatcher PlusTM, Schwarz and Dayhof (1979) Atlas of Protein Sequence and Structure, Dayhof, M. O., Ed pp 353-358; BLAST program (Basic Local Alignment Search Tool (Altschul, S. F., W. Gish, et al.
  • PapMV VLP and "PAL” are used interchangeably herein to refer to a Virus-Like Particle derived from the Papaya Mosaic Virus.
  • the PapMV VLP can be further engineered to present one or more antigens fused to the PapMV coat protein.
  • one or more epitopes from an influenza protein other than NP For example, and without limitation, one or more epitopes from an influenza protein other than NP.
  • the NLPs disclosed herein comprise recombinant orthomyxoviral nucleoprotein self-assembled with an ssRNA scaffold.
  • the recombinant nucleoprotein is an influenza nucleoprotein.
  • the NLPs are ordered, elongated structures having a greater length than width (referred to herein as "rod-shaped” or “rod- like”).
  • the structures are characterized by having a normal (bell-shaped) distribution when analyzed by dynamic light scattering (DLS) that may range from about lOnm to about 200nm, typically from about lOnm to about 150nm.
  • DLS dynamic light scattering
  • the average length of the NLPs when analyzed by DLS is typically between about 20nm and about lOOnm, for example, between about 20nm and about 80nm. As is evident from the distribution of lengths seen by DLS, longer NLP structures may form under certain conditions.
  • the average width of the NLPs is generally between about lOnm and about 20nm. The length of the NLP is believed to be dependent to some extent on the length of the ssRNA, with shorter ssRNA scaffolds tending to produce NLPs with a shorter average length, and longer ssRNAs tending to produce NLPs with a longer average length.
  • the NLPs typically comprise at least 10 monomer units, for example, at least 12, 14, 16, 18 or 20 monomer units. Without being bound by any particular theory or structure, it is believed that an NLP comprising about 20 monomer units would comprise two or more ring-like structures bound together in a NLP structure by the ssRNA scaffold.
  • EM electron microscopy
  • the appearance of the NLPs by electron microscopy (EM) can be characterized as elongated structures with irregular edges (see Figures 2, 5, 17, 18, 20, 24, and 29). Under EM some aggregation of the NLPs is observed, however, this is likely due to the EM conditions as the results of the DLS analysis suggests there are no aggregates present.
  • the recombinant nucleoprotein (NP) used to prepare the NLPs may be derived from one of a variety of orthomyxoviral NP sequences, for example, one of a variety of influenza virus NP sequences.
  • derived from it is meant that the recombinant NP has an amino acid sequence substantially identical to the sequence of the wild-type NP.
  • sequences of NPs various orthomyxoviral genera, influenza types, subtypes and strains are known in the art and are publicly available from databases such as the NCBI's GenBank database. Selection of an appropriate NP sequence will depend to some extent on the intended application of the final NLPs.
  • the NP sequence selected will be from a strain of influenza that commonly infects humans.
  • the NP sequence selected will be from a strain of orthomyxovirus, e.g., influenza, that commonly infects the target non-human animal.
  • the NLPs are formed from a recombinant NP derived from an influenza type A virus NP or an influenza type B virus NP. In some embodiments, the NLPs are formed from a recombinant NP derived from an influenza type A virus. In further embodiments, the NLPs are formed from a recombinant NP derived from an influenza type B virus.
  • influenza type A viruses are divided into subtypes based on the sequences of the hemagglutinin (HA) and neuraminidase (NA) proteins comprised by the virus.
  • HA hemagglutinin
  • NA neuraminidase
  • Many different combinations of HA and NA proteins are possible, however, only certain influenza A subtypes tend to infect humans, for example, HlNl, H1N2, and H3N2 subtypes, whereas other subtypes are found most commonly in other animal species.
  • the NLPs are formed from a recombinant NP derived from influenza A HlNl, H1N2 or H3N2 NP.
  • the NLPs are formed from a recombinant NP derived from influenza A HlNl NP.
  • FIG. 21 A Representative non-limiting examples of known influenza virus type B NP amino acid sequence are shown in Figure 21 A, B (GenBank Accession No. NP_056661 ; SEQ ID NO: 10, and GenBank Accession No. AFH57958 (B/Wisconsin/01/2010); SEQ ID NO:31), a representative non-limiting example of a known influenza A subtype HlNl NP amino acid sequence is shown in Figure 13A (A/California/04/2009; GenBank Accession No. ACP41106.1 ; SEQ ID NO:4), a representative non-limiting example of a known influenza A subtype H1N2 NP amino acid sequence is shown in Figure 21C (A/New York/78/2002; GenBank Accession No.
  • AAY78943.1 SEQ ID NO: 11
  • a representative non-limiting example of a known influenza A subtype H3N2 NP amino acid sequence is shown in Figure 21D (A/Switzerland/9243/99; GenBank Accession No. CAD30200.1; SEQ ID NO: 12). It is to be understood that these sequences are provided as examples only and that they are not limiting.
  • a large number of influenza type A and type B NP sequences are known and have been deposited in GenBank and other databases, and may be used as a basis for a recombinant NP in accordance with various embodiments of the present invention.
  • the NLPs may be formed from a recombinant NP derived from the sequence of an NP from an influenza A subtype that commonly infects a non-human animal. Such subtypes may or may not also infect humans.
  • subtype H1N1, H1N2 and H3N2 are prevalent; in horses, subtypes H7N7 and H3N8 are prevalent; in poultry subtypes H1N7, H2N2, H3N8, H4N2, H4N8, H5N1, H5N2, H5N9, H6N5, H7N2, H7N3, H9N2, H10N7, H11N6, H12N5, H13N6 and H14N5 have been reported; in domestic cats, H5N1 has been reported, and in dogs, H3N8 has been reported.
  • the NLPs may be formed from a recombinant NP derived from the sequence of an NP from an influenza A subtype that is considered to be a zoonotic, potential pandemic strain, such as H5N1, H9N2 or H7N7.
  • the recombinant NP used to prepare the NLPs may be a full-length NP, or a functional fragment thereof, or it can be a genetically modified version of the wild-type NP, for example, comprising one or more amino acid deletions, insertions, replacements and the like, provided that the NP retains its immunogenicity and the ability to self-assemble on ssRNA into an NLP as described herein.
  • the amino acid sequence of the recombinant NP need not correspond precisely to the parental (wild-type) sequence, i.e. it may be a "variant sequence.”
  • the NP may be mutagenized by substitution, insertion or deletion of one or more amino acid residues so that the residue at that site does not correspond to the parental sequence.
  • mutations will not be extensive and will not affect the immunogenicity of the recombinant NP or its ability to self-assemble on ssRNA into an NLP.
  • NPs that are functional fragments of the corresponding wild-type NP (i.e. that retain immunogenicity and the ability to self-assemble on ssRNA into an NLP) are contemplated in certain embodiments.
  • a functional fragment may comprise a deletion of one or more amino acids from the N-terminus, the C-terminus, or the interior of the protein, or a combination thereof.
  • Deletions typically consist of 50 amino acids or less, for example, 40 amino acids or less, 30 amino acids or less, 20 amino acids or less, or 10 amino acids or less.
  • Wild-type influenza B NP is typically about 560 amino acids in length.
  • functional fragments of influenza B NP are contemplated that are at least 500 amino acids in length, for example, at least 510 amino acids in length, at least 520 amino acids in length, at least 530 amino acids in length, at least 540 amino acids in length, at least 550 amino acids in length, at least 555 amino acids in length, or any amount therebetween.
  • Wild-type influenza A NP is typically about 498 amino acids in length.
  • functional fragments of influenza A NP are contemplated that are at least 450 amino acids in length, for example, at least 460 amino acids in length, at least 470 amino acids in length, at least 480 amino acids in length, at least 490 amino acids in length, at least 495 amino acids in length, or any amount therebetween.
  • the variant sequence when a recombinant NP comprises a variant sequence, is at least about 75% identical to the corresponding wild-type sequence. In some embodiments, the variant sequence is at least about 80% identical to the wild-type sequence, for example, at least about 85%, at least about 90%, at least about 95%, at least about 97% identical, at least about 98% identical to the wild-type sequence. In certain embodiments, the wild-type amino acid sequence is one of SEQ ID NOs: 10, 31, 4, 11 or 12.
  • any substitutions comprised by the recombinant NP are conservative substitutions.
  • a conservative substitution involves the replacement of one amino acid residue by another residue having similar side chain properties. As is known in the art, the twenty naturally occurring amino acids can be grouped according to the physicochemical properties of their side chains.
  • Suitable groupings include alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine and tryptophan (hydrophobic side chains); glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine (polar, uncharged side chains); aspartic acid and glutamic acid (acidic side chains) and lysine, arginine and histidine (basic side chains).
  • Another grouping of amino acids is phenylalanine, tryptophan, and tyrosine (aromatic side chains). A conservative substitution involves the substitution of an amino acid with another amino acid from the same group.
  • a non-conservative substitution involves the replacement of one amino acid residue by another residue having different side chain properties, for example, replacement of an acidic residue with a neutral or basic residue, replacement of a neutral residue with an acidic or basic residue, replacement of a hydrophobic residue with a hydrophilic residue, and the like.
  • the recombinant NP used in the preparation of the NLPs has a sequence that is substantially identical to SEQ ID NO:4, for example, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or any amount therebetween.
  • the recombinant NP used to prepare the NLPs is a fusion protein comprising a peptide fused to the NP sequence, in which the peptide comprises one or more epitopes from another orthomyxoviral protein, preferably an influenza protein.
  • the peptide may be fused at the N-terminus of the NP, at the C-terminus of the NP or at an internal site, provided that it does not interfere with the immunogenicity of the NP or with the ability of the NP to assemble with ssRNA to form NLPs.
  • the peptide may optionally replace one or more amino acids that form the N- or C- terminus of the wild-type protein.
  • the peptide is fused at the C- terminus of the NP and may optionally replace one or more amino acids that form the C- terminus of the wild-type protein.
  • the peptide comprising the one or more epitopes will be a short amino acid sequence, for example, between about 4 and about 27 amino acids in length. In certain embodiments, the peptide is between about 5 and about 27 amino acids in length, between about 6 and about 27 amino acids in length, between about 7 and about 27 amino acids in length, between about 8 and about 27 amino acids in length, or any amount therebetween. In some embodiments, the peptide has a minimum size corresponding to a CTL epitope. CTL epitopes are considered to be at least 9 amino acids in length.
  • the peptide is between about 9 and about 27 amino acids in length, for example, between about 9 and about 24 amino acids in length, between about 9 and about 22 amino acids in length, between about 9 and about 20 amino acids in length, between about 9 and about 18 amino acids in length, between about 9 and about 16 amino acids in length, between about 9 and about 14 amino acids in length, between about 9 and about 12 amino acids in length, or any amount therebetween.
  • Various antigenic peptides from influenza virus proteins are known in the art and may be used to prepare an NP-peptide fusion protein in accordance with certain embodiments of the invention.
  • Examples include, but are not limited to, antigenic fragments of HA or the matrix proteins such as, the haemagglutinin epitopes: HA 91-108, HA 307-319 and HA 306- 324 (Rothbard, 1988, Cell 52:515-523), HA 458-467 (Alexander et al, 1997, J. Immunol , 159(10): 4753-61), HA 213-227, HA 241-255, HA 529-543 and HA 533-547 (Gao, W. et al, 2006 J.
  • Ml matrix protein epitopes: Ml 2-22, Ml 2-12, Ml 3- 11, Ml 3-12, Ml 41-51, Ml 50-59, Ml 51-59, Ml 134-142, Ml 145-155, Ml 164-172, Ml 164-173 (Nijman, 1993, Eur. J.
  • M2e peptide fragments of the ion channel protein (M2) such as the M2e peptide (the extracellular domain of M2).
  • M2e peptide the extracellular domain of M2
  • SEQ ID NO: 13 An example of an M2e peptide sequence is shown in Table 1 as SEQ ID NO: 13. Variants of this sequence have been identified in the art and some are also shown in Table 1.
  • the entire M2e sequence or a partial M2e sequence may be used, for example, a partial sequence that is conserved across influenza variants, such as fragments within the region defined by amino acids 2 to 10, or the conserved epitope EVETPIRN [SEQ ID NO:23] (amino acids 6-13 of the M2e sequence).
  • the 6-13 epitope has been found to be invariable in 84% of human influenza A strains available in GenBank.
  • Variants of this sequence include EVETLTRN [SEQ ID NO:24] (9.6%), EVETPIRS [SEQ ID NO:25] (2.3%), EVETPTRN [SEQ ID NO:26] (1.1%), EVETPTKN [SEQ ID NO:27] (1.1%), EVDTLTRN [SEQ ID NO:28], and EVETPIRK [SEQ ID NO:29] and EVETLTKN [SEQ ID NO:30] (0.6% each) (see Zou, P., et al, 2005, Int Immunopharmacology, 5:631-635; Liu et al, 2005, Microbes and Infection, 7: 171-177).
  • M2e fragments include the sequence EVETPIRNE [SEQ ID NO:9].
  • the NLPs are prepared from an NP -peptide fusion protein comprising an M2e peptide or fragment thereof. In some embodiments, the NLPs are prepared from an NP -peptide fusion protein comprising an M2e peptide or fragment thereof in which the M2e peptide or fragment has a sequence as set forth in any one of SEQ ID NOs:9 and 13-30. In some embodiments, the NLPs are prepared from an NP-peptide fusion protein comprising an M2e peptide fragment in which the M2e peptide fragment has a sequence as set forth in any one of SEQ ID NOs:9 and 23-30.
  • the peptide comprised by an NP-peptide fusion proteins will comprise one or more epitopes from an influenza protein other than NP
  • certain embodiments contemplate that the peptide may comprise one or more epitopes from the NP in order to strengthen the anti-NP immune response generated by the resulting NLPs.
  • the NP from which the peptide is derived may be, for example, from a different strain, subtype or type of influenza virus in order to broaden the protection provided by the NLPs. Examples of known NP epitopes include, but are not limited to, NP 206-229 (Brett, 1991, J.
  • Recombinant NP and NP-peptide fusion proteins can be readily prepared by standard genetic engineering techniques by the skilled worker based on the known and publicly available sequences of various orthomyxoviral, preferably influenza, NPs and influenza antigenic peptides, such as those described above. Methods of genetically engineering proteins are well known in the art (see, for example, Ausubel et al. (1994 & updates) Current Protocols in Molecular Biology, John Wiley & Sons, New York). [00103] For example, isolation and cloning of the nucleic acid sequence encoding the wild- type protein can be achieved using standard techniques (see, for example, Ausubel et al, ibid).
  • the nucleic acid sequence can be obtained directly from the orthomyxovirus (e.g., influenza virus) or from cells infected by orthomyxovirus (e.g., influenza virus) by extracting RNA by standard techniques and then synthesizing cDNA from the RNA template (for example, by RT-PCR).
  • orthomyxovirus e.g., influenza virus
  • influenza virus e.g., influenza virus
  • RNA template for example, by RT-PCR
  • the nucleic acid sequence encoding NP is then inserted directly or after one or more subcloning steps into a suitable expression vector.
  • suitable vectors include, but are not limited to, plasmids, phagemids, cosmids, bacteriophage, baculoviruses, retroviruses or DNA viruses.
  • the NP can then be expressed and purified by standard techniques. Selection of appropriate vector and host cell combinations in this regard is well within the ordinary skills of a worker in the art.
  • the nucleic acid sequence encoding the NP can be further engineered to introduce one or more mutations, such as those described above, by standard in vitro site- directed mutagenesis techniques well-known in the art. Mutations can be introduced by deletion, insertion, substitution, inversion, or a combination thereof, of one or more of the appropriate nucleotides making up the coding sequence. This can be achieved, for example, by PCR-based techniques for which primers are designed that incorporate one or more nucleotide mismatches, insertions or deletions. The presence of the mutation can be verified by a number of standard techniques, for example by restriction analysis or by DNA sequencing.
  • DNA encoding the NP can be altered in various ways without affecting the activity of the encoded protein.
  • variations in DNA sequence may be used to optimize for codon preference in a host cell used to express the protein, or may contain other sequence changes that facilitate expression.
  • the nucleic acid sequence encoding the NP may also be engineered to include one or more heterologous sequences, such as a sequence encoding an influenza antigenic peptide as discussed above such that the expressed protein is a NP -peptide fusion protein, and/or a sequence encoding an affinity tag to facilitate purification.
  • heterologous sequences such as a sequence encoding an influenza antigenic peptide as discussed above such that the expressed protein is a NP -peptide fusion protein, and/or a sequence encoding an affinity tag to facilitate purification.
  • affinity tags include, but are not limited to, metal-affinity tags, histidine tags, avidin/streptavidin encoding sequences, glutathione-S-transferase (GST) encoding sequences and biotin encoding sequences.
  • the affinity tag may be removed from the expressed NP prior to use according to methods known in the art or may be retained on the NP provided that it does not interfere with the immunogenicity of the NP or its assembly into NLPs.
  • the expression vector may further include regulatory elements, such as transcriptional elements, required for efficient transcription of the DNA sequence encoding the NP or fusion protein. Examples of regulatory elements that can be incorporated into the vector include, but are not limited to, promoters, enhancers, terminators, and polyadenylation signals.
  • regulatory elements are dependent on the host cell chosen for expression of the recombinant NP or fusion protein and that such regulatory elements may be derived from a variety of sources, including bacterial, fungal, viral, mammalian or insect genes.
  • the expression vector may optionally contain heterologous nucleic acid sequences that facilitate the purification of the expressed protein.
  • heterologous nucleic acid sequences include, but are not limited to, affinity tags such as those described above.
  • the expression vector can be introduced into a suitable host cell or tissue by one of a variety of methods known in the art. Such methods can be found generally described in Ausubel et al. (ibid.) and include, for example, stable or transient transfection, lipofection, electroporation, and infection with recombinant viral vectors.
  • a suitable host cell or tissue such methods can be found generally described in Ausubel et al. (ibid.) and include, for example, stable or transient transfection, lipofection, electroporation, and infection with recombinant viral vectors.
  • host cells include, but are not limited to, bacterial, yeast, insect, plant and mammalian cells. The precise host cell used is not critical to the invention.
  • the rNPs can be produced in a prokaryotic host (e.g. E. coli, A.
  • a eukaryotic host e.g. Saccharomyces or Pichia; mammalian cells, e.g. COS, NIH 3T3, CHO, BHK, 293 or HeLa cells; insect cells or plant cells.
  • the NP is cloned into a vector that allows for expression in prokaryotic cells, such as E. coli.
  • the NP or NP-peptide fusion protein can be purified from the host cells by standard techniques known in the art (see, for example, in Current Protocols in Protein Science, ed. Coligan, J.E., et al, Wiley & Sons, New York, NY). Verification that the NP or NP-peptide fusion protein is in monomelic form may be made, for example, by analysis of a sample of the protein by size-exclusion chromatography. The protein may optionally be subjected to one or more additional purification steps to remove exotoxin when necessary. Exemplary protocols for the cloning, expression and purification of NP and NP-peptide fusion proteins are provided in the Examples.
  • ssRNA Single-Stranded RNA Scaffold
  • Various ssRNAs may be used as a scaffold for assembly of the NP or NP-peptide fusion protein into NLPs.
  • the ssRNA scaffold may be, for example, a synthetic ssRNA, a naturally occurring ssRNA, a modified naturally occurring ssRNA, a fragment of a naturally occurring ssRNA, or the like.
  • the ssRNA scaffold is a synthetic ssRNA.
  • the ssRNA scaffold is at least about 120 nucleotides in length and up to about 5000 nucleotides in length, for example, at least about 140, 160, 180, 200, 250, 300, 350, 400, 450 or 500 nucleotides in length and up to about 5000, 4500, 4000, 3500, 3000 or 2500 nucleotides in length.
  • the ssRNA for in vitro assembly is between about 500 and about 3000 nucleotides in length, for example, between about 500 and about 2500 nucleotides in length, or between about 500 and about 2000 nucleotides in length.
  • the exact sequence of the ssRNA used as a scaffold does not appear to be critical.
  • the sequence may be a random sequence, a naturally occurring sequence, or a modified naturally occurring sequence, such as a fragment of a naturally occurring sequence or one in which one or more nucleotides have been substituted.
  • the ssRNA may be composed of a single base, such as poly-U, poly-C, poly-A or poly-G.
  • the ssRNA sequence includes a modified naturally occurring sequence, for example, a modified version of a sequence from another virus.
  • the ssRNA sequence is a fragment of a naturally occurring viral ssRNA sequence, a naturally occurring viral ssRNA sequence that has been modified such that it does not encode any proteins (for example by introducing stop codons, frameshift mutations or exchanging any ATG codons for TAA codons), or a fragment of such a modified naturally occurring viral ssRNA sequence.
  • the ssRNA is a poly-U or poly-C ssRNA.
  • the ssRNA comprises a sequence corresponding to the sequence as set forth in SEQ ID NO: 7 or 8, or a fragment of SEQ ID NO: 7 or 8. Fragments may be, for example, between about 120 nucleotides and about 1000 nucleotides and may comprise the 5' end of SEQ ID NO:7 or 8, the 3' end of SEQ ID NO:7 or 8, or a central region of SEQ ID NO:7 or 8, for example, a fragment starting from nucleotide 17 or from nucleotide 55 of SEQ ID NO:7 or 8.
  • the ssRNA template can be isolated and/or prepared by standard techniques known in the art (see, for example, Ausubel et al. (1994 & updates) ibid.).
  • the sequence encoding the ssRNA template can be inserted into a suitable plasmid which can be used to transform an appropriate host cell.
  • plasmid DNA can be purified from the cell culture by standard molecular biology techniques and linearized by restriction enzyme digestion.
  • the ssRNA is then transcribed using a suitable RNA polymerase and the transcribed product purified by standard protocols.
  • Shorter ssRNA scaffolds may also be synthesized chemically using standard techniques. A number of commercial RNA synthesis services are also available.
  • the final ssRNA scaffold may optionally be sterilized prior to use.
  • the assembly reaction is conducted in vitro using the prepared recombinant NP or NP-peptide fusion protein and the ssRNA scaffold.
  • the assembly reaction may be conducted in a neutral aqueous solution and does not require any other particular ion. Typically, a buffer solution is used.
  • the pH should be in the range of about pH6.0 to about pH9.0, for example, between about pH6.5 and about pH9.0, between about pH7.0 and about pH9.0, between about pH6.0 and about pH8.5, between about pH6.5 and about pH8.5, or between about pH7.0 and about pH8.5.
  • the nature of the buffer is not critical to the assembly process provided that it can maintain the pH in the ranges described above.
  • buffers for use within the pH ranges described above include, but are not limited to, Tris buffer, phosphate buffer, citrate buffer and the like.
  • the presence of salt has been found to adversely affect the ability of the NP to bind ssRNA. Accordingly, in certain embodiments, the amount of salt present in the in vitro assembly reaction is less than about 150mM, for example, less than about 140mM, less than about 130mM, less than about 120mM, less than about 1 lOmM, or less than about lOOmM.
  • the assembly reaction can be conducted using various proteimssRNA ratios.
  • a proteimssRNA ratio between about 1 : 1 and about 50: 1 by weight may be used, for example, between at least about 1 : 1, 2: 1, 3: 1, 4: 1 or 5: 1 by weight and no more than about 50: 1, 40: 1 or 30: 1 by weight.
  • the protein:ssRNA ratio used in the assembly reaction is between about 5: 1 and about 50: 1 by weight, for example, between about 6: 1 and about 50: 1 by weight, between about 7: 1 and about 50: 1 by weight, between about 8: 1 and about 50: 1 by weight, between about 9: 1 and about 50: 1 by weight, or between about 10: 1 and about 50: 1 by weight.
  • the proteimssRNA ratio used in the assembly reaction is between about 5: 1 and about 40: 1 by weight, between about 5: 1 and about 30: 1 by weight, or between about 5: 1 and about 20: 1 by weight.
  • the assembly reaction can be conducted at temperatures varying from about 2°C to about 37°C, for example, between at least about 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C or 10°C and about 37°C, 35°C, 30°C or 28°C. In certain embodiments, the assembly reaction is conducted at a temperature between about 15°C and about 37°C, for example, between about 20°C and about 37°C.
  • the assembly reaction is allowed to proceed for a sufficient period of time to allow NLPs to form.
  • the time period will vary depending on the concentrations of recombinant NP or NP-peptide fusion protein and ssRNA employed, as well as the temperature of the reaction, and can be readily determined by the skilled worker. Typically time periods of at least about 60 minutes are employed, for example, between about 60 minutes and about 12 hours, between about 60 minutes and about 10 hours, between about 60 minutes and about 8 hours, or between about 60 minutes and about 5 hours. Formation of NLPs can be monitored if required by standard techniques, such as dynamic light scattering or electron microscopy.
  • the assembled NLPs can be purified from other reaction components including monomelic NP or NP-peptide fusion protein and ssRNA by standard techniques, such as dialysis, diafiltration or chromatography.
  • the NLP preparation can optionally be concentrated (or enriched) by, for example, ultracentrifugation or diafiltration, either before or after the purification step(s) if desired.
  • compositions comprising an effective amount of the disclosed NLPs and one or more pharmaceutically acceptable carriers, diluents and/or excipients. If desired, other active ingredients may be included in the compositions, for example, additional immune stimulating compounds, antigens, adjuvants, or the like.
  • the pharmaceutical composition of the present disclosure comprises an effective amount of a single type of NLP, i.e., an NLP comprising recombinant NP polypeptides having a sequence derived from the same orthomyxoviral NP.
  • the pharmaceutical composition comprises an effective amount of two or more types of NLP.
  • the pharmaceutical composition can comprise NLPs having NP polypeptides derived from one or more influenza type A strains and one or more influenza type B strains.
  • compositions can be formulated for administration by a variety of routes.
  • the compositions can be formulated for oral, topical, rectal, nasal or parenteral administration or for administration by inhalation or spray.
  • parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrathecal, intrasternal injection or infusion techniques.
  • Intranasal administration to the subject includes administering the composition to the mucous membranes of the nasal passage or nasal cavity of the subject.
  • compositions formulated as aqueous suspensions may contain the NLPs in admixture with one or more suitable excipients, for example, with suspending agents, such as sodium carboxymethylcellulose, methyl cellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, hydroxypropyl-P-cyclodextrin, gum tragacanth and gum acacia; dispersing or wetting agents such as a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example, polyoxyethyene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example, hepta-decaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol for example, polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from
  • the aqueous suspensions may also contain one or more preservatives, for example ethyl, or n- propyl /?-hydroxy-benzoate, one or more colouring agents, one or more flavouring agents or one or more sweetening agents, such as sucrose or saccharin.
  • preservatives for example ethyl, or n- propyl /?-hydroxy-benzoate
  • colouring agents for example ethyl, or n- propyl /?-hydroxy-benzoate
  • flavouring agents such as sucrose or saccharin.
  • sweetening agents such as sucrose or saccharin.
  • the pharmaceutical compositions may be formulated as oily suspensions by suspending the NLPs in a vegetable oil, for example, arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
  • the oily suspensions may contain a thickening agent, for example, beeswax, hard paraffin or cetyl alcohol. These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid.
  • the pharmaceutical compositions may be formulated as a dispersible powder or granules, which can subsequently be used to prepare an aqueous suspension by the addition of water.
  • Such dispersible powders or granules provide the NLPs in admixture with one or more dispersing or wetting agents, suspending agents and/or preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example, colouring agents, can also be included in these compositions.
  • Pharmaceutical compositions of the invention may also be formulated as oil-in- water emulsions in some embodiments.
  • the oil phase can be a vegetable oil, for example, olive oil or arachis oil, or a mineral oil, for example, liquid paraffin, or it may be a mixture of these oils.
  • Suitable emulsifying agents for inclusion in these compositions include naturally- occurring gums, for example, gum acacia or gum tragacanth; naturally-occurring phosphatides, for example, soy bean, lecithin; or esters or partial esters derived from fatty acids and hexitol, anhydrides, for example, sorbitan monoleate, and condensation products of the said partial esters with ethylene oxide, for example, polyoxyethylene sorbitan monoleate.
  • the pharmaceutical compositions may be formulated as a sterile injectable aqueous or oleaginous suspension according to methods known in the art and using one or more suitable dispersing or wetting agents and/or suspending agents, such as those mentioned above.
  • the sterile injectable preparation can be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
  • Acceptable vehicles and solvents that can be employed include, but are not limited to, water, Ringer's solution, lactated Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils which are conventionally employed as a solvent or suspending medium
  • a variety of bland fixed oils including, for example, synthetic mono- or diglycerides.
  • Fatty acids such as oleic acid can also be used in the preparation of injectables.
  • the pharmaceutical compositions may contain preservatives such as antimicrobial agents, anti-oxidants, chelating agents, and inert gases, and/or stabilizers such as a carbohydrate (e.g. sorbitol, mannitol, starch, sucrose, glucose, or dextran), a protein (e.g. albumin or casein), or a protein-containing agent (e.g. bovine serum or skimmed milk) together with a suitable buffer (e.g. phosphate buffer).
  • a suitable buffer e.g. phosphate buffer
  • Sterile compositions can be prepared for example by incorporating the NLPs in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by sterilization, for example, filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • some exemplary methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Contemplated for use in certain embodiments of the invention are various mechanical devices designed for pulmonary or intranasal delivery of therapeutic products, including but not limited to, nebulizers, metered dose inhalers, powder inhalers and nasal spray devices, all of which are familiar to those skilled in the art.
  • each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to the usual diluents, adjuvants and/or carriers useful in administration of therapeutic products as would be understood by a worker skilled in the art. Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated.
  • the pharmaceutical compositions may optionally further comprise an adjuvant.
  • adjuvants include, but are not limited to, alum adjuvants (such as aluminium hydroxide, phosphate or oxide), oil-emulsions (e.g. Bayol F® or Marcol 52®), saponins, vitamin-E solubilisate, monophosphoryl lipid A, CpG oligonucleotides, Resiqumod, and certain virus-like particles (VLPs), such as Papaya mosaic virus (PapMV) VLPs described in International Patent Application Publication Nos. WO 2004/004761 and WO 2012/155262.
  • alum adjuvants such as aluminium hydroxide, phosphate or oxide
  • oil-emulsions e.g. Bayol F® or Marcol 52®
  • saponins e.g. Bayol F® or Marcol 52®
  • saponins e.g. Bayol F® or Marcol 52®
  • saponins e.g. Bayol F® or Marcol 52®
  • the pharmaceutical compositions comprise PapMV VLPs as an adjuvant.
  • the PapMV VLPs can comprise one or more influenza antigens fused to the PapMV coat protein as described, for example, in International Patent Application Publication Nos. WO 2004/004761, WO 2012/155262, and WO 2013/149334.
  • the pharmaceutical compositions comprise PapMV VLPs that comprise one or more epitopes from another influenza protein fused to the PapMV VLP.
  • Various antigenic peptides from influenza virus proteins are known in the art and may be fused to the PapMV VLPs.
  • Examples include, but are not limited to, antigenic fragments of HA or the matrix proteins such as, the haemagglutinin epitopes: HA 91-108, HA 307-319 and HA 306-324 (Rothbard, 1988, Cell 52:515-523), HA 458-467 (Alexander et al, 1997, J. Immunol, 159(10): 4753-61), HA 213-227, HA 241-255, HA 529-543 and HA 533- 547 (Gao, W. et al, 2006 J.
  • Ml matrix protein epitopes: Ml 2- 22, Ml 2-12, Ml 3-11, Ml 3-12, Ml 41-51, Ml 50-59, Ml 51-59, Ml 134-142, Ml 145-155, Ml 164-172, Ml 164-173 (Nijman, 1993, Eur. J. Immunol.
  • Ml 17-31, Ml 55-73, Ml 57-68 Carreno, 1992, Mol Immunol, 29: 1131-1140
  • Ml 27-35, Ml 232-240 (DiBrino, 1993, PNAS, 90: 1508-12)
  • Ml 59-68, Ml 60-68 Connan et al, 1994, Eur. J. Immunol, 24(3):777-80
  • Ml 128-135 Dong et al, 1996, Eur. J. Immunol, 26(2): 335- 39.
  • Other antigenic regions and epitopes include fragments of the ion channel protein (M2) such as the M2e peptide (the extracellular domain of M2).
  • the pharmaceutical compositions comprise the disclosed NLPs in combination with PapMV VLPs as an adjuvant, the PapMV VLPs comprising one or more influenza antigens derived from the influenza M2 protein.
  • the PapMV VLPs comprise one or more influenza antigens derived from the M2e peptide or a fragment thereof.
  • compositions and methods of preparing pharmaceutical compositions are known in the art and are described, for example, in “Remington: The Science and Practice of Pharmacy” (formerly “Remingtons Pharmaceutical Sciences”); Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, PA (2000).
  • the NLPs disclosed herein may find use as vaccines to protect against influenza infection. Certain embodiments of the invention thus relate to methods for inducing an immune response against an influenza virus in an animal and to the use of the disclosed NLPs for the preparation of medicaments, including vaccines, for immunizing a subject against an influenza infection.
  • the NLPs may be used to induce an immune response to more than one influenza virus strain.
  • the NP protein is highly conserved between orthomyxoviruses, particularly between influenza virus types, and highly conserved between influenza A virus subtypes and strains, it is contemplated in certain embodiments that NLPs comprising a NP sequence derived from one type of orthomyxovirus will be able to provide protection against heterologous orthomyxoviruses.
  • NLPs comprising a NP sequence derived from one type of influenza virus strain will be able to provide protection against heterologous strains of influenza.
  • NLPs comprising a NP sequence derived from one influenza A virus strain will be able to provide protection against heterologous strains of influenza A, and potentially other subtypes and types of influenza virus.
  • the NLPs may be used to induce antibodies against influenza virus NP in a subject.
  • the NLPs may be used to induce a cellular immune response, such as a CTL response, against influenza virus NP in a subject.
  • the NLPs may be used to induce both antibodies and a CTL response against influenza virus NP in a subject.
  • the NLPs may be used to induce a humoral immune response and/or a cellular immune response against influenza virus NP in a subject that provides cross-protection against heterologous influenza A strains. In some embodiments, the NLPs may be used to induce a humoral immune response and/or a cellular immune response against influenza virus NP in a subject that provides cross- protection against heterologous influenza A subtypes. In some embodiments, the NLPs may be used to induce a humoral immune response and/or a cellular immune response against influenza virus NP in a subject that provides protection against a plurality of influenza virus types and subtypes.
  • Certain embodiments relate to the use of the NLPs as a vaccine in humans. Some embodiments relate to the use of the NLPs as a vaccine in non-human animals, including domestic and farm animals. Due to the conserved nature of the NP sequence, it is also contemplated in certain embodiments that the NLPs could be used to vaccinate both humans and non-humans even though the strains of influenza that typically infect humans and non- human animals may be different.
  • Certain embodiments relate to the use of the NLPs as an influenza vaccine for humans. Some embodiments relate to the use of NLPs comprising NP derived from the HlNl, H1N2 or H3N2 subtype of influenza as an influenza vaccine for humans. Further embodiments relate to the use of NLPs comprising NP derived from one or more subtypes of influenza type B virus as an influenza vaccine for humans. Other embodiments relate to the use of NLPs comprising NP derived from one or more subtypes of influenza type A virus in combination with NLPs derived from one or more subtypes of influenza type B virus as an influenza vaccine for humans. The vaccine may optionally be used for other non-human animals.
  • the administration regime for the NLPs need not differ from any other generally accepted vaccination programs. In certain embodiments, a single administration of the NLPs in an amount sufficient to elicit an effective immune response may be used. In some embodiments, an initial administration of the NLPs may be followed by one or more booster vaccinations. In some embodiments, the administration regime for the NLPs may comprise an initial dose plus a booster dose. In some embodiments, the administration regime for the NLPs may comprise an initial dose plus two or more booster doses. In some embodiments, the administration regime for the NLPs may comprise an initial dose plus three or more booster doses.
  • the NLPs are administered to the subject in combination with an adjuvant.
  • adjuvants are described above.
  • the NLPs in combination with one or more other influenza antigens.
  • the NLPs may be combined with an influenza M2 protein or Ml protein or a fragment of one of these proteins.
  • the NLPs may be combined with a seasonal influenza vaccine to provide broader cross-protection against heterologous influenza types and/or subtypes.
  • the NLPs may be administered concomitantly with the antigen or vaccine, or may be administered prior or subsequent to the administration of the antigen or vaccine.
  • kits comprising the NLPs for use as an influenza vaccine.
  • Individual components of the kit would be packaged in separate containers and, associated with such containers, can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale.
  • the kit may optionally contain instructions or directions outlining the method of use or administration regimen for the vaccine.
  • the container means may itself be an inhalant, syringe, pipette, eye dropper, or other such like apparatus, from which the solution may be administered to a subject or applied to and mixed with the other components of the kit.
  • the components of the kit may also be provided in dried or lyophilised form and the kit can additionally contain a suitable solvent for reconstitution of the lyophilised components.
  • the pack or kit may also comprise an instrument for assisting with the administration of the vaccine to a patient.
  • Such an instrument may be an inhalant, nasal spray device, syringe, pipette, measured spoon or similar medically approved delivery vehicle.
  • Influenza A/California/04/2009 [H1N1] NP gene (GenBank: ACP41106.1) with a C-terminal 6x-His-tag cloned into the vector pJExpress 411 was obtained from DNA2.0, Inc. (Menlo Park, CA). The coding sequence was optimized to maximize expression in E coli.
  • the gene was amplified by PCR (Kit PCR used: Phusion® Hot Start Flex DNA polymerase (New England Biolabs, Ipswich, MA)) with the following primers and conditions:
  • Step 1 98 ° C for 30 seconds
  • Step 2 98 " C for 10 seconds
  • Step 3 50 ° C for 30 seconds (gradient from 50 to 70 ° C)
  • Step 4 72 ° C for 2 min 30 seconds. 29 repetitions of Step 2 to 4 followed by: Step 5: 72 °C for 10 min, and Step 6: Pause at 16° C.
  • the resulting PCR product was digested with EcoRl and Kpnl enzymes, and cloned into an EcoRl/Kpnl linearized pQE-80L-KAN vector (Qiagen, Toronto, ON).
  • the Escherichia coli expression strain BD792 was transformed with the plasmid pQE-80L-KAN containing the A/California/04/2009 (H1N1) NP gene and maintained in 2xYT medium containing Kanamycin (25 ⁇ g/mL).
  • Bacterial cells were grown at 37°C to an optical density of 0.8 ⁇ 0.2 at 600nm and protein expression was induced with 1 mM isopropyl ⁇ -D- thiogalactopyranoside (IPTG). Induction was continued for 16h at 22°C. The bacteria were then collected and lysed using an Emulsifiex C5 (Avestin, Ottawa, Canada) in 50mM NaH 2 P0 4 , 125mM NaCl, pH 8 buffer. The lysate was treated with 100 U/mL of Benzonase Nuclease (Sigma-Aldrich, Oakville, Canada) for 20 min at RT and centrifuged.
  • IPTG isopropyl ⁇ -D- thiogalactopyranoside
  • the supernatant was then treated with Benzonase nuclease (Sigma-Aldrich, Oakville, Canada) for 30 min at RT and centrifuged. The resulting supernatant was filtered through a 0.22 ⁇ filter and kept overnight at 4°C. The NaCl concentration of the buffer was adjusted to 1 M NaCl before the protein purification, which was made on an IMAC column connected to an AKTA purifier 10 (GE Healthcare, Baie d'Urfe, Canada).
  • the lysate was loaded onto a XK26/20 chromatography column containing Ni Sepharose 6 Fast Flow resin (both GE Healthcare, Baie d'Urfe, Canada) and the beads were washed successively with 5 column volume (CV) of washing buffer 1 (lOmM Tris-HCl, lOOOmM NaCl, 25mM Imidazole, pH 8.0) and 5 CV of washing buffer 2 (lOmM Tris-HCl, 300mM NaCl, 40mM Imidazole, pH 8.0). Proteins were eluted in elution buffer (lOmM Tris-HCl, 300mM NaCl, 500mM Imidazole, pH 8.0).
  • the eluted proteins were then subjected to a tangential flow filtration (TFF) using a Sartocon Slice 200 Benchtop Complete and a Sartocon Slice 200 Hydrostart 30 kDa (both from Sartorius, Mississauga, Canada) to remove the imidazole and further improve the protein purity.
  • the protein solution was then filtered with a 0.22 ⁇ membrane and the purity of the proteins was evaluated by SDS/PAGE.
  • Protein concentrations were evaluated with a bicinchoninic acid protein (BCA) kit (Pierce, Rockford, IL) and the lipopolysaccharide (LPS) content in the purified proteins was calculated with the Limulus test according to the manufacturer's instructions (Lonza or Cambrex, Walkersville, MD) and was consistently below the limit acceptable for injection in mice ( ⁇ 50 endotoxin units/injection).
  • BCA bicinchoninic acid protein
  • LPS lipopolysaccharide
  • NLPs Nucleocapsid-Like Particles
  • rNP monomeric nucleoprotein
  • Poly-C Polycytidylic acid potassium salt
  • Poly-U Polyuridylic acid single-stranded RNA polymer
  • 10 mM Tris HC1 buffer pH8.0 for 2 hours at 22°C.
  • NLPs were then subjected to a tangential flow filtration (TFF) using a Sartocon Slice 200 Benchtop Complete and a Sartocon Slice 200 Hydrostart 100 kDa to remove the rNP that did not bind the RNA and to further purify the NLPs.
  • the NLPs were then filtered through a 0.22 ⁇ membrane.
  • NLPs were loaded on a Superdex 200 10/30 GL gel-filtration column. Proteins concentrations and LPS content were evaluated as described above. Electron Microscopy and Dynamic Light Scattering
  • rNP and NLPs were diluted in water to a concentration of 0.01 mg/ml and stained by mixing 10 of the sample with 10 of 3% acetate-uranyl for 7 minutes in the dark. 8 of this solution was then put on carbon-formvar grids for 5 minutes. Grids were observed with a FEI-Tecnai-G2 Spirit transmission electron microscope (FEI, Hillsboro, Oregon, USA). The size of the NLPs was recorded with a ZetaSizer Nano ZS (Malvern, Worcestershire, U.K.) at a temperature of 4°C and at a concentration of lmg/ml diluted in lOmM Tris-HCl pH 8.0, 125mM NaCl. The variation in the NLP size induced by temperature variations was measured according to the same experimental conditions. Immunization of Mice
  • mice 6-8-week-old BALB/c mice (5/group) (Charles River, Wilmington, MA) were immunized intramuscularly with 10 or 50 ⁇ g of rNP or either NLP-Poly-U or NLP-Poly-C. Primary immunization was followed by a boost dose given 2 weeks later. Blood was collected 14 days after each immunization.
  • mice 6-8-week-old BALB/c mice (10/group) were immunized intramuscularly with 0.5 ⁇ g of either rNP or NLP-Poly-U with or without 5, 10, 20 or 40 ⁇ g of Papaya mosaic virus (PapMV) virus-like particles (VLPs) as adjuvant (PAL adjuvant). Blood was collected 14 days after the immunization and an ELISPOT assay was performed using 5 mice per group.
  • PapMV Papaya mosaic virus
  • VLPs virus-like particles
  • a boost immunization using the same dose was made in each of the 4 groups (rNP, NLP-Poly-U, rNP + 40 ⁇ g PAL, NLP-Poly-U + 40 ⁇ g PAL) and an ELISPOT assay was performed one week later.
  • mice 6-8-week-old BALB/c mice (10/group) were immunized once intramuscularly with the vaccine formulations shown in Table 2. At day 14 post-immunization, blood was collected to measure the IgG2a titers against NP. Table 2: Vaccine formulations for the influenza challenge
  • rNP at 2 ⁇ g/mL was diluted in 0.1M NaHCOs buffer (pH 9.6) and 100 ⁇ , of the diluted NP was coated on a 96-well plates overnight at 4°C. Plates were blocked with 150 ⁇ of PBS/0.1% Tween-20/2% BSA for lh at 37°C and then washed three times with PBS/0.1% Tween-20. Sera from the immunized mice were added in 2-fold serial dilutions starting with 1 :50 and the plates were incubated for 90 min at 37°C.
  • NP-GST was used at 3 ⁇ g/ml instead of rNP to coat the 96-well plates since cross-reactivity was observed between the PAL antibody and the rNP used for the ELISA.
  • MultiScreen-IP opaque 96-well plates (Fischer Scientific, Ontario, Canada) were treated with ethanol 35% then coated with 100 ⁇ ⁇ of capture IFN- ⁇ diluted in sterile PBS as suggested in the murine interferon-gamma ELISPOT kit instructions (Abeam, Cambridge, MA, USA).
  • mice On the day of the splenocytes isolation, two weeks post-immunization, the mice were sacrificed and the spleens were removed and put in culture media (RPMI 1640) supplemented with 10% heat-inactivated fetal bovine serum, 2mM glutamine, ImM sodium pyruvate, 50 ⁇ ⁇ -mercaptoethanol, lOmM HEPES, 100 U/mL penicillin and 100 ⁇ g/mL streptomycin. Spleens were cut in culture medium and then mashed through a 100 ⁇ cell strainer. The cells were then centrifuged and incubated 3 min at room temperature with ammonium chloride-potassium (ACK) lysis buffer to remove the red blood cells.
  • RPMI 1640 RPMI 1640
  • fetal bovine serum 2mM glutamine
  • ImM sodium pyruvate 50 ⁇ ⁇ -mercaptoethanol
  • lOmM HEPES 100 U/mL penicillin and 100 ⁇ g/mL str
  • the plates were washed 3 times with 100 ⁇ ⁇ of PBS/0.1 % Tween-20.
  • 100 ⁇ /well of biotinylated detection anti-mouse IFN- ⁇ antibody diluted in PBS/1% BSA was added and the plates were incubated for 1.5 h at 37°C, 5% CO2.
  • Plates were once again washed 3 times with PBS/0.1 % Tween-20 and 100 ⁇ ⁇ of streptavidin-alkaline phosphatase conjugated secondary antibody diluted in PBS/1% BSA was added and the plates were incubated for 1.5 h at 37°C, 5% C0 2 .
  • mice were challenged 2 weeks after the last immunization with 120 or 240 plaque forming units (pfu) of A/WSN/33 (H1N1) influenza virus by 50 ⁇ intranasal instillation. Weight losses, survival and symptoms were monitored daily for 14 days post-infection. Symptoms are rated from 1 to 4, where 4 is the highest score and mice are euthanized (1 - lightly spiked fur; 2 - spiked fur, curved back; 3 - spiked fur, curved back, difficulty to move, light dehydration; 4 - spiked fur, curved back, difficulty to move, severe dehydration, closed eyes, ocular secretion). Mice that lost more than 20% of their initial weight are also euthanized.
  • NP nucleoprotein
  • H1N1 pandemic strain A/California/04/2009
  • SEQ ID NO:5 The sequence coding for the nucleoprotein (NP) of the pandemic strain A/California/04/2009 (H1N1) combined with a 6xHis tag at the C-terminal end (Fig. 1A and Fig. 13B) [SEQ ID NO:5] was amplified and cloned into the expression vector pQE-80-L- KAN. The expression vector was then cloned into an E. coli K12 strain (BD792) and a parental cell bank was produced. The day of production, a vial from the cell bank was put into culture until the optical density at 600 nm of the culture got to 1.0.
  • BD792 E. coli K12 strain
  • the recombinant protein was induced with the addition of ImM IPTG and incubation at 22°C for 16h.
  • the biomass was harvested by centrifugation and the pellet was resuspended in lysis buffer.
  • the cells were broken mechanically by homogenization and the cell lysate was liquefied with nuclease treatment, and clarified by centrifugation and filtration.
  • the clarified cell lysate was then loaded on an IMAC column. The column was washed with a set of buffers and the recombinant protein was eluted with high concentrations of imidazole.
  • the eluted protein was dialysed with a 30 kDa MWCO membrane using Tangential Flow Filtration to further purify the protein, remove the imidazole and decrease the LPS content below 50 EU/injection.
  • the yield of the purified and filtered recombinant NP was consistent between batches and ranged between 80 - 100 mg per litre of culture.
  • the SDS-PAGE profile showed that the rNP is highly purified (Fig. IB) and a Western blot assay (Fig. 1C) using a polyclonal antibody reacting specifically to the nucleoprotein of influenza A virus confirmed the identity of the recombinant protein.
  • the rNP was multimerized into nucleocapsid-like-particles (NLPs) using two different single-stranded synthetic RNAs as a scaffold: polycytidylic acid potassium salt (NLP-Poly-C) or polyuridylic acid single-stranded RNA polymer (NLP-Poly-U). Briefly, the monomeric rNP was mixed in a 10: 1 ratio with either Poly-C or Poly-U, incubated for 2 hours and dialysed using Tangential Flow Filtration with a 100 kDa MWCO membrane to remove any monomeric rNP that did not bind to the RNA.
  • NLP-Poly-C polycytidylic acid potassium salt
  • NLP-Poly-U polyuridylic acid single-stranded RNA polymer
  • the NLPs were analyzed on a Superdex 200 to evaluate their molecular weight and to confirm that there was no monomeric rNP left after the dialysis.
  • the elution profile of each NLP (Fig. 2A) showed a heterogeneous population of NLPs with a higher molecular weight than the monomeric rNP.
  • the elution profile also showed that the dialysis efficiently removed any monomeric rNP that could have been associated with the NLPs since there was no protein eluted between 15 and 18 mL.
  • DLS dynamic light scattering
  • NLP-Poly-C was stable throughout the temperature gradient while the NLP-Poly-U was stable up to 37°C when aggregation was initiated (Fig. 2B, right panel).
  • Transmission electron microscopy of both NLPs showed a population of elongated structures having different lengths and irregular edges (Fig. 2C, middle and right panel), while no visible monomeric rNP (Fig. 2C, left panel) was observed.
  • FIG. 14 A flow-chart summarizing the preparation of the NP NLPs using the His-tagged NP is provided in Fig. 14.
  • EXAMPLE 2 Immune Response Against Multimerized NP
  • Example 1 As the two different NLPs produced in Example 1 showed different behaviour under a temperature gradient, an investigation was made into whether there was a difference between the two NLPs in eliciting a humoral immune response against NP.
  • Mice were immunized twice at a 14-day interval by the intramuscular route with two different doses of monomeric rNP or with either NLP-Poly-U or NLP-Poly-C.
  • Antibody levels against NP in the blood were measured by ELISA at day 14 and 28.
  • Total IgG titers Fig. 3A
  • IgG2a titers Fig.
  • NLPs had a length of 27 nm (Fig. 5B, left panel) and that they were stable throughout the temperature gradient (Fig. 5B, right panel). Transmission electron microscopy of the NLPs still showed elongated structures of various lengths and having irregular edges (Fig. 5C).
  • NP is one of the main targets of the cellular immune response against influenza and that it contains multiple conserved MHC class I and class II epitopes (Lee LY-H, et al, 2008, J Clin Invest, 118:3478-90).
  • an ELISPOT assay was performed on the mice immunized with either rNP or NLP-Poly-U.
  • the IFN- ⁇ secretion of T cells was evaluated using the H-2K D Influenza NP peptide TYQRTRALV [SEQ ID NO:3] or NLP-Poly-U to reactivate splenocytes that were harvested 2 weeks post-immunization.
  • the ELISPOT assay (Fig. 6B) showed that the multimerization did not improve the number of T cells secreting IFN- ⁇ . Since no differences were observed even with the control group that received formulation buffer, it appears that a single immunization was not enough to induce a potent anti-NP cellular response although it induced a significant humoral response to NP. As such, a boost immunization was performed and splenocytes harvested 1 week after.
  • WO2012/155262 was administered in combination with monomeric rNP or the NLP-Poly-U to determine whether there is still a benefit to the multimerization of NP when used with an adjuvant, or whether similar results could be achieved with adjuvanted monomeric rNP.
  • BALB/c mice (10/group) were immunized once intramuscularly with 0.5 ⁇ g of either rNP or NLP-Poly-U adjuvanted with 5, 10, 20 or 40 ⁇ g of PapMV VLPs ("PAL") and the humoral response to NP was measured by ELISA 14 days post-immunization.
  • Total IgG and IgG2a titers Fig.
  • EXAMPLE 5 Boost-Immunization Increases Both Humoral and Cellular Responses to NP
  • Splenocytes were harvested 7 days after the last immunization as described in the previous Example and reactivated with the NLP-Poly-U or the H-2K D peptide to evaluate the IFN- ⁇ secretion of the T cells.
  • the boost immunization significantly increased (p ⁇ 0.001) the number of T cells secreting IFN- ⁇ (Fig. 8B) when stimulated with the NLP or the H-2K D peptide.
  • the results also show that, after one or two immunizations, the combination of PAL to NLP did not significantly increase the cellular immmune response when compared to NLP alone (Fig. 8C).
  • EXAMPLE 6 Challenge Experiments With Influenza Virus
  • This Example investigated whether the combination of NLP with PAL adjuvant was able to protect mice from a lethal challenge with a H1N1 influenza strain after a single immunization.
  • Mice (10/group) were immunized with the vaccine formulations listed in Table 2 above.
  • blood was collected to measure the IgG2a titers to NP.
  • NP-GST was used as the capture antigen to prevent putative cross-reactivity to PAL and rNP antigens that both were harboring a His-Tag when used for immunization.
  • IgG2a titers (Fig. 9) showed that groups immunized with NLP + PAL had a significantly increased humoral response against NP (10-fold increase) when compared to the groups immunized with NLP alone.
  • mice were challenged with 1 or 2 lethal dose (LD50) of the mouse-adapted influenza strain A/WSN/33 (H1N1) and monitored daily for weight loss, clinical symptoms and survival.
  • LD50 lethal dose
  • the results of the infectious challenge showed that a single immunization with NLP-Poly-U + PAL was not sufficient to provide a complete protection from a lethal challenge with the A/WSN/33 (H1N1) strain.
  • Fig. 10A There were no significant difference in the weight loss between the different groups challenged with 1 or 2 LD50 (Fig. 10A), and no significant differences of survival (Fig. 10B, left panel) or symptoms (Fig. IOC, left panel) between the groups challenged with 1 LD50.
  • mice immunized with 20 ⁇ g NLP + 80 ⁇ g PAL showed significantly (p ⁇ 0.001) better survival (Fig. 10B, right panel) and exhibited significantly (p ⁇ 0.05) less symptoms (Fig. IOC, right panel) at the peak of infection when compared with mice that did not receive the NLP vaccine.
  • the significant difference (p ⁇ 0.01) in survival between the mice immunized with 20 ⁇ g of NLP alone and the mice immunized with 20 ⁇ g of NLP combined with 80 ⁇ g of PAL also showed the importance of the adjuvant in the protection against an influenza challenge.
  • Blood was collected from mice surviving the ILD50 infectious challenge and the IgG2a response against NP was measured.
  • Fig. 11A An increase in the humoral response to NP is a mechanism that could explain the protection against infection in the surviving mice.
  • the ELISA results (Fig. 11A) showed a significant (pO.001) increase in the NP-specific IgG2a humoral response before and after the challenge in all animal groups. Seven days post- challenge, the cellular response in the formulation buffer group (5 mice) was also measured, as well as in the 10 ⁇ g NLP + 40 ⁇ g PAL group (5 mice) using the ELISPOT assay described in the previous Examples.
  • This assay (Fig. 11B) showed that mice immunized with NLP + PAL had a 3-fold increase (p ⁇ 0.001) in the number of IFN- ⁇ secreting T-cells when compared to the group immunized with formulation buffer only. While the increased cellular response did not enhance the survival of the mice immunized with the vaccine, it is possible that the enhanced cellular response could lead to a better protection against a subsequent infection.
  • Examples 1 to 6 demonstrate that multimerization of NP enhances the immunogenicity of the protein. It is known that influenza NP can bind single-stranded RNA with a high affinity and that this binding requires little or no sequence specificity (Kingsbury D, et al. , 1987, Virology, 156:396-403; Portela A, and Digard P., 2002, J Gen Virol, 83:723- 34). This capacity was leveraged to multimerize NP with a synthetic ssRNA (Poly-U) to produce Nucleocapsid-like-particles (NLPs). The NLPs are nanoparticles that look like elongated structures with irregular edges and are composed of multiple copies of the same protein assembled in a more ordered and repetitive form with an RNA scaffold.
  • PAMPs pathogen-associated molecular patterns
  • PRRs pathogen-recognition receptors
  • TLRs Toll-like receptors
  • Poly-U has also been shown to be a potent inducer of a cytotoxic immune response mediated by CD8+ T cells and can generate a Thl response (Crespo MI, et al, 2013, J Immunol, 190:948-60).
  • the increased cellular response observed with the NLP may be at least partially due to these effects.
  • Adjuvants can be used to increase the protective antibody response, lower the vaccine dose to allow dose sparing and enhance the generation of a T-cell response.
  • PapMV VLPs PAL
  • PAL enhanced both the IgG2a and total IgG response against the nucleoprotein after one intramuscular immunization.
  • Examples 1 to 6 show that one immunization with NLP + PAL induced NP-specific IFN- ⁇ production and an especially potent humoral response to NP, however, a single immunization was not able to fully protect mice against a lethal influenza challenge.
  • Antibodies to NP are non-neutralizing and thus cannot stop the viral infection, however, they are still essential for rNP-elicited protection from influenza virus (Carragher DM, et al, 2008, J Immunol, 181 :4168-76). Huang et al.
  • the efficacy of the multimerized NP with or without adjuvant may be improved using one or more of different doses of NLP in combination with the PAL adjuvant, combining the NLP (and optionally the PAL adjuvant) with another conserved influenza antigen, such as the matrix protein 2 ectodomain (M2e).
  • NLP and optionally the PAL adjuvant
  • M2e matrix protein 2 ectodomain
  • Graphs summarizing the in vivo effect of NLP with and without PAL adjuvant on IgG2a titers are provided in Fig. 12 and show that multimerization of NP into NLPs increases the immune response (Fig. 12A), the addition of the PAL adjuvant to the NLPs increases the immune response (Fig. 12B), and the combination of PAL adjuvant with NLPs produces a stronger immune response than the combination of PAL with monomeric NP (Fig. 12C).
  • NP fusion protein that contained a fusion of the M2e peptide EVETPIRNE [SEQ ID NO: 9] at the C -terminus was constructed as follows.
  • the plasmid pQE80-NP-Cterm.6his is composed of the pQE80-KAN expression vector in which the recombinant NP gene (H1N1 strain) fused at the 3 '-end to a sequence encoding 6his-tag has been inserted in front of a RNA polymerase promoter.
  • pQE80-NP-Cterm.6his ssDNA was incubated with two 33-mers primers complementary to sequences located at the 3'-end of the recombinant NP gene.
  • One oligonucleotide harbored the additional nucleotide codons 5'-CCG-ATC-CGT-AAC-GAA-3', whereas the other harbored the additional codons 5'-GAA-GTT-GAA-3'.
  • Each oligonucleotide was used to prime, in opposite direction, PCR replication of the plasmid DNA producing a double- stranded recombinant plasmid harboring a NP gene fused at its 3'-end to sequences encoding the M2e peptide EVETPIRNE followed by 6xhis-tag.
  • the amino acid sequence of the fusion protein is shown in Fig. 13C [SEQ ID NO:6].
  • NPM2ec protein Monomeric fusion protein
  • the NPM2ec protein was able to multimerize into NLPs.
  • the NLPs showed a tendency to aggregate under the conditions used for the EM. Optimization of the EM conditions should improve the quality of the micrograph.
  • Dynamic light scattering indicated that the NLPs formed from the NPM2ec protein had an average particle length of approximately 30 nm when using SRT500 as RNA scaffold and 50 nm when SRT1517 (Fig. 20B).
  • the average width of both NLPs was between about 13 nm and about 18 nm.
  • NPM2ec NLPs are very similar to those made with the wild-type (WT) NP, it is anticipated that NPM2ec NLPs will induce a comparable CTL response (for example, as measured by ELISPOT) against the NP component of the NPM2e protein and a comparable IgG and IgG2a response toward the WT NP protein, as was observed for the NLPs using the WT protein.
  • CTL response for example, as measured by ELISPOT
  • IgG and IgG2a response toward the M2e peptide will be observed, and will enhance the protection against influenza after an infectious challenge such as those described above.
  • NPM2e protein will be more robust than that obtained with the WT NP NLPs alone because the immune response directed to the M2e peptide will enhance the protection elicited in the vaccinated animals.
  • NPM2ec or NPM2ec NLPs are combined with the PAL adjuvant.
  • FIG. 31 A flow chart showing an overview of the steps for the production of influenza B NP is provided in Fig. 31.
  • an optimized influenza B amino acid sequence that is a consensus between several strains of influenza B [SEQ ID NO: 34] (Fig. 30) was amplified and cloned into the expression vector pQE-80-L-KAN. The expression vector was then cloned into an E.coli K12 strain (BD792) and a parental cell bank was produced.
  • the eluted protein was dialysed with a 8 kDa MWCO membrane to remove imidazole and filtered to sterilize the protein.
  • the SDS-PAGE profile showed that the rNP had associated with the bacterial RNA to multimerize into NLPs (Fig. 28).
  • Dynamic light scattering (DLS) was used to measure the average length of the NLPs (Fig. 29A) which showed an average particle length of approximately 80 nm and a width of between 12 and 15nm in diameter. Transmission electron microscopy confirmed that the NLPs had an elongated rod-like structure with irregular edges (Fig. 29B).
  • NLPs formed from the NPs derived from influenza B were shown to be very similar to those made with NPs derived from influenza A, it is anticipated that NLPs comprising NPs derived from influenza B will have comparable immunogenic properties to that observed with NLPs comprising NPs derived from influenza A. This preliminary study further confirms that rod-like NLPs can be multimerized from NPs derived from other influenza genera. It is further anticipated that comparably immunogenic NLPs may also be prepared with NPs derived from other orthomyxoviridae.
  • EXAMPLE 10 Comparison of Immunogenicity of NLPs and Closed Ring Structures
  • the structures formed were closed ring structures and the multimers were generally trimers or tetramers. These multimeric forms of NP were also described as being in dynamic equilibrium with the monomeric form.
  • the immunogenicity of the closed ring structures previously described was tested and compared to that of representative rod-like NLPs disclosed herein. The immunogenicity of the closed ring structures was shown to be lower than the NLP structures, likely the result of the NLP structures of the present disclosure being more ordered and stable.
  • NP protein was purified from ii. coli as described in Example 1 with the update described in Example 3.
  • a flow chart showing an overview of the steps for the production of NP protein and NLPs is provided in Fig. 23.
  • NP protein was cloned into a bacterial expression vector (pQE-80) and the protein was expressed through induction of the T7 promoter using IPTG.
  • E coli strain BD-792
  • the bacteria are lysed using an homogenizer.
  • the lysate was treated with a nuclease, clarified and passed on an immobilized metal ion chromatography (IMAC) for purification.
  • IMAC immobilized metal ion chromatography
  • the 6xH tag located at the C terminus of the protein facilitated its purification.
  • the protein was eluted, LPS removed, passed through a tangential flow filtration 30kDa to remove the imidazole and filtered.
  • the NP protein was then used for the assembly reaction for preparation
  • NLPs were assembled with the monomeric NPs prepared as described above and SRT1517 ssRNA (Fig. 22) [SEQ ID NO:32]. Similar to the protocol described in Example 1, the NP proteins were combined with the ssRNA in an optimal ratio of protein:RNA of 7.5: 1. The assembly reaction was either passed through a TFF lOOkDa (RS100) or not (FSAR).
  • Electron micrographs were taken on a FEI Technai Spirit G2, and negative staining was used using uranyl acetate 2% dissolved in lOmM Tris/HCl pH 8.0.
  • mice (5 per group) were immunized once at day 0 by the intramuscular route with 10 ⁇ g of either the monomeric form of the influenza NP (NPcH monomer (SR30)), the closed ring form previously disclosed (NPcH Ring (SRS)), the NLPs that have been filtered after the assembly reaction (NPcH NLPs (FSAR)) and the NLPs that have been passed through tangential flow filtration (lOOkDa) before filtering (NPcH NLPs (SRI 00)). Bleeding was performed at day 14 and the serum was used to make the ELISA against influenza NP fused to the GST protein (without 6xH tag). Total IgG and IgG2a titers were measured.
  • NLPs NP Nanoparticles
  • the assembly reaction was performed using purified influenza NP from the pandemic strain H1N1 and two different substrates, a ssRNA of 1517 nt long (SRT1517) [SEQ ID NO:32] to make NLP nanoparticles, or a ssDNA of 76 nt long [SEQ ID NO:33] to make the closed ring structure.
  • Fig. 24A transmission electron microscopy showed long rod-shaped nanoparticles assembled around the SRT1517 RNA.
  • Dynamic light scattering (DLS) indicated that the NLPs had an average particle length of approximately 80nm (Fig. 24B).
  • the NLPs were analyzed on a Superdex 200 to evaluate their molecular weight. The NLPs were excluded from the gel filtration due to their large size and were found in the exclusion with a peak at 9.07 mL (Fig. 24C).
  • the immunogenicity of the multimerized rod-like NLP structure was compared to the immunogenicity of the monomeric form of NP and the closed ring structures.
  • Balb/C mice (5 per group) were immunized once with 10 ⁇ g of either the monomeric form of NP [NPcH monomer (SR30)], the NP ring [NPcH Ring (SRS)] or the NP nanoparticles that have been filtered after the assembly reaction [NPcH NLPs (FSAR)] or passed through tangential flow filtration lOOkDa before filtering [NPcH NLPs (SRI 00)].
  • the serum was harvested at 14 days after immunization.
  • NLPs were combined with PapMV VLP adjuvant or PapMV VLPs fused with a short version of the M2e peptide (Carignan et al, 2015). Fusion of the PapMV VLP with M2e peptide introduced a second conserved influenza antigen, i.e., the matrix protein 2 ectodomain.
  • mice were immunized twice at day 0 and 21 by the intramuscular route with either buffer control, 10 ⁇ g of the rod-like NLP alone (NP), 10 ⁇ g of the rod-like NLP + 30 or 60 or c ⁇ g of PapMV VLP (NP + PapMV 30 or 60 or 90), ⁇ g of the rod-like NLP + 30 or 60 or 90 ⁇ g of engineered PapMV VLPs presenting at their surface the influenza M2e peptide (Carignan et al, 2015)(NP+PapMV-M2e 30, 60 or 90).
  • mice were challenged with influenza WSN/33 at day 42 and the infection protocol terminated at day 56. Mice were infected with either 300 or 600 pfu (plaque forming unit) that correspond to 3 and 6 LD50 respectively. During those 14 days (42-56), scoring was conducted for the development of symptoms, weight loss, and survival. Symptoms were scored as follows: 0. No symptoms. 1. Lightly spiked fur, slightly curved back. 2. Spiked fur, curved back. 3 Spiked fur, curved back, difficulty in moving and mild dehydration. 4. Spiked fur, curved back, difficulty in moving, severe dehydration, closed eyes and ocular secretion.
  • a second challenge was performed on animals immunized with the same vaccine formulations as well as with four additional groups of 10 mice immunized with 10 ⁇ g of rod- like NP nanoparticles+30, 60 or ⁇ g of PapMV-M2e nanoparticles (NP+PapMV -M2e 30, 60 or 90) and PapMV-M2e ⁇ g (Fig. 27B-D).
  • the best performing group was the group immunized with rod-like NP (10 ⁇ g) + PapMV-M2e nanoparticles (90 ⁇ g).
  • This vaccinated group contains 2 antigens, the NP and the M2e that together provided the best survival (100%) (Fig. 27B), the least symptoms (Fig. 27C) and the least weight loss (Fig. 27D).

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Abstract

L'invention concerne des particules de type nucléocapside (NLP) formées à partir de nucléoprotéine (NP) d'orthomyxovirus recombinée et d'ARN monocaténaire (ARNss) et ayant une structure de type bâtonnet, qui sont capables d'induire une réponse anticorpale contre la NP améliorée par rapport à celle de la NP monomère. La NP comprise par les NLP peut être dérivée du virus de la grippe et éventuellement être fusionnée à un peptide qui comprend un ou plusieurs épitopes provenant d'une autre protéine de la grippe, tels que le peptide M2e. Les NLP peuvent être utilisées comme vaccins pour une protection contre des infections par le virus de la grippe.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2835972A1 (fr) * 2011-05-13 2012-11-22 Folia Biotech Inc. Particules pseudo-virales (vlp)et leur procede de preparation
CA2908414A1 (fr) * 2012-04-02 2013-10-10 Folia Biotech Inc. Proteines de revetement du virus recombinant de la mosaique de la papaye et utilisation de celles-ci dans des vaccins contre la grippe

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Publication number Priority date Publication date Assignee Title
CA2835972A1 (fr) * 2011-05-13 2012-11-22 Folia Biotech Inc. Particules pseudo-virales (vlp)et leur procede de preparation
CA2908414A1 (fr) * 2012-04-02 2013-10-10 Folia Biotech Inc. Proteines de revetement du virus recombinant de la mosaique de la papaye et utilisation de celles-ci dans des vaccins contre la grippe

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