WO2016127262A1 - Multimerized orthomyxovirus nucleoprotein and uses thereof - Google Patents

Abstract

Nucleocapsid-like particles (NLPs) formed from recombinant orthomyxoviral nucleoprotein (NP) and single-stranded RNA (ssRNA) and having a rod-like structure are provided which are capable of eliciting an improved antibody response against NP when compared to monomeric NP. The NP comprised by the NLPs may be derived from influenza virus and optionally be fused to a peptide that comprises one or more epitopes from another influenza protein, such as the M2e peptide. The NLPs have use as vaccines for protection against influenza virus infections.

Description

MULTIMERIZED ORTHOMYXOVIRUS NUCLEOPROTEIN AND

USES THEREOF

FIELD OF THE INVENTION

[0001] The present invention relates to the field of influenza vaccines and, in particular, to multimerized orthomyxovirus nucleoprotein and influenza vaccines comprising same.

BACKGROUND OF THE INVENTION

[0002] Each year, influenza epidemics are responsible for the infection of 5 to 15% of the worldwide population which result in 3 to 5 million cases of severe illness and up to 500 000 deaths (World Health Organization. Influenza seasonal fact sheet. Fact Sheet N°211 2014). Trivalent inactivated vaccine (TIV), which results in a neutralising antibody-based response directed to the variable surface proteins hemagglutinin (HA) and neuraminidase (NA) has been used for more than 60 years and is still the most efficient way to prevent infection with the virus (Sigel MM., 1948, J Am Med Assoc, 136:437). However, 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. This response is associated with a more rapid viral clearance and a reduction in both morbidity and mortality (Epstein SL., 2003, Expert Rev Anti Infect Ther, 1 :627-38; Grebe KM, et al, 2008, Microbes Infect, 10: 1024-9; Tamura S, et al, 1996, J Immunol, 156:3892-900; Ulmer JB, et al, 1998, J Virol, 72:5648-53).

[0004] 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). Studies on the oligomerization paths of influenza NP indicate that it oligomerizes into trimers and tetramers in the presence of short RNA sequences (24-mers) and into ring-like structures of 8-10 NP molecules in the presence of longer RNA or DNA sequences (51- to 55-mers) (Tarus et al, 2012, Biochimie, 94:776-785). [0005] 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). A number of studies have shown that 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). The humoral response against NP has long been disregarded but recent studies have shown that immunization with NP results in antibodies that can also contribute to cross-protection (Lamere MW, et al, 2011, J Virol, 85:5027-35; Carragher DM, et al, 2008, J Immunol, 181 :4168-76) by a mechanism involving both FcRs and CD8+ cells (LaMere MW, et al, 2011, J Immunol, 186:4331-9). Immunization with soluble recombinant NP seems to be an efficient way to protect against infection but high doses, multiple immunizations or the use of an adjuvant is required since soluble recombinant proteins generally have a low immunogenicity (Guo L, et al, 2010, ibid. ; Wang W, et al, 2012, ibid ; Tite JP, et al, 1990, Immunology, 71 :202-7; Macleod MKL, et al, 2013, PLoS One, 8:e61775; Jelinek I, et al, 2011, J Immunol, 186:2422-9; Wang W, et al, 2014, Virology, 468-470C:265-73; Huang B, et al, 2012, Virol J, 9:322; Diego E, et al, 2012, J Microbiol Biotechnol, 22:416-21).

[0006] 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.

[0007] 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. [0008] 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. [0009] 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.

[0010] Combination of the influenza NP with other influenza proteins has been proposed as a means for eliciting an improved immune response. Fusions of NP to the M2e peptide from influenza matrix 2 protein (M2) have been described. Wang W, et al, 2012, PLoS One, 7:e52488 describe a recombinant NP-M2e fusion protein expressed in E. coli, which when administered in combination with an adjuvant such as alum or CpG, was able to induce an immune response to a heterologous influenza virus strain. [0011] International (PCT) Patent Application Publication No. WO 2009/073330 describes DNA vaccines that are capable of expressing a consensus influenza antigen, the consensus influenza antigen comprising consensus HA, NA, matrix protein, NP, M2e-NP or a combination thereof.

[0012] 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.

[0013] International (PCT) Patent Application Publication No. WO 2009/026465 describes compositions of influenza proteins, including NP fused to multiple copies of M2e, that are useful as vaccines. [0014] 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). Studies have shown that 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). This technique has been applied to a variety of nucleocapsids (Lopez C, et al, 2009, Arch Virol, 154:695-8; Kunkel M, et al, 2001, J Virol, 75:2119-29; Tellinghuisen TL, et al, 1999, J Virol, 73:5309-19; Kho C, et al, 2001, J Microbiol, 39:293-9) to produce nucleocapsid-like-particles (NLPs) which result in an enhancement of antigen immunogenicity (Gil L, et al, 2012, J Gen Virol, 93: 1204-14; Gil L, et al, 2009, Int Immunol, 21 : 1175-83).

[0015] 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

L, et al, 2009, ibid.; Lazo L, et al, 2007, Vaccine, 25: 1064-70). [0016] 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. [0017] Chinese Patent No. CN101643721 describes a recombinant virus-like particle containing influenza matrix protein Ml, NA, HA and an M2eNP fusion protein.

[0018] This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.

SUMMARY OF THE INVENTION

[0019] 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.

[0020] According to certain embodiments described herein, NLPs of the present disclosure can comprise recombinant NP polypeptides having a sequence derived from an orthomyxovirus. According to a preferred embodiment, the NLPs can comprise recombinant NP polypeptides having a sequence derived from influenza virus. According to further embodiments, the NLPs can comprise recombinant NP polypeptides having a sequence derived from an influenza virus type B or type A NP.

[0021] Another aspect of the invention relates to a pharmaceutical composition comprising an NLP as described above and a pharmaceutically acceptable carrier or diluent.

[0022] According to certain embodiments described herein, pharmaceutical compositions of the present disclosure can further comprise an adjuvant. In some such embodiments, the adjuvant comprises Papaya mosaic virus (PapMV) virus-like particles (VLPs). According to further embodiments, the PapMV VLPs comprise one or more influenza antigens fused to the PapMV coat protein. In this way, the PapMV VLPs can be used as a vaccine platform that presents the one or more influenza antigens to immune cells. In other embodiments, the one or more influenza antigens are derived from the influenza M2 protein, for example, the M2e peptide or a fragment thereof.

[0023] Another aspect of the invention relates to a vaccine comprising the pharmaceutical composition.

[0024] Another aspect of the invention relates to an in vitro process for preparing a nucleocapsid-like particle (NLP) comprising a plurality of recombinant influenza nucleoprotein (NP) polypeptides and single-stranded RNA (ssRNA) comprising: a) combining recombinant NP polypeptide and ssRNA at a protein:RNA ratio of between about 1 : 1 and 50: 1 by weight, and a temperature between about 2°C and about 37°C, for a time sufficient to allow assembly of NLPs, the ssRNA being between about 120 and about 5000 nucleotides in length, and b) separating the NLPs from other process components.

[0025] 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. According to a preferred embodiment, the immune response is against influenza virus infection.

[0026] 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.

[0027] 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].

[0028] Another aspect of the invention relates to a nucleocapsid-like particle (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. [0029] 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. [0030] 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.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] These and other features of the invention will become more apparent following detailed description in which reference is made to the appended drawings. [0032] 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: purified NP protein, and (D) shows the elution profile of the purified rNP on a Superdex 200 Size-exclusion chromatography (SEC) column. The molecular weight of the different proteins used to calibrate the column is shown above the graph. The absorbance at 280 nm and 254 nm is used to reveal protein. [0033] 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.

[0034] Figure 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.

[0035] 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. [0036] 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). Right panel: Size of NLP-Poly-U measured under a temperature gradient. C. Transmission electron microscope images of NLP-Poly-U at magnification 49 OOOx. [0037] Figure 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 μg 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-γ 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.

[0038] 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 μg of either rNP or NLP-Poly-U adjuv anted with 5, 10, 20 or 40 μg 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. The number of IFN-γ 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.

[0039] 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 μg of NLP alone or combined with 40 μg 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-γ secretion by the T cells. Splenocytes of mice immunized twice were harvested 7 days after the last immunization and reactivated with the H-2K^D peptide (C: left panel) or NLP-Poly-U (C: right panel) to evaluate the IFN-γ secretion by the T cells. ** P < 0.01, *** P < 0.001. [0040] Figure 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. [0041] 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 LD₅₀) or 240 pfu (2 LD₅₀) of A/WSN/33 (H1N1) influenza virus and monitored for weight loss (A), survival (B) and clinical symptoms (C) for 14 days. 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.

[0042] 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 μg NLP + 40 μg PAL group were harvested 7 days post-infection and re-stimulated with the H-2K^D peptide to evaluate IFN-γ secretion by T cells. ND: non-detected. *** P < 0.001. [0043] 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). Each point corresponds to the Geometric Mean Titer (GMT) for 10 mice. Bleeding was at day 14 post-immunization. No adverse event was observed for any of the tested formulations.

[0044] 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].

[0045] Figure 14 presents a flow-chart summarizing the process for the production and purification of NLP. [0046] Figure 15 is a SDS-PAGE gel showing the purification of the His-tagged recombinant rNP protein from E. coli.

[0047] 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. [0048] 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).

[0049] 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).

[0050] 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"). [0051] 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).

[0052] 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].

[0053] 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].

[0054] Figure 23 presents a flow-chart summarizing the process for the production of rodlike NLPs using the SRT1517 ssRNA scaffold of Fig. 22A.

[0055] 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.

[0056] 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.

[0057] 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. [0058] Figure 27 shows the effect of different vaccine formulations. BALB/c mice (10/group) were immunized twice i.m. and challenged with either 300pfu (3LDso) of WSN/33 or 600pfu (6LD50) and monitored for survival, symptoms, and weight loss during 14 days after infection: (A) survival after challenge with 300 pfu or 3LD50 of WSN33; (B) survival after challenge with 600 pfu or 6LD50 of WSN33; (C) symptoms after challenge with 600 pfu or 6LD50 of WSN33; (D) weight loss after challenge with 600 pfu or 6LD50 of WSN33. 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. [0059] Figure 28 shows a SDS-PAGE evaluation of the production and purification of NP derived from influenza B.

[0060] 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).

[0061] Figure 30 presents the RNA sequence encoding the optimized influenza B amino acid sequence of influenza B [SEQ ID NO:34] .

[0062] Figure 31 presents a flow-chart summarizing the process for the production of influenza B NP.

DETAILED DESCRIPTION OF THE INVENTION [0063] 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. According to preferred embodiments, the 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).

[0064] 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. Thus, the NLPs described herein have potential use in vaccines for protection against influenza virus infections. As the 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). [0065] In certain embodiments, the NLPs are also capable of improving the CTL response to NP when compared to the monomeric recombinant NP. [0066] As demonstrated herein, fusion of the NP at the C-terminus to a heterologous peptide does not interfere with the ability of the NP to form NLPs. In certain embodiments, therefore, 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.

[0067] While 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. As demonstrated, 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.

Definitions

[0068] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0069] As used herein, 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. [0070] 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."

[0071] As used herein, 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. A 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. [0072] The terms "subject" and "patient" as used herein refer to an animal in need of treatment.

[0073] The term "animal," as used herein, 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.

[0074] 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).

[0075] The terms "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.

[0076] The term "substantially identical" as used herein in relation to a 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"). "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 Plus™, 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. (1990) J Mol Biol 215: 403-10), and variations thereof including BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, and Megalign (DNASTAR) software. In addition, those skilled in the art can determine appropriate parameters for measuring alignment, including algorithms needed to achieve maximal alignment over the length of the sequences being compared.

[0077] The term "PapMV VLP" and "PAL" are used interchangeably herein to refer to a Virus-Like Particle derived from the Papaya Mosaic Virus. As known to those skilled in the art, and as described herein, the PapMV VLP can be further engineered to present one or more antigens fused to the PapMV coat protein. For example, and without limitation, one or more epitopes from an influenza protein other than NP.

[0078] It is contemplated that any embodiment disclosed herein can be implemented with respect to any method, use or composition of the invention, and vice versa. Furthermore, the disclosed compositions and kits can be used to achieve methods and uses of the invention.

NUCLEOCAPSID-LIKE PARTICLES (NLPs)

[0079] The NLPs disclosed herein comprise recombinant orthomyxoviral nucleoprotein self-assembled with an ssRNA scaffold. According to preferred embodiments, 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. 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.

[0080] In general, 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. [0081] 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.

Nucleoprotein [0082] 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. By "derived from" it is meant that the recombinant NP has an amino acid sequence substantially identical to the sequence of the wild-type NP. The 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. For example, if the NLPs are intended for use as a human vaccine against influenza, then the NP sequence selected will be from a strain of influenza that commonly infects humans. Likewise, if the intended use of the NLPs is as a vaccine for a non-human animal, then the NP sequence selected will be from a strain of orthomyxovirus, e.g., influenza, that commonly infects the target non-human animal.

[0083] In certain embodiments, 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.

[0084] As is known in the art, influenza type A viruses are divided into subtypes based on the sequences of the hemagglutinin (HA) and neuraminidase (NA) proteins comprised by the virus. 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. In certain embodiments, the NLPs are formed from a recombinant NP derived from influenza A HlNl, H1N2 or H3N2 NP. In some embodiments, the NLPs are formed from a recombinant NP derived from influenza A HlNl NP.

[0085] 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), and 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. [0086] In certain embodiments, 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. For example, in pigs, 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.

[0087] In certain embodiments, 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.

[0088] 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.

[0089] Thus in certain embodiments, 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." For example, 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. One skilled in the art will appreciate, however, that such 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.

[0090] Recombinant 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. For example, 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. In certain embodiments, 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. In certain embodiments, 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.

[0091] In certain embodiments of the present invention, when a recombinant NP comprises a variant sequence, the 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.

[0092] When the recombinant NP contains one or more amino acid substitutions, these can be "conservative" substitutions or "non-conservative" substitutions. In certain embodiments, 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.

[0093] In certain embodiments, 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.

Fusion Proteins

[0094] In certain embodiments, 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. When fused at the N- or C- terminus, the peptide may optionally replace one or more amino acids that form the N- or C- terminus of the wild-type protein. In some embodiments, 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.

[0095] Typically, 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. Accordingly, in some embodiments, 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. [0096] 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. Virol, 80: 1959-1964); the matrix protein (Ml) 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 23: 1215-1219); 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); and Ml 128-135 (Dong et al, 1996, Eur. J. Immunol, 26(2): 335-39).

[0097] Other antigenic regions and epitopes include fragments of the ion channel protein (M2) such as the M2e peptide (the extracellular domain of M2). The sequence of this peptide is highly conserved across different strains of influenza. 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.

Table 1: M2e Peptide Sequences

Region of Sequence Viral Strain SEQ ID M2 NO

1-24 MS LLTEVETPTRNGWGCRC SD S SD Avian H9N2 21

1-24 MSLLTEVETPTRNEWGCRCSDSSD Mutant H1N1 22

* see U.S. Patent Application Publication No. 2006/0246092

[0098] 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 that were also identified 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).

[0099] Other useful M2e fragments include the sequence EVETPIRNE [SEQ ID NO:9].

[00100] In certain embodiments, 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. [00101] While in general, 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. Immunol, 147:984- 991), NP 335-350 and NP 380-393 (Dyer and Middleton, 1993, In: Histocompatibility Testing, A Practical Approach (Ed. : Rickwood, D. and Hames, B. D.) IRL Press, Oxford, p. 292; Gulukota and DeLisi, 1996, Genetic Analysis: Biomolecular Engineering, 13:81), NP 305-313 (DiBrino, 1993, ibid.); NP 384-394 (Kvist, 1991, Nature 348:446-448); NP 89-101 (Cerundolo, 1991, Proc. R. Soc. Lon. 244: 169-7); NP 91-99 (Silver et al, 1993, Nature 360: 367-369); NP 380-388 (Suhrbier, 1993, J. Immunol. 79: 171-173); NP 44-52 and NP 265-273 (DiBrino, 1993, ibid.); and NP 365-380 (Townsend, 1986, Cell 44:959-968).

Preparation of NP and NP-Peptide Fusion Proteins

[00102] 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). For example, 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).

[00104] The nucleic acid sequence encoding NP is then inserted directly or after one or more subcloning steps into a suitable expression vector. One skilled in the art will appreciate that the precise vector used is not critical to the instant invention. Examples of 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.

[00105] Alternatively, 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.

[00106] One of ordinary skill in the art will appreciate that the DNA encoding the NP can be altered in various ways without affecting the activity of the encoded protein. For example, 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.

[00107] 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. Examples of 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. [00108] One skilled in the art will understand that 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. One skilled in the art will appreciate that selection of suitable regulatory elements is 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.

[00109] The expression vector may optionally contain heterologous nucleic acid sequences that facilitate the purification of the expressed protein. Examples of such heterologous nucleic acid sequences include, but are not limited to, affinity tags such as those described above.

[00110] 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. One skilled in the art will understand that selection of the appropriate host cell for expression of the NP or fusion protein will be dependent upon the vector being used. Examples of 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. salmonicida or B. subtilis) or in 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).

[00111] In certain embodiments, the NP is cloned into a vector that allows for expression in prokaryotic cells, such as E. coli.

[00112] 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.

Single-Stranded RNA (ssRNA) Scaffold [00113] 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. In certain embodiments, the ssRNA scaffold is a synthetic ssRNA. [00114] Typically, 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. In certain embodiments, 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.

[00115] The exact sequence of the ssRNA used as a scaffold does not appear to be critical. Thus 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. In certain embodiments, the ssRNA may be composed of a single base, such as poly-U, poly-C, poly-A or poly-G. In some embodiments, the ssRNA sequence includes a modified naturally occurring sequence, for example, a modified version of a sequence from another virus. In certain embodiments, 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.

[00116] In certain embodiments, the ssRNA is a poly-U or poly-C ssRNA. In some embodiments, 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.

[00117] 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.).

[00118] For example, for synthetic ssRNA, the sequence encoding the ssRNA template can be inserted into a suitable plasmid which can be used to transform an appropriate host cell. After culture of the transformed host cells under appropriate cell culture conditions, 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.

[00119] Shorter ssRNA scaffolds may also be synthesized chemically using standard techniques. A number of commercial RNA synthesis services are also available.

[00120] The final ssRNA scaffold may optionally be sterilized prior to use.

Assembly of NLPs

[00121] 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.

[00122] 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.

[00123] The nature of the buffer is not critical to the assembly process provided that it can maintain the pH in the ranges described above. Examples of buffers for use within the pH ranges described above include, but are not limited to, Tris buffer, phosphate buffer, citrate buffer and the like. [00124] 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.

[00125] The assembly reaction can be conducted using various proteimssRNA ratios. In general, 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. In certain embodiments, 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. In some embodiments, 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.

[00126] 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.

[00127] 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.

[00128] 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.

[00129] 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.

PHARMACEUTICAL COMPOSITIONS [00130] Certain embodiments of the invention relate to pharmaceutical 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. According to certain embodiments, 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. According to other embodiments, the pharmaceutical composition comprises an effective amount of two or more types of NLP. For example, 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.

[00131] The pharmaceutical compositions can be formulated for administration by a variety of routes. For example, the compositions can be formulated for oral, topical, rectal, nasal or parenteral administration or for administration by inhalation or spray. The term 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.

[00132] 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 fatty acids and hexitol anhydrides, for example, polyethylene sorbitan monooleate. 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.

[00133] In certain embodiments, 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.

[00134] In certain embodiments, 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. [00135] 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.

[00136] In certain embodiments, 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. Other examples include, sterile, fixed oils, which are conventionally employed as a solvent or suspending medium, and 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.

[00137] Optionally, 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). The pH and exact concentration of the various components of the composition may be adjusted according to well-known parameters.

[00138] 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. Generally, 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. In the case of sterile powders for the preparation of sterile compositions, 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. [00139] 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.

[00140] All such devices require the use of formulations suitable for the dispensing of the NLPs. Typically, 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. [00141] The pharmaceutical compositions may optionally further comprise an adjuvant. Various adjuvants are known in the art and 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.

[00142] In certain embodiments, the pharmaceutical compositions comprise PapMV VLPs as an adjuvant. According to some embodiments, 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. In certain embodiments, 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. Virol, 80: 1959-1964); the matrix protein (Ml) 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. 23: 1215-1219); 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); and 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 sequence of this peptide is highly conserved across different strains of influenza. [00143] In certain embodiments, 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. According to other embodiments, the PapMV VLPs comprise one or more influenza antigens derived from the M2e peptide or a fragment thereof.

[00144] Other pharmaceutical 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).

USES

[00145] 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.

[00146] In certain embodiments, the NLPs may be used to induce an immune response to more than one influenza virus strain. As 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. In particular, it is contemplated that NLPs comprising a NP sequence derived from one type of influenza virus strain will be able to provide protection against heterologous strains of influenza. More particularly, it is contemplated that 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. [00147] In certain embodiments, the NLPs may be used to induce antibodies against influenza virus NP in a subject. In some embodiments, the NLPs may be used to induce a cellular immune response, such as a CTL response, against influenza virus NP in a subject. In certain embodiments, the NLPs may be used to induce both antibodies and a CTL response against influenza virus NP in a subject. 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 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.

[00148] 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.

[00149] 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.

[00150] 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.

[00151] In certain embodiments, the NLPs are administered to the subject in combination with an adjuvant. Examples of adjuvants are described above.

[00152] Certain embodiments contemplate the use of the NLPs in combination with one or more other influenza antigens. For example, the NLPs may be combined with an influenza M2 protein or Ml protein or a fragment of one of these proteins. In some embodiments, it is contemplated that the NLPs may be combined with a seasonal influenza vaccine to provide broader cross-protection against heterologous influenza types and/or subtypes. When the NLPs are administered in combination with another antigen or vaccine, 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.

PHARMACEUTICAL KITS

[00153] Certain embodiments of the invention relate to pharmaceutical packs and 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. [00154] When one or more components of the kit are provided as solutions, for example an aqueous solution, or a sterile aqueous solution, 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.

[00155] 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. Irrespective of the number or type of containers, 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. [00156] To gain a better understanding of the invention described herein, the following examples are set forth. It will be understood that these examples are intended to describe illustrative embodiments of the invention and are not intended to limit the scope of the invention in any way. EXAMPLES

[00157] The following materials and methods were employed for Examples 1 to 6:

Expression and Purification of Recombinant Nucleoprotein (NP) froni E. coli

[00158] 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.

[00159] 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:

[00160] Primers: 5'- ATC-GGA-ATT-CAG-GAG-GTA-AAA-ACC-ATG-G-3' [SEQ ID NO: 1], and 5 ' -ATC-GGG-TAC-C AG- ATC-TTT- AGT-GGT-GAT-G-3 ' [SEQ ID NO:2].

[00161] 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.

[00162] 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 μg/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 NaH₂P0₄, 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. 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 μηι 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). Briefly, 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 μΜ membrane and the purity of the proteins was evaluated by SDS/PAGE.

[00163] To verify if the recombinant nucleoprotein (rNP) was in a monomeric state, purified protein was loaded on a Superdex 200 10/300 GL gel -filtration column (GE Healthcare, Baie d'Urfe, Canada) and a set of control proteins were used to calculate the apparent molecular weight of the rNP. 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).

Production and Purification of Nucleocapsid-Like Particles (NLPs)

[00164] Purified recombinant monomeric nucleoprotein (rNP) was mixed at a 10: 1 ratio with Polycytidylic acid potassium salt (Poly-C) (Sigma-Aldrich, Oakville, Canada) or with Polyuridylic acid single-stranded RNA polymer (Poly-U) (800-1000kDa) (Cedarlane, Burlington, Canada) in 10 mM Tris HC1 buffer at 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 μΜ membrane. To validate that the multimerization of the monomeric NP was successful, 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

[00165] 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

[00166] For the first immunization protocol, 6-8-week-old BALB/c mice (5/group) (Charles River, Wilmington, MA) were immunized intramuscularly with 10 or 50 μg 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. [00167] For the second immunization protocol, 6-8-week-old BALB/c mice (10/group) were immunized intramuscularly with 0.5 μg of either rNP or NLP-Poly-U with or without 5, 10, 20 or 40 μg 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. A boost immunization using the same dose was made in each of the 4 groups (rNP, NLP-Poly-U, rNP + 40 μg PAL, NLP-Poly-U + 40 μg PAL) and an ELISPOT assay was performed one week later.

[00168] For the third immunization protocol, 6-8 week-old BALB/c mice (5/group) were immunized once or twice intramuscularly at 14 days interval with 10 μg of NLP alone or combined with 40 μg of PAL. Blood was collected on day 13 (one day before the boost- immunization) and on day 21 (7 days after the boost). An ELISPOT assay was performed one week after the last immunization.

[00169] For the last immunization protocol (challenge), 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

Antibody Titration by ELISA

[00170] rNP at 2 μg/mL was diluted in 0.1M NaHCOs buffer (pH 9.6) and 100 μΐ, of the diluted NP was coated on a 96-well plates overnight at 4°C. Plates were blocked with 150 ΐνννβΐΐ 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. Plates were washed 4 times in PBS/0.1% Tween-20 and then 100 μίΛνβΙΙ of peroxidase-conjugated goat anti- mouse IgG or IgG2a (both from Jackson Immunoresearch, Baltimore, PA), diluted 1/10,000 in PBS/0.1% Tween-20/2% BSA, was added and the plates were incubated lh at 37°C. After another 4 washes in PBS/0.1% Tween-20, presence of antibodies was detected by adding 100 μίΛνβΙΙ of TMB-S (Ultra-TMB-S, Research Diagnostics, Flanders, NJ) according the manufacturer's instructions. After 8 minutes, the reaction was stopped by adding 100 μΐ. of 0.18M H2SO4. The OD of each plate was read at 450nm and the results were expressed as an antibody endpoint titre, determined when the OD value is 3 -fold greater than the background value obtained with the same serum dilution from a pre-immune mouse. For the last ELISA assay (see Fig. 9), NP-GST was used at 3μg/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.

ELISPOT

[00171] On the day before splenocytes isolation, MultiScreen-IP opaque 96-well plates (Fischer Scientific, Ontario, Canada) were treated with ethanol 35% then coated with 100 μΐ ννβΐΐ of capture IFN-γ diluted in sterile PBS as suggested in the murine interferon-gamma ELISPOT kit instructions (Abeam, Cambridge, MA, USA). 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 μΜ β-mercaptoethanol, lOmM HEPES, 100 U/mL penicillin and 100 μg/mL streptomycin. Spleens were cut in culture medium and then mashed through a 100 μΜ 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. While the splenocytes were centrifuged, resuspended in culture media and counted, the plates that were previously coated with capture IFN-γ the day before were washed three times with 100 μΐ. of PBS IX and blocked with 100 μίΛ εΙΙ of PBS-2% skimmed dry milk for 2 h at 37°C, 5% C0₂. After a wash with PBS, duplicates of 2.5 x 10⁵ cells/well were reactivated with 50 μg/mL of NLP, 10 μΜ of the H- 2K^D Influenza NP peptide TYQRTRALV [SEQ ID NO:3], 2 μg/mL of Concavanine A or culture media for 18 h at 37°C, 5% CO2. At the end of the incubation period, the plates were washed 3 times with 100 μίΛ εΙΙ of PBS/0.1 % Tween-20. 100 μΕ/well of biotinylated detection anti-mouse IFN-γ 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 μΐ ννβΐΐ 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% C0₂. After 3 washes with PBS/0.1% Tween-20, 100 μΐ, of ready-to-use BCIP/NBT substrate buffer was added to each wells and after an incubation of 2 to 15 min, distilled water was added to stop the enzymatic reaction. Plates were observed with a Nikon SMZ800 stereomicroscope and photographed with a Nikon Digital Sight DS-L2. Spots were counted using the ImageJ Software (freeware from author Wayne Rasban) to determine the number of spots per well. Threshold was adjusted before the analysis of the particles (spots) and the number of IFN-γ secreting cells was determined by subtracting the background spots in media only from the spots in wells reactivated with either NLP or the peptide. After initial results using this ELISPOT protocol, the day of splenocytes isolation and re-stimulation was performed one week post-immunization instead of two weeks.

Challenge with Influenza Virus

[00172] 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 μΐ 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.

EXAMPLE 1: Preparation of Multimerized NP

Production and Purification of the Monomeric NP Recombinant Protein

[00173] 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. Then, 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.

[00174] Next, it was confirmed that the recombinant NP was in a monomeric form and not associated with bacterial nucleic acids. A Superdex 200 size-exclusion chromatography column on the FPLC system was used to detect any possible contamination with nucleic acids, as this column is suitable for both protein and nucleic acid analysis. The FPLC profile (Fig. ID) showed that the rNP, which is eluted between 15 mL and 18 mL, is highly purified and not contaminated with nucleic acids. The Superdex 200 column was also used to measure the apparent molecular weight of the rNP. Using a set of reference proteins to calibrate the Superdex 200 (Fig. ID, shown as arrows), a protein eluting between 15 mL and 18 mL was estimated to have an apparent molecular weight between 29 and 76 kDa. It was concluded, therefore, that the purified rNP was in a monomeric form as the influenza nucleoprotein is known to be 56 kDa (Baudin F, et al, 1994, EMBO J 13:3158-65).

Multimerization of the Monomeric NP Recombinant Protein using Different RNA Templates

[00175] 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. 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. When compared to the monomeric rNP (Fig. ID), 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. To further the biochemical characterization of the NLPs, dynamic light scattering (DLS) was used to measure the average length and stability of the NLPs under the influence of temperature. Both NLPs were found to have approximately the same average length: 22.1 nm for NLP-Poly-C and 23.9 for NLP-Poly-U (Fig. 2B, left panel). 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.

[00176] A flow-chart summarizing the preparation of the NP NLPs using the His-tagged NP is provided in Fig. 14. An SDS-PAGE gel showing the purification of the His-tagged NP ("NP-cH") protein from E. coli is shown in Fig. 15. EXAMPLE 2: Immune Response Against Multimerized NP

[00177] 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) and IgG2a titers (Fig. 3B) showed that NLP immunization led to a humoral response against NP, but there was no significant difference in the humoral response between the two different NLPs at days 14 and 28. [00178] The stability of the NLPs was also evaluated using the Superdex 200 column and showed that the elution profile of the NLPs had changed over the time course of the above experiment from the original elution profile (Fig. 4 cf. Fig. 2A). This change was especially noteworthy for NLP-Poly-C for which the appearance of a new eluted peak between 19 and 23 mL was observed. The appearance of aggregates in the NLP solution was also observed. Based on the fact that there was no difference in the humoral response between the two NLPs and that NLP-Poly-U was more stable than NLP-Poly-C, subsequent experiments as detailed in Examples 3 to 6 were conducted using NLP-Poly-U. EXAMPLE 3: Importance of Multimerization on Nucleoprotein Immunogenicity

[00179] For this Example and Examples 4 to 6, the following change was made to the protocol used to produce the monomeric rNP and the NLP-Poly-U in order to improve the stability in solution. Before the Tangential Flow Filtration dialysis, the eluted recombinant nucleoprotein was passed through a Sartobind capsule to remove the endotoxin contaminants. The LPS concentration was reduced by this procedure from 50 EU/injection to below the detection limit of the Limulus test. A full biochemical characterization was made to see if the modifications to the production protocol affected the NLP. The monomeric rNP still eluted at the same volume on the Superdex 200 and the elution profile of the NLP-Poly-U was consistent with previous results shown in Fig. 2A. DLS analysis showed that 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).

[00180] After selection of the NLP-Poly-U as a candidate influenza vaccine, an immunization program to validate that the multimerization of the monomeric rNP into a NLP led to increase in the NP immunogenicity was set up. BALB/c mice (10/group) were immunized once intramuscularly with formulation buffer or 0.5 μg of either rNP or NLP- Poly-U. At day 14 post-immunization, the humoral response to NP was measured by ELISA. As seen in Fig. 6A and 6B, the multimerization of the monomeric rNP into a NLP led to a significant (pO.001) increase in both total IgG (32-fold increase) (Fig. 6A, left panel) and IgG2a (128-fold increase) (Fig. 6A, right panel) titers.

[00181] It has been established that 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). To evaluate if the multimerization of the rNP into a NLP led to a better cellular immune response, an ELISPOT assay was performed on the mice immunized with either rNP or NLP-Poly-U. Briefly, the IFN-γ 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-γ. 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. This ELISPOT assay (Fig. 6C, left panel) showed that the boost immunization with the NLP-Poly-U led to a significant increase (p<0.01) in NP-specific CD8+ T-cells when compared with the group immunized with the monomeric rNP. The same increase was not observed for the splenocytes stimulated with NLP-Poly-U (Fig. 6C, right panel). The results of the ELISPOT and ELISA assays confirmed that the multimerization of the monomeric rNP into a NLP significantly increased both the humoral and cellular response against the influenza nucleoprotein. EXAMPLE 4: Combination of Monomeric or Multimerized NP with an Adjuvant

[00182] Immunization with recombinant purified antigens frequently results in the induction of a modest antibody response with little or no T cell response. To bypass the weak immunogenicity of the antigen, either a high dose of antigen or an adjuvant may be employed as a means to enhance the efficacy and trigger the appropriate immune response (Reed SG, et al , 2013, Nat Med, 19: 1597-608). In this experiment, Papaya mosaic virus (PapMV) viruslike particles (VLPs) adjuvant (International Patent Application Publication No. 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. [00183] BALB/c mice (10/group) were immunized once intramuscularly with 0.5 μg of either rNP or NLP-Poly-U adjuvanted with 5, 10, 20 or 40 μg of PapMV VLPs ("PAL") and the humoral response to NP was measured by ELISA 14 days post-immunization. Total IgG and IgG2a titers (Fig. 7 A) showed that the multimerization of NP was still beneficial on the NP-specific immunity even with the addition of the PAL adjuvant. The addition of PAL also improved the humoral response against NP with 40 μg of PAL leading to the most substantial increase in mean titers of total IgG (12.4x) and of IgG2a (12.8x) when compared to NLP alone (Fig. 7 A, Total IgG: 8.8x, IgG2a: 8.3x).

[00184] The effect of the addition of PAL on the cellular response against NP was also evaluated to determine whether the multimerization of the NP was still beneficial for the cellular response when compared to adjuvanted rNP. The ELISPOT assay described in the earlier Examples was used. The results showed no difference in IFN-γ secretion between the adjuv anted monomeric rNP and the adjuv anted NLP groups after a single immunization (Fig. 7B). An increase in the number of T cells secreting IFN-γ was, however, observed in the adjuv anted NLP group after a boost immunization (Fig. 7C) suggesting that multimerization of NP even with an adjuvant can increase the cellular response against the influenza nucleoprotein.

EXAMPLE 5: Boost-Immunization Increases Both Humoral and Cellular Responses to NP

[00185] As shown in Fig. 6B and 6C, immunization with 0.5 μg of NP induced only a weak cellular response after one immunization and a boost was needed to see a significant difference in the T-cell response between rNP and the NLP. As such, the use of a higher quantity of antigen was tested to determine whether higher doses could improve the cellular response after a single immunization. The ability of a prime-boost immunization with this higher dose to increase both the humoral and cellular response was also tested. BALB/c mice (5/group) were immunized once or twice at a 14-day interval with 10 μg of NLP alone or combined with 40 μg of PAL adjuvant. Blood was collected on day 13 (one day before the boost-immunization) and at day 21 (7 days after the boost) and the humoral response to NP was assessed by ELISA. The boost immunization (Fig. 8A) led to a 15-fold increase in total IgG titers and a 21 -fold increase in IgG2a titers for the NLP group, while mice immunized with NLP + PAL had a 9-fold and 10-fold increase in their total IgG and IgG2a titers (p <0.001), respectively. In brief, two immunizations with PAL-NLP was the best condition tested to stimulate an anti-NP humoral response.

[00186] 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-γ secretion of the T cells. The boost immunization significantly increased (p<0.001) the number of T cells secreting IFN-γ (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

[00187] 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. At day 14 post-immunization, blood was collected to measure the IgG2a titers to NP. During ELISA assessment, 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.

[00188] Then, 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. 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. 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. However, after an infectious challenge with 2 LD50, mice immunized with 20 μg NLP + 80 μg 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 μg of NLP alone and the mice immunized with 20 μg of NLP combined with 80 μg of PAL also showed the importance of the adjuvant in the protection against an influenza challenge. [00189] Blood was collected from mice surviving the ILD50 infectious challenge and the IgG2a response against NP was measured. 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 μg NLP + 40 μg 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-γ 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.

Discussion

[00190] 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.

[00191] The results described in Examples 1 to 6 show that the multimerization of the monomeric rNP into NLPs led to an increase in both the humoral response and the cellular response to NP. One major difference between monomeric NP and the NLP is the presence of the synthetic ssRNA used as a scaffold for the multimerization of the NP. It has been shown that the presence of single-stranded RNA inside a VLP can activate TLR7 present in the B- cells and that the activation of this receptor enhanced B-cell responses and class switching (IgG2a) when compared to VLPs lacking a TLR7 agonist (Jegerlehner A., et al , 2007, J Immunol, 178:2415-20). Therefore induction of innate immunity through TLR7 could be the basis for the increase in IgG2a when mice were immunized with the NLP (Fig. 6A, right panel). It is known that priming of naive T cells can only be acheived by professional antigen-presenting cells (APCs) such as dendritic cells (DCs), but these APCs need to be activated to gain their full T-cell stimulatory activity (Diebold SS., 2009, Handb Exp Pharmacol, 3-30). The most common way to activate APCs is through the recognition of pathogen-associated molecular patterns (PAMPs) by the pathogen-recognition receptors (PRRs) such as Toll-like receptors (TLRs) (Atmar RL, and Keitel WA, 2009, Curr Top Microbiol Immunol, 333:323-44). For the multimerized NP, the single-stranded Poly-U of the NLP could potentially play this role by binding TLR7, which would lead to the activation of the APCs and subsequent expression of co-stimulatory molecules and cytokines necessary to generate a strong and effective T-cell response (Jennings G, and Bachmann M, 2008, Biol Chem, 389:521-36). 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.

[00192] It is possible that the size difference between the monomeric NP and the NLP plays a role in the increased immune response against NP since it is known that VLPs are more efficiently taken up by dendritic cells (DCs) due to their particulate nature and dimensions (Jennings G, ibid. ; Plummer EM, and Manchester M., 2011, Wiley Inter discip Rev Nanomed Nanobiotechnol, 3: 174-96).

[00193] 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. In the above Examples, PapMV VLPs (PAL) were used as an adjuvant. A recent report has shown that PAL is a TLR7 ligand, a receptor that activates innate immunity (Lebel M-E, et al., 2014, J Immunol, 192: 1071-8). When used in combination with the NLP, PAL enhanced both the IgG2a and total IgG response against the nucleoprotein after one intramuscular immunization. Previous results have shown that subcutaneous administration of PAL improved and broadened the immune response towards the trivalent inactivated influenza vaccine (TIV) in mouse and ferret animal models (Savard C, et al. , 2011, PLoS One, 6:e21522) while intranasal administration of TIV combined with PAL increased the IgG, IgG2a and IgA response in lungs of vaccinated mice when compared to TIV alone (Rioux G, et al , 2014, J Nanobiotechnology, 12: 19). While PAL enhanced the humoral response to NP, the addition of PAL did not increase the cellular response against NP, even after boost immunization. This result was surprising as previous reports have shown that PAL is able to trigger a cellular mediated response against the conserved influenza epitopes present in the TIV (Savard C, et al., ibid.).

[00194] Examples 1 to 6 show that one immunization with NLP + PAL induced NP-specific IFN-γ 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. have showed that mice immunized 3 times with 90 μg of rNP had a strong humoral response against NP and survived a 10 LD50 heterotypic infectious challenge despite the fact that they had low levels of CTL (Huang B, et al, 2012, Virol J 9:322). Given that the above Examples demonstrate that the multimerized NP improved the CTL response (Fig. 8B) after two immunizations with 10 μg of NLP, similar positive results with respect to a challenge are expected after a prime-boost immunization protocol. In addition, 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).

[00195] 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).

EXAMPLE 7: Multimerization of NP Using Other ssRNA Scaffolds

[00196] The ability of the monomeric NP to multimerize into NLPs using ssRNA scaffolds other than poly-U and poly-C was tested. Monomeric NP prepared as generally described in Example 1 with the update described in Example 3 was combined with two different ssRNAs, SRT500 (Fig. 16A) [SEQ ID NO:7] or SRT1517 (Fig. 16B) [SEQ ID NO:8], using the procedure described in Example 1 and the same protein: ssRNA ratio.

[00197] Both ssRNA scaffolds resulted in the production of NLPs (see Fig. 17B and 18B), suggesting that the nature of the ssRNA is not critical to the multimerization of NP into NLPs. Dynamic light scattering analysis indicated that the NLPs formed with SRT1517 had an average particle length of approximately 50 nm (Fig. 17A), and the NLPs formed with SRT500 had an average particle length of approximately 30 nm (Fig. 18A). Both types of NLPs had an average width of between about 13 nm and about 18 nm. EXAMPLE 8: Preparation of NP Fused to an M2e Peptide and Multimerization into NLPs

[00198] An NP fusion protein that contained a fusion of the M2e peptide EVETPIRNE [SEQ ID NO: 9] at the C -terminus was constructed as follows. [00199] In brief, 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. [00200] The amino acid sequence of the fusion protein is shown in Fig. 13C [SEQ ID NO:6].

[00201] Monomeric fusion protein ("NPM2ec protein") was purified from E. coli as described in Example 1 with the update described in Example 3 and multimerized into NLPs using SRT500 or SRT1517 ssRNA and the protocol described in Example 3. A flow chart showing an overview of the steps for the production of NPM2ec protein and NLPs is provided in Fig. 19. An SDS-PAGE gel showing the analysis of in process samples taken from the assembly reaction during the production of NLP-rNPM2e is provided in Fig. 20A.

[00202] As shown in Fig. 20C, 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.

[00203] As NLPs formed from the NPM2ec protein 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. In addition, it is expected that an 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. It is also anticipated that the protection obtained with the 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. Finally, an improvement in the M2e response is expected when NPM2ec or NPM2ec NLPs are combined with the PAL adjuvant.

EXAMPLE 9: Multimerization of Orthomyxoviral NP

[00204] The ability of other genera of orthomyxovirus NP to multimerize into NLPs was tested using an abbreviated NLP assembly process comprising a plurality of orthomyxoviridae nucleoprotein and bacterial RNA where the self-assembly of the NP occurs after lysis of the bacteria expressing the recombinant NP. The purified NP assembles into an NLP due to the high affinity of the NP protein for RNA liberated by the lysis of the bacteria.

[00205] A flow chart showing an overview of the steps for the production of influenza B NP is provided in Fig. 31. In brief, 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.

[00206] 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. Then, 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 8 kDa MWCO membrane to remove imidazole and filtered to sterilize the protein. [00207] 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).

[00208] As the 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 [00209] While the multimerization of NP in the presence of ssRNA has been previously reported (Ye et al, 2006, ibid. ; Chenavas et al, 2013, ibid. ; Tarus et al, 2012, ibid.), 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. [00210] 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.

[00211] The following materials and methods were employed: Preparation of NLPs and Closed Ring Structures

[00212] Monomeric NP 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. In brief, NP protein was cloned into a bacterial expression vector (pQE-80) and the protein was expressed through induction of the T7 promoter using IPTG. After overexpression of the protein in 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. 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 of the NLPs or the closed ring structures.

[00213] 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).

[00214] Closed ring structures were prepared using the same method with the exception that a ssDNA of 76 nucleotides was used to trigger assembly. The sequence of this ssDNA is 5'- ATC CCT AGG TTA ATG ATG ATG ATG ATG ATG ACG CGT AGC CGG CGC GCC ACC AAC TCT ATA GTG CTT AAT GTC AGA C -3' (Fig. 22) [SEQ ID NO:33].

Dynamic light scattering

[00215] The size of nanoparticles and ring structures were recorded with a ZetaSizer Nano ZS (Malvern, Worcestershire, United Kingdom) at a temperature of 10°C and at a concentration of 0.1 mg/ml diluted in lOmM Tris pH8 or PBS buffer.

Electron microscopy

[00216] 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.

Immunization of Mice

[00217] Balb/C mice (5 per group) were immunized once at day 0 by the intramuscular route with 10μg 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.

Results

Assembly of NP Nanoparticles (NLPs) and NP Closed Ring Structures

[00218] 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.

[00219] As shown in 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).

[00220] Transmission electron microscopy showed assembly of closed ring structures with the small ssDNA of 76nt [SEQ ID NO: 33] (Fig. 25A). The closed ring structures appeared as small circles (Fig. 25 A) and DLS showed an average size of 18nm (Fig. 25B). The elution profile showed an homogenous peak by gel filtration (Superdex 200) at 10.32 mL of column volume (Fig. 25C). Comparison of Immunogenicity

[00221] 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 10μg 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. [00222] Antibody levels against NP in the blood were measured by ELISA at day 14. Total IgG titers (Fig. 26A) and IgG2a titers (Fig. 26B) showed that the rod-like NLP form is significantly more immunogenic than both the closed ring and the monomelic forms. EXAMPLE 11: Combination of Multimerized NP with Antigen-Presenting Adjuvant

[00223] The effect of combining the rod-like NLPs with adjuvant was further examined. 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.

Challenge with Influenza Virus

[00224] Balb/C mice were immunized twice at day 0 and 21 by the intramuscular route with either buffer control, 10μg of the rod-like NLP alone (NP), 10μg 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 90μg of engineered PapMV VLPs presenting at their surface the influenza M2e peptide (Carignan et al, 2015)(NP+PapMV-M2e 30, 60 or 90).

[00225] The 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.

Results

[00226] The capacity of animals immunized twice at 21 day intervals with 10μg of rod-like NP nanoparticles (NP), ^g of rod-like NP nanoparticles+30, 60, 90μg of PapMV nanoparticles (NP+PapMV 30,60 or 90), buffer, or PapMV nanoparticles 90μg, was evaluated. The animals were challenged at day 42 with 3LD50 of influenza WSN/33 and scored for survival during the following 14 days (Fig. 27 A).

[00227] 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 10μg 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).

[00228] The results from the challenge revealed that rod-like NP nanoparticles alone can provide a partial protection to a challenge with 3LD50 of the influenza virus WSN/33 in a mouse model (Fig. 27 A). However, it is also shown that the addition of either 30, 60 or 90 μg of PapMV nanoparticle (adjuvant) improves the protection significantly. The group adjuv anted with the highest amount of PapMV nanoparticles (90μg) was the only group showing 100% survival.

[00229] When 600pfu or 6LD50 of virus was used for the challenge, the best performing group was the group immunized with rod-like NP (10μg) + PapMV-M2e nanoparticles (90μg). 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).

[00230] The disclosures of all patents, patent applications, publications and database entries referenced in this specification are hereby specifically incorporated by reference in their entirety to the same extent as if each such individual patent, patent application, publication and database entry were specifically and individually indicated to be incorporated by reference.

[00231] Although the invention has been described with reference to certain specific embodiments, various modifications thereof will be apparent to those skilled in the art without departing from the spirit and scope of the invention. All such modifications as would be apparent to one skilled in the art are intended to be included within the scope of the following claims.

Claims

WE CLAIM:

1. A nucleocapsid-like particle (NLP) having a rod-like shape and comprising a plurality of recombinant orthomyxoviral nucleoprotein (NP) polypeptides assembled on a single- stranded RNA (ssRNA) scaffold, the ssRNA being between about 120 and about 5000 nucleotides in length.

2. The NLP according to claim 1, wherein the ssRNA is a poly-U ssRNA or a poly-C ssRNA.

3. The NLP according to claim 1, wherein the ssRNA has a sequence corresponding to the sequence as set forth in SEQ ID NO:7 or 8, or a fragment thereof.

4. The NLP according to any one of claims 1 to 3, wherein the recombinant NP polypeptides have a sequence derived from an influenza virus.

5. The NLP according to claim 4, wherein the recombinant NP polypeptides have a sequence derived from an influenza virus type B NP.

6. The NLP according to claim 4, wherein the recombinant NP polypeptides have a sequence derived from an influenza virus type A NP.

7. The NLP according to claim 6, wherein the recombinant NP polypeptides have a sequence derived from an NP of influenza virus A subtype H1N1.

8. The NLP according to claim 7, wherein the recombinant NP polypeptides comprise a sequence at least 90% identical to the sequence set forth in SEQ ID NO:4.

9. The NLP according to claim 7, wherein the recombinant NP polypeptides comprise a sequence as set forth in SEQ ID NO:4.

10. The NLP according to any one of claims 1 to 9, wherein the recombinant NP polypeptides are fused to one or more other influenza antigens.

11. The NLP according to claim 10, wherein the one or more other influenza antigens are fused at the C-terminus of each recombinant NP polypeptide.

12. The NLP according to claim 10 or 11, wherein the one or more other influenza antigens are derived from the influenza M2 protein.

13. The NLP according to claim 12, wherein the one or more other influenza antigens comprise the M2e peptide or a fragment thereof.

14. The NLP according to claim 12 or 13, wherein the one or more other influenza antigens comprise a sequence as set forth in SEQ ID NO:9.

15. The NLP according to claim 12 or 13, wherein the one or more other influenza antigens consist of a sequence as set forth in SEQ ID NO:9.

16. A pharmaceutical composition comprising one or more types of NLP according to any one of claims 1 to 15 and a pharmaceutically acceptable carrier or diluent.

17. The pharmaceutical composition according to claim 16 further comprising an adjuvant.

18. The pharmaceutical composition according to claim 17, wherein the adjuvant comprises Papaya mosaic virus (PapMV) virus-like particles (VLPs).

19. The pharmaceutical composition according to claim 18, wherein the PapMV VLPs comprise one or more influenza antigens fused to the PapMV coat protein.

20. The pharmaceutical composition according to claim 19, wherein the one or more influenza antigens are derived from the influenza M2 protein.

21. The pharmaceutical composition according to claim 20, wherein the one or more influenza antigens comprise the M2e peptide or a fragment thereof.

22. A vaccine comprising the pharmaceutical composition according to any one of claims 16 to 21.

23. The vaccine according to claim 22, further comprising one or more additional antigenic influenza proteins or protein fragments.

24. An in vitro process for preparing a nucleocapsid-like particle (NLP) comprising a plurality of recombinant orthomyxoviral nucleoprotein (NP) polypeptides and single-stranded RNA (ssRNA) comprising: a) combining recombinant NP polypeptide and ssRNA at a protein: RNA ratio of between about 1 : 1 and 50: 1 by weight, and a temperature between about 2°C and about 37°C, for a time sufficient to allow assembly of NLPs, the ssRNA being between about 120 and about 5000 nucleotides in length, and b) separating the NLPs from other process components.

25. The process according to claim 24, wherein the recombinant NP polypeptides have a sequence derived from an influenza virus.

26. A method of inducing an immune response against an orthomyxovirus in a subject comprising administering to the subject an effective amount of the NLP according to any one of claims 1 to 15, the pharmaceutical composition according to any one of claims 16 to 21, or the vaccine according to claim 22 or 23.

27. The method according to claim 26, wherein the orthomyxovirus is an influenza virus.

28. The method according to claim 26 or 27, wherein the immune response comprises the production of antibodies and/or a CTL response.

29. A method of vaccinating a subject against an orthomyxoviral infection comprising administering to the subject an effective amount of the NLP according to any one of claims 1 to 15, the pharmaceutical composition according to any one of claims 16 to 21, or the vaccine according to claim 22 or 23.

30. The method according to claim 29, wherein the orthomyxoviral infection is an influenza virus infection.

31. The method according to any one of claims 26 to 29, wherein the method comprises a first administration of an initial dose of the NLP and a second administration of a booster dose of the NLP.

32. The method according to any one of claims 26 to 31, wherein administration of the NLP provides protection against a plurality of influenza virus A strains.

33. 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].

34. The fusion protein according to claim 33, comprising a sequence as set forth in SEQ ID NO:6.

35. A nucleocapsid-like particle (NLP) having a rod-like shape and comprising a plurality of the fusion proteins according to claim 33 or 34 assembled on a single-stranded RNA (ssRNA) scaffold, the ssRNA being between about 120 and about 5000 nucleotides in length.

36. A method of inducing an immune response against influenza virus in a subject comprising administering to the subject an effective amount of the NLP according to claim 35.

37. A method of vaccinating a subject against influenza virus infection comprising administering to the subject an effective amount of the NLP according to claim 35.