WO2012048430A1 - Affinity-conjugated nucleoprotein-papaya mosaic virus-like particles and uses thereof - Google Patents

Abstract

An affinity-conjugated nucleoprotein-PapMV virus-like particle (ANP) system is provided. The ANP system comprises a virus-like particle (VLP) derived from the coat protein of PapMV which has been modified by the addition of one or more "affinity peptides." The affinity peptides are short peptide sequences capable of specifically binding to influenza nucleoprotein (NP). The ANP system further comprises influenza NP conjugated via the one or more affinity peptides to the VLP. By "derived from" it is meant that the VLP comprises coat proteins that have an amino acid sequence substantially identical to the sequence of the wild-type coat protein. The one or more affinity peptides are attached, for example by chemical or genetic means, to the coat protein of the PapMV to form a Pap MV High Affinity VLP (Pap MV HAV). The ANP system is suitable for use as a vaccine.

Description

AFFINITY-CONJUGATED NUCLEOPROTEIN-PAPAYA MOSAIC VIRUS-LIKE PARTICLES AND USES THEREOF

FIELD OF THE INVENTION

[001] The present invention relates to the field of vaccine formulations and adjuvants and, in particular to influenza vaccines based on plant virus particles that elicit an immune response to the influenza nucleoprotein.

BACKGROUND OF THE INVENTION

[002] Influenza remains a major cause of morbidity and mortality. Annual epidemics are thought to result in between three to five million cases of severe illness and between 250,000 and 500,000 deaths every year around the world (see "Fact Sheet on Influenza" provided on website maintained by the World Health Organization, at www.who.int) . Despite significant success in controlling the emergence of this disease via vaccination, well-known deficiencies in current existing vaccines has long made their improvement a crucial research and public health priority. [ Ilyinskii et al. Int Rev Immunol 2008;27(6) :392-426] . Inactivated influenza vaccines have been available for more than 50 years and since 2003 a live attenuated influenza vaccine has also been available in the USA [Nichol et al. Vaccine 2008 Sep 12;26 Suppl 4 :D 17-22] . The principal disadvantage of existing influenza vaccines is their failure to provide protection to the strains other than those used to make the vaccine. In fact, persistent (drift) and dramatic (shift) antigenic changes on the major surface proteins necessitate annual repeated immunizations against seasonal viral stains. The efficacy and effectiveness of traditional vaccines in a given year will depend on many factors, but mainly on the degree of vaccine circulating match. This can be explained by the fact that neutralizing antibody titers against highly variable external glycoproteins of virus, namely hemagglutinin (HA) and neuraminidase (NA) are considered to be the gold standard correlate of vaccine-induced protection [ Palladino et al. J Virol 1995 Apr;69(4) :2075-81 , Rimmelzwaan et al. Vaccine 2008 Sep 12;26 Suppl 4:D41-4] . Because of the accumulation of mutations in HA and NA genes, influenza vaccine must be reformulated each year to include the HA and NA proteins predicted to dominate in the following influenza season. Also, since they only protect against viral serotypes that express the same HA and NA proteins contained in the vaccine, these vaccines are less effective against the appearance of new HA and NA proteins in naive populations causing the potential risk of a pandemic disease with high mortality like the striking 1918 "Spanish Flu".

[003] By contrast, vaccinations with a more conserved protein, like the nucleoprotein (NP) stimulate immunity against multiple serotypes [Schulman et al. J Bacteriol 1965 Jan;89: 170- 4; Liang et al. J Immunol 1994 Feb 15; 152(4) : 1653-61] . Such immunity has long been studied in animals and there is growing evidence that it may exist in humans. [Epstein et al. Expert Rev Anti Infect Ther 2003 Dec; l (4) :627-38] . This form of immunity does not generally prevent all infection by heterosubtypic virus but it leads to more rapid viral clearance and to reduction in morbidity and mortality [ Epstein et al. Expert Rev Anti Infect Ther 2003 Dec; l (4) :627-38] . The antigenic changes of NP are rare and only occur to a minor extent. The protein NP exhibits more than 90% protein sequence identity among influenza A isolates [ Altmuller et al. J Gen Virol 1989 Aug; 70 ( Pt 8) :21 11-9; Gorman et al. J Virol 1990 Apr;64(4) : 1487-97; Scholtissek et al. Arch Virol 1993; 131 (3-4) :237-50; Shu et al. J Virol 1993 May;67(5) :2723-9.] and also contains dominant CTL target epitopes [Townsend et al. J Exp Med 1984 Aug 1 ; 160(2) :552-63; Yewdell et al. Proc Natl Acad Sci U S A 1985 Mar;82 (6) : 1785-9; McMichael et al. J Gen Virol 1986 Apr;67 ( Pt 4) :719-26; Chen et al. Immunity 2000 Jan; 12 (l) :83-93] that are directed against different variants of NP [Haanen et al. J Exp Med 1999 Nov 1 ; 190(9) : 1319-28] . NP vaccination was formerly thought to confer protection primarily via CD8 effectors mechanisms [ Taylor et al. Immunology 1986 Jul;58(3) :417-20; Gschoesser et al. Vaccine 2002 Nov l ;20(31-32) :3731-8] because restimulated T cells can transfer protection to naive mice [ Yap et al. Scand J Immunol 1978;8(5) :413-20; Wells et al. J Immunol 1981 Mar; 126(3) : 1042-6; Lukacher et al. J Exp Med 1984 Sep 1 ; 160(3) :814-26] and because T cell depletion in the vaccinated mice can abrogate protection [ Liang et al. J Immunol 1994 Feb 15; 152 (4) : 1653-61 , Epstein et al. J Immunol 1997 Feb 1 ; 158(3) : 1222-30] . Many studies have also established that both CD4+ T cells secreting Τπι-type cytokines and CD8⁺ CTL play important roles in protection obtained with NP protein [ Ulmer et al. J Virol 1998 Jul; 72 (7) : 5648-53.] . In fact, mice immunized with influenza NP (as soluble protein or using DNA vector) have higher frequencies of NP- specific CD8 T cells before infection and have a better control of viral titer after challenge with H3N2 and H1N1 strains of influenza virus. In these studies, the involvement of antibodies in protection has largely been underestimated. On the other hand, recent studies [Carragher et al. J Immunol 2008 Sep 15; 181 (6) :4168-76] suggest that soluble NP immunization may promote both antibodies and T-cell protective response to this conserved internal protein. Immunization with soluble recombinant NP protein is efficient but necessitates the uses of adjuvant in all the studies [Carragher et al. J Immunol 2008 Sep 15; 181 (6) :4168-76; Tite et al. Immunology 1990 Oct;71 (2) :202-7; Tamura et al. J Immunol 1996 May 15; 156(10) :3892-900; Guo et al. Arch Virol 2010 Jul 22] .

[004] The adjuvant capacity of PapMV VLPs to carry selected B-cell and CTL epitopes has been previously shown [Denis et al. Virology 2007 Jun 20;363(l) :59-68; Leclerc et al. J Virol 2007 Feb;81 (3) : 1319-26; Lacasse et al. J Virol 2008 Jan;82 (2) : 785-94] . PapMV VLPs, like many other VLP carriers, are restricted in the size and the nature of epitopes that can be inserted into their C-terminal region [Tremblay et al. Febs J 2006 Jan; 273(1) : 14-25] . Nevertheless, PapMV VLPs increase the immunogenicity of peptides carried on heterologous PapMV VLPs [ Denis et al Vaccine 2008 Jun 25;26(27-28) :3395-403] , as well as some components of the whole influenza inactivated vaccine (Savard et al. (201 1) ; Plus One 6(6) :e21522). The multimerisation of peptides selected by phage display has been shown to be an efficient method to improve the avidity of the peptide for its target (Terskikh et al. Proc Natl Acad Sci USA. 1997 Mar 4;94 (5) : 1663-8).

[005] PapMV VLPs have been used as a platform for the fusion of affinity peptides and high avidity VLPs (HAV) have been generated directed to the resting spores of the fungus Plasmodiophora brassicae (Morin et al. J Biotechnol 2007 Feb 1 ; 128(2) :423-34.) .

[006] 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

[007] An object of the present invention is to provide an affinity-conjugated nucleoprotein- papaya mosaic virus-like particles and uses thereof. In accordance with an aspect of the invention, there is provided an affinity-conjugated nucleoprotein-PapMV virus-like particle system comprising an influenza nucleoprotein (NP) and a virus-like particle (VLP) derived from PapMV coat protein, said PapMV coat protein modified by the addition of one or more peptides capable of specifically binding to influenza NP, wherein said system is capable of inducing an immune response in an animal.

[008] In accordance with another aspect of the invention, there is provided an immunogenic composition comprising the affinity-conjugated nucleoprotein- PapMV virus-like particle system according to the invention, and a pharmaceutically acceptable carrier.

[009] In accordance with another aspect of the invention, there is provided a method of inducing an immune response to influenza nucleoprotein in an animal comprising administering to said animal an effective amount of the affinity-conjugated nucleoprotein- PapMV virus-like particle system according to the invention.

[010] In accordance with another aspect of the invention, there is provided a method of preventing or treating influenza in an animal, said method comprising administering to said animal an effective amount of the affinity-conjugated nucleoprotein-PapMV virus-like particle system according to the invention.

[011] In accordance with another aspect of the invention, there is provided a use of an effective amount of the affinity-conjugated nucleoprotein-PapMV virus-like particle system according to the invention, to induce an immune response to influenza nucleoprotein in an animal in need thereof.

[012] In accordance with another aspect of the invention, there is provided a use of an effective amount of the affinity-conjugated nucleoprotein-PapMV virus-like particle system according to the invention, to prevent or treat influenza in an animal in need thereof. [013] In accordance with another aspect of the invention, there is provided a use of the affinity-conjugated nucleoprotein-PapMV virus-like particle system of the invention, in the manufacture of a medicament.

[014] In accordance with another aspect of the invention, there is provided a method of preparing an immunogenic composition comprising admixing influenza nucleoprotein with a papaya mosaic virus (PapMV) virus-like particle (VLP) derived from PapMV coat protein, said PapMV VLP comprising one or more peptides attached to coat proteins of said PapMV VLP, said peptides capable of specifically binding to influenza nucleoprotein.

[015] In accordance with another aspect of the invention, there is provided an immunogenic composition prepared by the method according to the invention.

[016] In accordance with another aspect of the invention, there is provided a fusion protein comprising a papaya mosaic virus (PapMV) coat protein fused to one or more peptides capable of specifically binding to influenza nucleoprotein.

[017] In accordance with another aspect of the invention, there is provided an isolated polynucleotide encoding the fusion protein according to the invention.

[018] In accordance with another aspect of the invention, there is provided a use of the fusion protein according to the invention, or a polynucleotide according to the invention, to prepare a virus-like particle.

BRIEF DESCRIPTION OF THE DRAWINGS

[019] These and other features of the invention will become more apparent in the following detailed description in which reference is made to the appended drawings.

[020] Figure 1 presents data relating to the selection of affinity peptides against NP protein. A) SDS-Page evaluation of influenza A/WSN/33 (H1N1) NP protein. Lane 1 : broad range protein marker. Lane 2: Bacterial lysate before induction. Lane 3: Bacterial lysate after induction. Lane 4: Purified protein. B) Selected peptides against target NP protein by phage display after 5 rounds of panning. [021] Figure 2 presents data relating to the characterization of the coat proteins fused to affinity peptides. A) SDS-Page evaluation of adjuvant protein PapMV and high avidity PapMV (PapMV HAV-ANP1 and PapMV HAV-ANP2). Lane 1 : broad range protein marker. Lane 2: Bacterial lysate after induction. Lane 3: Purified protein after elution. B) Morphologic evaluation of VLPs by Electron microscopy.

[022] Figure 3 presents measurement of the affinity of PapMV VLPs against the target NP. A) ELISA technique. Numbers are expressed as the ratio between the absorbance at 450nm of NP coated plated versus control plate. B) Silicone nano-porous biosensor analysis.

[023] Figure 4 presents data showing the immune response generated against NP protein. Mice, 10 per groups, were vaccinated three times with 10 μg of purified recombinant NP (NP) with or without 30 μg of recombinant PapMV VLPs or high avidity PapMV HAV (PapMV HAV-ANP1 and PapMV HAV-ANP2). Serum titer, 2 weeks after the last injection (Data are representative of three experiments) . A) IgGl serum titer against NP. B) IgG2a serum titer against NP. ** E 0 .01 vs NP and NP+PapMV HAV-ANP1 , number represent the time increase versus the NP alone. C) IgGl/IgG2a ratio serum titer against NP. * P < 0 .05 vs. all groups. D) Mice, 5 per groups, were vaccinated three times as described above. 2 weeks after the last injection, the spleen was removed to obtain a cell suspension and 2.5x105 cells were reactivated with rNP protein. INF-g secreting cells following reactivation were revealed by ELISPOT assay. Background Spots obtained by reactivation with medium alone were removed from data obtained by reactivation with NP protein. * F 0.05 vs. all groups (data are representative of two experiments) , number represent the time increase versus the NP alone.

[024] Figure 5 presents the effect of adjuvants on mouse influenza challenge with homologous strains A(H lNl)/WSN/33. Mice, 10 per groups, were vaccinated three times with 10 μg of purified NP (NP) with or without 30 μg of PapMV VLPs or PapMV HAVs (PapMV HAV-ANP2. Mice were challenged with 2LD50 of A(HlNl)/WSN/33 influenza virus, 2 weeks after the last boost and were sacrificed at day 7. A) Measure of the body weight losses of mice at day 7 B) Symptoms observed on each infected mice were scored at day 7 after the challenge. 1. Lightly spiked fur, lightly curved back. 2. Spiked fur, curved back. 3 Spiked fur, curved back, difficulty to move and light dehydratation. 4. Spiked fur, curved back, difficulty to move and severe dehydratation, closed eyes and ocular secretion. ** P < 0.05 vs all groups. C) In vitro influenza virus titration of infected mouse homogenized lungs in MDCK cells. Data are expressed as Log 10 of plaque forming units (PFU). D) 10 mice per groups were immunized as above and were challenged with 2LD50 of A(HlNl)/WSN/33 influenza virus, 2 weeks after the last boost. Body weight of each mouse was monitored for a 14 days period. Mice that lost more than 20% of their initial weight were sacrificed. Survival curve of infected mice express as percentage of mice who losses less than 20% of their initial weight.

[025] Figure 6 presents the immune response generated against NP protein. Mice, 15 per groups (5 were not challenged) , were vaccinated three times with 10 μg of purified NP (NP) with or without 30 μg of PapMV VLPs (PapMV VLP) or high avidity PapMV HAVs ( PapMV HAV-ANP2) . Serum titer, 2 weeks after the last injection) A) IgGl serum titer against NP. B) IgG2a serum titer against NP. * B .05 vs NP, *** F< 0 .001 vs NP. Numbers represent the time increase versus the NP alone. C) Total IgG serum titer against PapMV. Both adjuvanted groups, F< 0 .001 vs NP, ** F< 0 .01 vs NP + PapMV HAV- ANP2. D) Curve of IgG2a serum titer against NP protein in time following immunization. Arrows represent each injection.

[026] Figure 7 presents the biochemical characterization of the PapMV VLPs. A) SDS- PAGE showing the expression and purification profile of the PapMV CP. Lane 1 : broad range protein marker. Lane 2: Bacterial lysate before induction. Lane 3: Bacterial lysate after induction. Lane 4: Purified protein after elution. B) Morphologic evaluation of the adjuvant PapMV VLPs by Electron microscopy, bar is 0.2μιη and C) dynamic light scattering of the PapMV VLPs showing the average length the different populations of the VLPs found in solution.

[027] Figure 8 presents PapMV VLPs stimulate the secretion of THI-TH2 cytokines. A) In vivo imaging of fluorescently labeled PapMV VLPs. The data are presented as pseudocolor images indicating fluorescence (Alexa@680) intensity, with a graduation from red (more intense) to yellow, which were superimposed over gray-scale reference photographs of left inferior member of the treated mouse. Imaging was taken at 24, 48 and 72h post-injection. B) Cytokine/chemokine profile of reactivated splenocytes with PapMV VLPs (10C^g/ml) isolated after one subcutaneous injection. C) after 2 subcutaneous immunizations.

[028] Figure 9 presents (A) the amino acid sequence for the papaya mosaic virus (PapMV) coat protein (GenBank Accession No. NP_044334.1 ; SEQ ID NO: l l), (B) the nucleotide sequence encoding the PapMV coat protein (GenBank Accession No. NC_001748 (nucleotides 5889-6536) ; SEQ ID NO: 12), (C) the amino acid sequence of the modified PapMV coat protein ΟΡΔΝ5 (SEQ ID NO: 13) and the amino acid sequence of the modified PapMV coat protein PapMV CPsm [SEQ ID NO: 14.

[029] Figure 10 presents (A) the nucleotide sequence encoding the NP protein from influenza virus strain A/WSN/33 [SEQ ID NO: 15] , and (B) the amino acid sequence of the NP protein [SEQ ID NO: 16] encoded by the sequence provided in (A) .

DETAILED DESCRIPTION OF THE INVENTION

[030] An affinity-conjugated nucleoprotein-PapMV virus-like particle (ANP) system is provided. The ANP system comprises a virus-like particle (VLP) derived from the coat protein of PapMV which has been modified by the addition of one or more "affinity peptides." The affinity peptides are short peptide sequences capable of specifically binding to influenza nucleoprotein (NP) . The ANP system further comprises influenza NP conjugated via the one or more affinity peptides to the VLP. By "derived from" it is meant that the VLP comprises coat proteins that have an amino acid sequence substantially identical to the sequence of the wild-type coat protein. The one or more affinity peptides are attached, for example by chemical or genetic means, to the coat protein of the PapMV to form a PapMV High Affinity VLP (PapMV HAV). In accordance with one embodiment of the present invention, the ANP system is capable of inducing a humoral immune response, a cellular immune response, or both, to the NP protein in an animal. The ANP system is thus suitable for use as a vaccine, which may require an active participation of one or both of these two branches of the immune system. Definitions

[031] 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.

[032] As used herein, the term "about" refers to approximately a +/-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.

[033] The term "adjuvant," as used herein, refers to an agent that augments, stimulates, actuates, potentiates and/or modulates an immune response in an animal. An adjuvant may or may not have an effect on the immune response in itself.

[034] The term "immune response," as used herein, refers to an alteration in the reactivity of the immune system of an animal in response to an antigen or antigenic material and may involve antibody production, induction of cell-mediated immunity, complement activation, development of immunological tolerance, or a combination thereof.

[035] The terms "effective immunoprotective response," "effective immune response," and "immunoprotection," as used herein, mean an immune response that is directed against one or more antigen so as to protect partially or completely against disease and/or infection by a pathogen in a vaccinated animal. For purposes of the present invention, protection against disease and/or infection by a pathogen thus includes not only the absolute prevention of the disease or infection, but also any detectable reduction in the degree or rate of disease or infection, or any detectable reduction in the severity of the disease or any symptom or condition resulting from infection by the pathogen in the vaccinated animal as compared to an unvaccinated infected or diseased animal. An effective immune response can be induced in animals that were not previously suffering from the disease, have not previously been infected with the pathogen and/or do not have the disease or infection at the time of vaccination. An effective immune response can also be induced in an animal already suffering from the disease or infected with the pathogen at the time of vaccination. Immunoprotection can be the result of one or more mechanisms, including humoral and/or cellular immunity. [036] The terms "immune stimulation" and "immunostimulation" as used interchangeably herein, refer to the ability of a molecule, such as a PapMV or PapMV VLP, that is unrelated to an animal pathogen or disease to provide protection to against infection by the pathogen or against the disease by stimulating the immune system and/or improving the capacity of the immune system to respond to the infection or disease. Immunostimulation may have a prophylactic effect, a therapeutic effect, or a combination thereof.

[037] A "recombinant virus" is one in which the genetic material of a naturally-occurring virus has combined with other genetic material.

[038] "Naturally-occurring," as used herein, as applied to an object, refers to the fact that an object can be found in nature. For example, an organism (including a virus) , or a polypeptide or polynucleotide sequence that is present in an organism that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally- occurring.

[039] The terms "polypeptide" or "peptide" as used herein is intended to mean a molecule in which there is at least four amino acids linked by peptide bonds.

[040] The expression "viral nucleic acid," as used herein, may be the genome (or a majority thereof) of a virus, or a nucleic acid molecule complementary in base sequence to that genome. A DNA molecule that is complementary to viral RNA is also considered viral nucleic acid, as is a RNA molecule that is complementary in base sequence to viral DNA.

[041] The term "virus-like particle" (VLP), as used herein, refers to a self-assembling particle which has a similar physical appearance to a virus particle. The VLP may or may not comprise viral nucleic acids. VLPs are generally incapable of replication.

[042] The term "pseudovirus," as used herein, refers to a VLP that comprises nucleic acid sequences, such as DNA or RNA, including nucleic acids in plasmid form. Pseudoviruses are generally incapable of replication.

[043] The term "vaccine," as used herein, refers to a material capable of producing an effective immune response. [044] The terms "immunogen" and "antigen" as used herein refer to a molecule, molecules, a portion or portions of a molecule, or a combination of molecules, up to and including whole cells and tissues, which are capable of inducing an immune response in a subject alone or in combination with an adjuvant. The immunogen/antigen may comprise a single epitope or may comprise a plurality of epitopes. The term thus encompasses peptides, carbohydrates, proteins, nucleic acids, and various microorganisms, in whole or in part, including viruses, bacteria and parasites. Haptens are also considered to be encompassed by the terms "immunogen" and "antigen" as used herein.

[045] 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 an effective immune response and can have a prophylactic effect, a therapeutic effect, or a combination thereof. 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.

[046] As used herein, the terms "treat," "treated," or "treating" when used with respect to a disease or pathogen refers to a treatment which increases the resistance of a subject to the disease or to infection with a pathogen {i.e. decreases the likelihood that the subject will contract the disease or become infected with the pathogen) as well as a treatment after the subject has contracted the disease or become infected in order to fight a disease or infection (for example, reduce, eliminate, ameliorate or stabilise a disease or infection).

[047] The term "prime" and grammatical variations thereof, as used herein, means to stimulate and/or actuate an immune response against an antigen in an animal prior to administering a booster vaccination with the antigen.

[048] The term "subject" or "patient" as used herein refers to an animal in need of treatment.

[049] 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 or other hoofed animals, dogs, cats, chickens, ducks, non-human primates, guinea pigs, rabbits, ferrets, rats, hamsters and mice.

[050] 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 70%, 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 (or "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 (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. In general, for amino acid sequences, the length of comparison sequences will be at least 10 amino acids. One skilled in the art will understand that the actual length will depend on the overall length of the sequences being compared and may be at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 1 10, at least 120, at least 130, at least 140, at least 150, or at least 200 amino acids, or it may be the full-length of the amino acid sequence. For nucleic acids, the length of comparison sequences will generally be at least 25 nucleotides, but may be at least 50, at least 100, at least 125, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, or at least 600 nucleotides, or it may be the full-length of the nucleic acid sequence. [051] The terms "corresponding to" or "corresponds to" indicate that a nucleic acid sequence is identical to all or a portion of a reference nucleic acid sequence. In contradistinction, the term "complementary to" is used herein to indicate that the nucleic acid sequence is identical to all or a portion of the complementary strand of a reference nucleic acid sequence. For illustration, the nucleic acid sequence "TATAC" corresponds to a reference sequence "TATAC" and is complementary to a reference sequence "GTATA."

AFFINITY-CONJUGATED NUCLEOPROTEIN-PAPMV VLP (ANP) SYSTEM

[052] As indicated above, the ANP system comprises a virus-like particle (VLP) derived from the coat protein of PapMV which has been modified by the addition of one or more "affinity peptides. " The affinity peptides are short peptide sequences capable of specifically binding to influenza nucleoprotein (NP). The ANP system further comprises influenza NP conjugated via the one or more affinity peptides to the VLP.

PAPMV VLPs

[053] The ANP system of the present invention comprises PapMV VLPs formed from recombinant PapMV coat proteins that have multimerised and self-assembled to form a VLP. When assembled, each VLP comprises a long helical array of coat protein subunits. The wild- type virus comprises over 1200 coat protein subunits and is about 500nm in length. PapMV VLPs that are either shorter or longer than the wild-type virus can still, however, be effective. In one embodiment of the present invention, the VLP comprises at least 40 coat protein subunits. In another embodiment, the VLP comprises between about 40 and about 1600 coat protein subunits. In an alternative embodiment, the VLP is at least 40nm in length. In another embodiment, the VLP is between about 40nm and about 600nm in length.

[054] The VLPs of the present invention can be prepared from a plurality of recombinant coat proteins having identical amino acid sequences, such that the final VLP when assembled comprises identical coat protein subunits, or the VLP can be prepared from a plurality of recombinant coat proteins having different amino acid sequences, such that the final VLP when assembled comprises variations in its coat protein subunits. [055] The coat protein used to form the VLP can be the entire PapMV coat protein, or part thereof, or it can be a genetically modified version of the PapMV coat protein, for example, comprising one or more amino acid deletions, insertions, replacements and the like, provided that the coat protein retains the ability to multimerise and assemble into a VLP. The amino acid sequence of the wild-type PapMV coat (or capsid) protein is known in the art (see, Sit, et al, 1989, J. Gen. Virol, 70:2325-2331, and GenBank Accession No. NP_044334.1) and is provided herein as SEQ ID NO: 11 (see Figure 9A) . The nucleotide sequence of the PapMV coat protein is also known in the art (see, Sit, et al, ibid., and GenBank Accession No. NC_001748 (nucleotides 5889-6536)) and is provided herein as SEQ ID NO: 12 (see Figure 9B).

[056] As noted above, the amino acid sequence of the recombinant PapMV coat protein comprised by the VLP need not correspond precisely to the parental (wild-type) sequence, i.e. it may be a "variant sequence." For example, the recombinant protein 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 either the parental (reference) sequence. One skilled in the art will appreciate, however, that such mutations will not be extensive and will not dramatically affect the ability of the recombinant coat protein to multimerise and assemble into a VLP. The ability of a variant version of the PapMV coat protein to assemble into multimers and VLPs can be assessed, for example, by electron microscopy following standard techniques, such as the exemplary methods set out in the Examples provided herein.

[057] Recombinant coat proteins that are fragments of the wild-type protein that retain the ability to multimerise and assemble into a VLP {i.e. are "functional" fragments) are, therefore, also contemplated by the present invention. For example, a 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. In general, functional fragments are at least 100 amino acids in length. In one embodiment of the present invention, functional fragments are at least 150 amino acids, at least 160 amino acids, at least 170 amino acids, at least 180 amino acids, and at least 190 amino acids in length. Deletions made at the N-terminus of the protein should generally delete fewer than 25 amino acids in order to retain the ability of the protein to multimerise. [058] In accordance with the present invention, when a recombinant coat protein comprises a variant sequence, the variant sequence is at least about 70% identical to the reference sequence. In one embodiment, the variant sequence is at least about 75% identical to the reference sequence. In other embodiments, the variant sequence is at least about 80%, at least about 85%, at least about 90%, at least about 95%, and at least about 97% identical to the reference sequence. In a specific embodiment, the reference amino acid sequence is SEQ ID NO: l l.

[059] In one embodiment of the present invention, the VLP comprises a genetically modified (i.e. variant) version of the PapMV coat protein. In another embodiment, the PapMV coat protein has been genetically modified to delete amino acids from the N- or C- terminus of the protein and/or to include one or more amino acid substitutions. In a further embodiment, the PapMV coat protein has been genetically modified to delete between about 1 and about 10 amino acids from the N- or C-terminus of the protein.

[060] In a specific embodiment, the PapMV coat protein has been genetically modified to remove one of the two methionine codons that occur proximal to the N-terminus of the protein (i.e. at positions 1 and 6 of SEQ ID NO: 1 1) and can initiate translation. Removal of one of the translation initiation codons allows a homogeneous population of proteins to be produced. The selected methionine codon can be removed, for example, by substituting one or more of the nucleotides that make up the codon such that the codon codes for an amino acid other than methionine, or becomes a nonsense codon. Alternatively all or part of the codon, or the 5' region of the nucleic acid encoding the protein that includes the selected codon, can be deleted. In a specific embodiment of the present invention, the PapMV coat protein has been genetically modified to delete between 1 and 5 amino acids from the N- terminus of the protein. In a further embodiment, the genetically modified PapMV coat protein has an amino acid sequence substantially identical to SEQ ID NO: 13. In a further embodiment, the PapMV coat protein that has been genetically modified to include additional amino acids (for example between about 1 and about 8 amino acids) at the C-terminus that result from the inclusion of one or more specific restriction enzyme sites into the encoding nucleotide sequence. In a specific embodiment, the PapMV coat protein has an amino acid sequence substantially identical to SEQ ID NO: 14. [061] When the recombinant coat protein comprises a variant sequence that contains one or more amino acid substitutions, these can be "conservative" substitutions or "non- 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.

[062] In one embodiment of the present invention, the variant sequence comprises one or more non-conservative substitutions. Replacement of one amino acid with another having different properties may improve the properties of the coat protein. For example, as described herein, mutation of residue 128 of the coat protein improves assembly of the protein into VLPs. In one embodiment of the present invention, therefore, the coat protein comprises a mutation at residue 128 of the coat protein in which the glutamic residue at this position is substituted with a neutral residue. In a further embodiment, the glutamic residue at position 128 is substituted with an alanine residue.

[063] Likewise, the nucleic acid sequence encoding the recombinant coat protein need not correspond precisely to the parental reference sequence but may vary by virtue of the degeneracy of the genetic code and/or such that it encodes a variant amino acid sequence as described above. In one embodiment of the present invention, therefore, the nucleic acid sequence encoding a the recombinant coat protein is at least about 70% identical to the reference sequence. In another embodiment, the nucleic acid sequence encoding the recombinant coat protein is at least about 75% identical to the reference sequence. In other embodiments, the nucleic acid sequence encoding the recombinant coat protein is at least about 80%, at least about 85% or at least about 90% identical to the reference sequence. In a specific embodiment, the reference nucleic acid sequence is SEQ ID NO: 12.

AFFINITY PEPTIDES FOR NUCLEOPROTEIN

[064] The PapMV VLP coat protein is attached, for example, genetically fused to one or more affinity peptides that have a high avidity for the NP protein, to form a PapMV High Affinity VLP (PapMV HAV) as described in more detail below.

[065] The affinity peptides selected for use in the ANP system of the present invention are preferably capable of specifically binding to the NP protein and of being attached, for example by chemical or genetic means, to a PapMV coat protein. Exemplary peptides are described in the Examples provided herein. Other affinity peptides that bind influenza NP can be identified using methods such as those described below or are known in the art.

[066] Suitable affinity peptides can be selected by art-known techniques, such as phage or yeast display techniques. The peptides can be naturally occurring, recombinant, synthetic, or a combination of these. For example, the peptide can be a fragment of a naturally occurring protein or polypeptide. The term peptide as used herein also encompasses peptide analogues, peptide derivatives and peptidomimetic compounds. Such compounds are well known in the art and may have advantages over naturally occurring peptides, including, for example, greater chemical stability, increased resistance to proteolytic degradation, enhanced pharmacological properties (such as, half-life, absorption, potency and efficacy) and/or reduced antigenicity.

[067] Suitable peptides can range from about 3 amino acids in length to about 50 amino acids in length. In accordance with one embodiment of the invention, an affinity peptide suitable for use in the ANP system is at least 5 amino acids in length. In accordance with another embodiment of the invention, an affinity peptide suitable for use in the ANP system is at least 7 amino acids in length. In accordance with another embodiment of the invention, an affinity peptide suitable for use in the ANP system is between about 5 and about 50 amino acids in length. In accordance with another embodiment of the invention, an affinity peptide suitable for use in the ANP system is between about 7 and about 50 amino acids in length. In other embodiments of the present invention, an affinity peptide suitable for use in the ANP system between about 5 and about 45 amino acids in length, between about 5 and about 40 amino acids in length, between about 5 and about 35 amino acids in length and between about 5 and about 30 amino acids in length. In accordance with a specific embodiment of the invention, an affinity peptide suitable for use in the ANP system is 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14 or 15 amino acids in length. As would be understood by a worker skilled in the art, when the peptide is to be genetically fused to the PapMV coat protein, the length of the peptide selected should not interfere with the ability of the coat protein to self-assemble into VLPs.

[068] The affinity peptide comprised by the PapMV or VLP can be a single peptide or it can comprise a tandem or multiple arrangement of peptides.

[069] In one embodiment, the affinity peptide can be attached by chemical or genetic means to the C-terminus of the PapMV coat protein. In another embodiment, the affinity peptide is attached to the N-terminus of the PapMV coat protein. In yet another embodiment, the affinity peptide is attached to an internal loop of the PapMV coat protein that is exposed on the surface of the coat protein.

[070] A spacer can be included between the affinity peptide and the coat protein if desired in order to facilitate the binding of the NP protein. Suitable spacers include short stretches of neutral amino acids, such as glycine, for example, a stretch of between about 3 and about 10 neutral amino acids. In one embodiment, a stretch of between about 3 and about 10 amino acids is inserted between the PapMV coat protein and the affinity peptide.

[071] As noted above, phage display can be used to select specific peptides that bind to an antigenic protein of interest using standard techniques (see, for example, Current Protocols in Immunology, ed. Coligan et al, J. Wiley & Sons, New York, NY) and/or commercially available phage display kits (for example, the Ph.D. series of kits available from New England Biolabs, and the T7-Select® kit available from Novagen). An example of selection of peptides by phage display is also provided in Example 2, below.

[072] Representative peptides that bind NP that were identified by phage display include: FHEFWPT [SEQ ID NO:4] , FHENWPT [SEQ ID NO:5] , KVWQIPH [SEQ ID NO:6] and LPTPPWQ [SEQ ID NO:7] . One skilled in the art will appreciate that these peptides are examples only and that other peptides having an affinity for NP can be readily identified using art-known techniques. Truncated versions, for example comprising at least 4 consecutive amino acids, of the SEQ ID NOs:4 to 7 are also contemplated. In accordance with one embodiment of the present invention, there is provided an ANP system comprising a PapMV VLP that includes one or more affinity peptides comprising all or a part of the sequence set forth in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7.

INFLUENZA NUCLEOPROTEIN

[073] As indicated above, the ANP system of the present invention comprises an NP protein derived from an influenza virus. The ANP system may comprise polypeptide fragments of the NP protein and/or antigenic regions or fragments of the NP protein. The NP protein can be purified from the influenza virus, or expressed recombinantly.

[074] In the ANP system, the NP protein is combined with the PapMV VLP. In certain embodiments, it is contemplated that the NP protein may be conjugated to the affinity peptide added to the coat protein of the PapMV VLP. Conjugation can be, for example, binding via covalent or non-covalent means.

[075] In one embodiment, the NP protein of the ANP system is derived from an influenza A strain. Generally, influenza A strains are capable of infecting a large number of vertebrates including humans, domestic and farm animals, marine mammals, and various birds. In another embodiment, the NP protein of the ANP system is derived from an Influenza B strain. Typically, influenza B strains are capable of infecting humans and pigs. In another embodiment, the NP protein of the ANP system is derived from an influenza C strain. The influenza C strain has been observed to infect humans and seals.

[076] In one embodiment, the NP protein of the ANP system may be derived from an influenza A strain that infects humans, pigs, poultry. Humans are infected by a variety of influenza A strains, the most common strains being HlNl, H1N2 and H3N2. In pigs, strains HlNl , H1N2 and H3N2 are prevalent, whereas in horses, strains H7N7 and H3N8 are prevalent. Poultry are also affected by a wide variety of strains including H1N7, H2N2, H3N8, H4N2, H4N8, H5N1, H5N2, H5N9, H6N5, H7N2, H7N3, H9N2, H10N7, H11N6, H12N5, H13N6 and H14N5, many of which have also been reported in humans. In one embodiment, the NP protein of the ANP system may be derived from an influenza A strain that is a zoonotic, potential pandemic strain. Strains H5N1 , H9N2 and H7N7 are considered to be zoonotic, potential pandemic strains and are capable of affecting a variety of vertebrates. H5N1 has been reported to infect domestic cats and H3N8 has been reported in dogs. In one embodiment, the NP protein of the ANP system is derived from one of the following influenza A strains: H1N1 , H1N2 and H3N2.

[077] The sequences of the influenza virus NP protein from various influenza strains are known in the art and are readily accessible from GenBank database maintained by the National Center for Biotechnology Information (NCBI) . For example, the amino acid sequence of the NP protein from the influenza A strain A/WSN/33 is provided in Fig. 10 [SEQ ID NO: 16] . Suitable NP proteins for inclusion in the ANP system can, therefore, be readily selected by the skilled worker based on the knowledge in the art of antigenic regions of the influenza proteins and taking into consideration the animal in which an immune response is to be raised with the final ANP system.

[078] Various antigenic regions of the NP protein have been identified and are suitable for use in the ANP of the present invention. As indicated above, the NP protein comprised by the ANP of the present invention can be full-length proteins, fragments thereof, or antigenic fragments thereof. Examples include truncated versions of the NP protein, such as N-terminal or C-terminal truncations, as well as known antigenic fragments. Modified version of the NP protein, for example, NP protein that has been modified to facilitate expression or purification, are also contemplated.

[079] For humans, antigenic fragments of NP proteins include, but are not limited to, the nucleoprotein epitopes: NP 206-229 (Brett, 1991 , J. Immunol. 147:984-991), NP335-350 and NP380-393 (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, PNAS 90: 1508- 12) ; 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).

[080] In one embodiment of the present invention, the ANP system comprises a full-length NP protein. In another embodiment, the ANP system comprises a C-terminally or N- terminally truncated NP protein, or a fragment of NP that comprises a plurality of epitopes. In a further embodiment, the ANP system comprises a fragment of NP that comprises a plurality of the epitopes listed above.

PREPARATION OF THE ANP SYSTEM

[081] The present invention provides an ANP system that comprises PapMV VLPs derived from a recombinant PapMV coat protein that has been modified by the addition of one or more affinity peptides for the NP protein, and an NP protein. The recombinant coat proteins are capable of multimerisation and assembly into VLPs. Methods of genetically fusing the affinity peptides for linking to NP, to the coat protein are known in the art and some are described below and in the Examples. Methods of chemically cross-linking various molecules to proteins are well known in the art and can be employed.

PapMV VLPs

[082] The recombinant coat proteins for use to prepare the VLPs of the present invention can be readily prepared by standard genetic engineering techniques by the skilled worker provided with the sequence of the wild-type protein. 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), as is the sequence of the wild-type PapMV coat protein (see SEQ ID NOs: l 1 and 12).

[083] 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 PapMV by extracting RNA by standard techniques and then synthesizing cDNA from the RNA template (for example, by RT-PCR) . PapMV can be purified from infected plant leaves that show mosaic symptoms by standard techniques. [084] The nucleic acid sequence encoding the coat protein 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 coat protein can then be expressed and purified as described in more detail below.

[085] Alternatively, the nucleic acid sequence encoding the coat protein 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.

[086] As noted above, the coat proteins can also be engineered to produce fusion proteins comprising one or more affinity peptides fused to the coat protein. Methods for making fusion proteins are well known to those skilled in the art. DNA sequences encoding a fusion protein can be inserted into a suitable expression vector as noted above.

[087] One of ordinary skill in the art will appreciate that the DNA encoding the coat protein or fusion protein 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.

[088] 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 coat 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. The present invention, therefore, provides vectors comprising a regulatory element operatively linked to a nucleic acid sequence encoding a genetically engineered coat protein. One skilled in the art will appreciate that selection of suitable regulatory elements is dependent on the host cell chosen for expression of the genetically engineered coat protein and that such regulatory elements may be derived from a variety of sources, including bacterial, fungal, viral, mammalian or insect genes.

[089] In the context of the present invention, the expression vector may additionally 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 metal-affinity tags, histidine tags, avidin / streptavidin encoding sequences, glutathione-S-transferase (GST) encoding sequences and biotin encoding sequences. The amino acids corresponding to expression of the nucleic acids can be removed from the expressed coat protein prior to use according to methods known in the art. Alternatively, the amino acids corresponding to expression of heterologous nucleic acid sequences can be retained on the coat protein if they do not interfere with its subsequent assembly into VLPs.

[090] In one embodiment of the present invention, the coat protein is expressed as a histidine tagged protein. The histidine tag can be located at the carboxyl terminus or the amino terminus of the coat protein.

[091] 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 coat protein will be dependent upon the vector chosen. 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 coat proteins 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; or insect cells). In one embodiment, the coat proteins are expressed in prokaryotic cells.

[092] If desired, the coat proteins 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) and sequenced by standard peptide sequencing techniques using either the intact protein or proteolytic fragments thereof to confirm the identity of the protein.

[093] The recombinant coat proteins of the present invention comprising the affinity peptides are capable of multimerisation and assembly into VLPs. In general, assembly takes place in the host cell expressing the coat protein. The VLPs can be isolated from the host cells by standard techniques, such as those described in Denis et al. 2007, 2008, and Tremblay et al, 2006. In general, the isolate obtained from the host cells contains a mixture of VLPs, discs, less organised forms of the coat protein (for example, monomers and dimers) . The VLPs can be separated from the other coat protein components by, for example, ultracentrifugation or gel filtration chromatography (for example, using Superdex G-200) to provide a substantially pure VLP preparation. In this context, by "substantially pure" it is meant that the preparation contains 70% or greater of VLPs. Alternatively, a mixture of the various forms of coat protein can be used in the final vaccine compositions. When such a mixture us employed, the VLP content should be 40% or greater. In one embodiment, preparations containing 50% or more of VLPs are used in the final vaccine compositions. In another embodiment, preparations containing 60% or more of VLPs are used in the final vaccine compositions. In a further embodiment, preparations containing 70% or more of VLPs are used in the final vaccine compositions. In another embodiment, preparations containing 80% or more of VLPs are used in the final vaccine compositions.

[094] The VLPs can be further purified by standard techniques, such as chromatography, to remove contaminating host cell proteins or other compounds, such as LPS. In one embodiment of the present invention, the VLPs are purified to remove LPS.

[095] In one embodiment of the present invention, the coat proteins assemble to provide a recombinant virus in the host cell and can be used to produce infective virus particles which comprise nucleic acid and fusion protein. This can enable the infection of adjacent cells by the infective virus particle and expression of the fusion protein therein. In this embodiment, the host cell used to replicate the virus can be a plant cell, insect cell, mammalian cell or bacterial cell that will allow the virus to replicate. The cell may be a natural host cell for the virus from which the virus-like particle is derived, but this is not necessary. The host cell can be infected initially with virus in particle form {i.e. in assembled rods comprising nucleic acid and a protein) or alternatively in nucleic acid form {i.e. RNA such as viral RNA; cDNA or run-off transcripts prepared from cDNA) provided that the virus nucleic acid used for initial infection can replicate and cause production of whole virus particles having the chimeric protein.

Characteristics of Recombinant Coat Proteins

[096] The recombinant coat proteins can be analyzed for their ability to multimerize and self-assemble into a VLP by standard techniques. For example, by visualising the purified recombinant protein by electron microscopy (see, for example, Example 4). VLP formation may also be determined by ultracentrifugation, and circular dichroism (CD) spectrophotometry may be used to compare the secondary structure of the recombinant proteins with the WT virus.

[097] Stability of the VLPs can be determined if desired by techniques known in the art, for example, by SDS-PAGE and proteinase K degradation analyses. According to one embodiment of the present invention, the PapMV VLPs of the invention are stable at elevated temperatures and can be stored easily at room temperature.

Combination of the PapMV VLPs with NP

[098] The NP protein can be combined with the PapMV VLPs in the ANP system by bringing the NP protein into contact with the PapMV VLP. In certain embodiments, conjugation can occur between the affinity peptides on the PapMV VLPs and the NP protein, for example, via the formation of at least one non-covalent chemical bond, for example, a hydrogen bond, an ionic bond, a hydrophobic interaction or van der Waals interaction. Covalent attachment of the NP protein to the affinity peptide attached to the PapMV coat protein is also contemplated.

The PapMV VLPs and NP protein can be combined to provide the ANP system, for example, by simple mixing of the NP protein and the PapMV VLPs in solution with or without agitation.

If covalent attachment of the NP protein to the affinity peptide is to be carried out, an appropriate chemical agent, as is known in the art, can be added to the PapMV VLPs-NP protein mixture to induce formation of covalent bounds between the PapMV VLPs and the NP protein, and thereby improve the strength of attachment between the PapMV VLP and the NP protein. After conjugation any unconjugated NP protein and/or PapMV VLP and/or cross linking agent(s) can optionally be removed using standard techniques, for example, chromatography gel filtration technique that will separate the larger conjugated proteins from the unconjugated partners. Ultracentrifugation can also be used to separate the NP protein from the PapMV VLPs and the conjugated complex.

Optimal ratios of NP protein:PapMV VLP for inclusion in the ANP system can be readily determined by the skilled worker. For example, ratios of NP protein:PapMV VLP of between about 10: 1 and 1 : 10 on a weight:weight basis may be useful. In one embodiment, ratios of NP protein:PapMV VLP of between about 9: 1 and 1 :9 on a weight:weight basis are used to form the ANP system. In another embodiment, ratios of NP protein:PapMV VLP of between about 8: 1 and 1 :8 on a weight: weight basis are used to form the ANP system. In other embodiments, ratios of NP protein:PapMV VLP of between about 7: 1 and 1 :7, of about 6: 1 to 1 :6, and of about 5: 1 and 1 :5 on a weight:weight basis are used to form the ANP system.

EVALUATION OF EFFICACY

[099] In order to evaluate the efficacy of the ANP system of the present invention as a vaccine, challenge studies can be conducted. Such studies involve the inoculation of groups of test animals (such as mice) with an ANP system of the present invention by standard techniques. Control groups comprising non-inoculated animals and/or animals inoculated with a commercially available vaccine, or other positive control, are set up in parallel. After an appropriate period of time post-vaccination, the animals are challenged with an influenza virus. Blood samples collected from the animals pre- and post-inoculation, as well as post- challenge are then analyzed for an antibody response to the virus. Suitable tests for the antibody response include, but are not limited to, Western blot analysis and Enzyme-Linked Immunosorbent Assay (ELISA) . The animals can also be monitored for development of other conditions associated with infection with influenza virus including, for example, body temperature, weight, and the like. For certain strains of influenza, survival is also a suitable marker. [0100] Cellular immune responses can also be assessed by techniques known in the art, including those described in the Examples presented herein. For example, through processing and cross-presentation of an epitope expressed on a PapMV VLP to specific T lymphocytes by dendritic cells in vitro and in vivo. Other useful techniques for assessing induction of cellular immunity (T lymphocyte) include monitoring T cell expansion and IFN-γ secretion release, for example, by ELISA to monitor induction of cytokines (see Example 10).

[0101] The extent of infection can also be assessed by measurement of lung viral titer using standard techniques after sacrifice of the animal.

Production of stock PapMV or VLP

[0102] Stocks of recombinant PapMV or VLP can be prepared by standard techniques. For example, a recombinant virus can be propagated in an appropriate host, such as Carica papaya or Antirrhinum majus, such that sufficient recombinant virus can be harvested.

[0103] Stocks of PapMV VLPs can be prepared from an appropriate host cell, such as E. coli transformed or transfected with an expression vector encoding the recombinant coat protein that makes up the VLP. The host cells are then cultured under conditions that favor the expression of the encoded protein, as is known in the art. The expressed coat protein will multimerise and assemble into VLPs in the host cell and can be isolated from the cells by standard techniques, for example, by rupturing the cells and submitting the cell lysate to one or more chromatographic purification step.

[0104] PapMV VLPs are stable structures and stocks of the VLPs can, therefore, be stored easily at room temperature or in a refrigerator.

VACCINE COMPOSITIONS

[0105] The present invention provides for compositions suitable for use as influenza vaccines comprising the ANP system of the invention together with one or more non-toxic pharmaceutically acceptable carriers, diluents and/or excipients. If desired, other active ingredients, adjuvants and/or immunopotentiators may be included in the compositions.

[0106] The 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 pharmaceutical composition to the mucous membranes of the nasal passage or nasal cavity of the subject. In one embodiment of the present invention, the compositions are formulated for topical, rectal or parenteral administration or for administration by inhalation or spray, for example by an intranasal route. In another embodiment, the compositions are formulated for parenteral administration.

[0107] The compositions preferably comprise an effective amount of one or more ANP systems of the invention. The term "effective amount" as used herein refers to an amount of the ANP system required to induce a detectable immune response. The effective amount of ANP system for a given indication can be estimated initially, for example, either in cell culture assays or in animal models, usually in rodents, rabbits, dogs, pigs or primates. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in the animal to be treated, including humans. In one embodiment of the present invention, the unit dose comprises between about lC^g to about lOmg of protein. In another embodiment, the unit dose comprises between about lC^g to about 5mg of protein. In a further embodiment, the unit dose comprises between about 4C^g to about 2 mg of protein. One or more doses may be used to immunise the animal, and these may be administered on the same day or over the course of several days or weeks. In one embodiment of the invention, two or more doses of the composition are administered to the animal to be treated. In another embodiment, three or more doses of the composition are administered to the animal to be treated.

[0108] Compositions for oral use can be formulated, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion hard or soft capsules, or syrups or elixirs. Such compositions can be prepared according to standard methods known to the art for the manufacture of pharmaceutical compositions and may contain one or more agents selected from the group of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the ANP in admixture with suitable non-toxic pharmaceutically acceptable excipients including, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, such as corn starch, or alginic acid; binding agents, such as starch, gelatine or acacia, and lubricating agents, such as magnesium stearate, stearic acid or talc. The tablets can be uncoated, or they may be coated by known techniques in order to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate may be employed.

[0109] Compositions for oral use can also be presented as hard gelatine capsules wherein the ANP system is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatine capsules wherein the active ingredient is mixed with water or an oil medium such as peanut oil, liquid paraffin or olive oil.

[0110] Compositions for nasal administration can include, for example, nasal spray, nasal drops, suspensions, solutions, gels, ointments, creams, and powders. The compositions can be formulated for administration through a suitable commercially available nasal spray device, such as Accuspray™ (Becton Dickinson) . Other methods of nasal administration are known in the art.

[0111] Compositions formulated as aqueous suspensions contain the ANP 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 /j-propyl p- hydroxy-benzoate, one or more colouring agents, one or more flavouring agents or one or more sweetening agents, such as sucrose or saccharin.

[0112] Compositions can be formulated as oily suspensions by suspending the ANP 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. Sweetening agents such as those set forth above, and/or flavouring agents may optionally be added to provide palatable oral preparations. These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid.

[0113] The compositions can 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 ANP 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, sweetening, flavouring and colouring agents, can also be included in these compositions.

[0114] Compositions of the invention can also be formulated as oil-in-water emulsions. The oil phase can be a vegetable oil, for example, olive oil or arachis oil, or a mineral oil, for example, liquid paraffin, or it may be a mixture of these oils. Suitable emulsifying agents for inclusion in these compositions include naturally- occurring gums, for example, gum acacia or gum tragacanth; naturally-occurring phosphatides, for example, soy bean, lecithin; or esters or partial esters derived from fatty acids and hexitol, anhydrides, for example, sorbitan monoleate, and condensation products of the said partial esters with ethylene oxide, for example, polyoxyethylene sorbitan monoleate. The emulsions can also optionally contain sweetening and flavouring agents.

[0115] Compositions can be formulated as a syrup or elixir by combining the ANP with one or more sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations can also optionally contain one or more demulcents, preservatives, flavouring agents and/or colouring agents. [0116] The compositions can be formulated as a sterile injectable aqueous or oleaginous suspension according to methods known in the art and using suitable one or more 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.

[0117] Optionally the composition of the present invention 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.

[0118] Further, one or more compounds having adjuvant activity may be optionally added to the vaccine composition. Suitable adjuvants include, for example, alum adjuvants (such as aluminium hydroxide, phosphate or oxide) ; oil-emulsions (e.g. of Bayol F® or Marcol52®) ; saponins, or vitamin-E solubilisate. Virosomes are also known to have adjuvant properties (Adjuvant and Antigen Delivery Properties of Virosomes, Gliick, R., et al, 2005, Current Drug Delivery, 2:395-400) and can be used in conjunction with an ANP of the invention.

[0119] As previously demonstrated, PapMV and PapMV VLPs have adjuvant properties. Accordingly, in one embodiment of the invention, the vaccine compositions comprise additional PapMV or PapMV VLPs as an adjuvant. In some embodiments, use of PapMV or PapMV VLPs may provide advantages over commercially available adjuvants in that it has been observed that PapMV or PapMV VLPs do not cause obvious local toxicity when administered by injection (see, for example, International Patent Publication No. WO2008/058396) .

[0120] Also encompassed by the present invention are vaccine compositions comprising an ANP system of the present invention in combination with a commercially available influenza vaccine.

[0121] 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) .

APPLICATIONS & USES

[0122] The present invention provides for a number of applications and uses of the ANP system described herein. In one embodiment, the ANP system can be used as a vaccine against influenza. In another embodiment, the ANP system can be used to induce an immune response against the NP protein. In the latter embodiment, the VLP acts to potentiate the immune response to the NP protein. The present invention thus also provides methods for potentiating and/or inducing an immune response to the NP protein in an animal. As well, the use of the ANP system of the invention for the preparation of medicaments, including vaccines, and/or pharmaceutical compositions is within the scope of the present invention.

[0123] The ANP system of the present invention can be used to induce an immune response to one or more than one strain of influenza virus. The ANP system is suitable for use in humans as well as non-human animals, including domestic and farm animals. The administration regime for the ANP system need not differ from any other generally accepted vaccination programs. A single administration of the ANP system in an amount sufficient to elicit an effective immune response may be used or, alternatively, other regimes of initial administration of the ANP system followed by boosting, once or more than once, with NP alone or with the ANP system may be used. Similarly, boosting with either the ANP system or NP may occur at times that take place well after the initial administration if antibody titers fall below acceptable levels. In one embodiment of the invention, the administration regime for the ANP system comprises an initial dose of the ANP system plus a booster dose of the ANP system. In another embodiment, the administration regime for the ANP system comprises an initial dose of the ANP system plus two or more booster doses of the ANP system. In a further embodiment, the administration regime for the ANP system comprises an initial dose of the ANP system plus three or more booster doses of the ANP. Appropriate dosing regimens can be readily determined by the skilled practitioner.

[0124] When the ANP system comprises non-covalently linked NP protein, the PapMV VLP component of the ANP system can be administered concomitantly with the NP protein, or it can be administered prior or subsequent to the administration of the NP protein, depending on the needs of the human or non-human animal in which an immune response is desired.

[0125] Another embodiment provides for the use of a vaccine comprising the ANP system in conjunction with conventional influenza vaccines. In accordance with this embodiment, the ANP system vaccine may be administered concomitantly with the conventional vaccine (for example, by combining the two compositions) , it can be administered prior or subsequent to the administration of the conventional vaccine.

[0126] One embodiment of the present invention provides for the use of the ANP system as an influenza vaccine for humans. Another embodiment of the present invention provides for the use of an ANP system comprising NP protein from the H1N1 and/or H3N2 strains of influenza as an influenza vaccine for humans. In a specific embodiment of the present invention, there is provided an ANP system for use as a human influenza vaccine wherein the PapMV VLP is modified by the addition of at least one or more affinity peptides for NP protein.

[0127] An alternative embodiment of the present invention provides for the use of the ANP system as an influenza vaccine for non-humans. Another embodiment provides for the use of an ANP system comprising NP protein from the H3N8, H7N7, H9N2 and/or H5N1 strains of influenza as an influenza vaccine for non-humans. A further embodiment provides for the use of the ANP system as an influenza vaccine for non-human mammals. Another embodiment provides for the use of the ANP system as an influenza vaccine for birds.

KITS [0128] The present invention additionally provides for kits comprising one or more ANP system 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.

[0129] 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.

[0130] 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 kits of the invention also may comprise an instrument for assisting with the administration of the composition to a patient. Such an instrument may be an inhalant, nasal spray device, syringe, pipette, forceps, measured spoon, eye dropper or similar medically approved delivery vehicle.

[0131] 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

EXAMPLE 1 : EXPRESSION AND PURIFICATION OF RECOMBINANT NP PROTEINS FROM E. COLI

[0132] Recombinant NP was prepared as follows. DNA encoding the influenza A/WSN/33 (H1N1) NP gene was amplified from a cDNA clone of this NP gene (provided by Dr. Guy Boivin of the Infectious Disease Research Centre, Quebec City, Canada) by PCR with the following primers 5'-GAC-TCC-ATG-GCG-ACC-AAA-GGC-ACC-AAA-CGA-3' [SEQ ID NO: l] and 5 'GAT-CCT-CGA-GTT-AGT-GGT-GGT-GGT-GGT-GGT-GAT-TGT-CGT- ACT-CCT-C-3' [SEQ ID NO:2] . The resulting PCR product was digested with NCOl and XHOl enzymes, and ligated into a NCOl/XHOl linearized Pet24d vector.

[0133] Briefly, the E. coli expression strain BL21 (DE3) RIL was transformed with the plasmid pET-24d containing A/WSN/33 (HlNl) NP protein constructs, and maintained in 2xYT medium containing Kanamycin (30 μg·mL·-l). Bacterial cells were grown at 37 °C t o an optical density of 0.6 ± 0.1 at 600 nm and protein expression was induced with 1 mm isopropyl^-d-thiogalactopyranoside (IPTG). Induction was continued for 16 h at 22°C. Bacteria were harvested by centrifugation for 15 min at 8,983g. The pellet was resuspended in ice-cold lysis buffer (50mM NaH₂P0₄ (pH 8.0) , 300mM NaCl, 5mM imidazole, 20 μΜ phenylmethanesulfonyl fluoride) and bacteria were lysed by one passage through a French press at 750 PSIG. The lysate was centrifuged twice for 30 min at 20442xg to eliminate cellular debris. The supernatant was incubated overnight with 2 mL Ni-NTA beads (Qiagen, Mississauga, On, Canada) under gentle agitation at 4°C. Lysates were loaded onto a column and the beads were washed with 2 x 20 mL washing buffer (50mM NaF^PC^ (pH 8.0), 500mM NaCl, 5mM imidazole). At the end of this washing procedure, an additional washing step was performed with 40ml of buffer containing 10mM imidazole. A washing step to remove lipopolysaccharide contaminants from our preparations was then performed with 20 ml of (50mM NaH₂P0₄ (pH8.0), 500mM NaCl, lOmM imidazole and 0.5% Triton X- 100) . At the end of these steps, the beads were washed with 40 mL of working buffer (50mM NaH₂P0₄ (pH 8.0), 500mM NaCl, 20mM imidazole). Proteins were eluted in working buffer containing 0.5M imidazole. The eluted proteins were subjected to a step by step dialysis procedure with phosphate-buffered saline (PBS) containing decreasing concentration of imidazole (500, 250, 100, 0 mM) for a minimum of 2 hours with 8,000 kda cutoff. The resultant protein solution was filtered with a 0.45-μιη filter. The purity of the proteins was determined by SDS/PAGE and protein concentrations were evaluated by use of a bicinchoninic acid protein kit (Pierce, Rockford, IL) . The lipopolysaccharide (LPS) content in the purified proteins was evaluated with the Limulus test according to the manufacturer's instructions (Cambrex, Walkersville, MD) and was below 5 endotoxin units/mg of protein.

[0134] Results: [0135] The recombinant NP protein, fused to a 6xH tag at its C-terminus, was expressed in E. coli and purified by affinity chromatography using a nickel column, as shown in (Fig 1A).

EXAMPLE 2 : SELECTION OF AFFINITY PEPTIDES FOR NP

[0136] The Ph.D. -7™ Phage display peptide library kit (New England Biolabs, Berverly, MA, USA) was used for the selection of peptides having an affinity for NP. Target protein (NP) was coated at 10(^g/ml in 0.1M NaHC0₃ pH 8.6 on MaxiSorp™ plates (Nunc, Roskilde, Denmark) , overnight at 4°C. Coating solution was poured off and the plates were blocked with 0.5% BSA in 0.1M NaHC0₃ pH 8,6 supplemented with 0.02% NaN₃ for 1 hour at 4°C. After blocking, the plates were washed three times with TBS (50mM Tris (pH 7.5) , 150 mM NaCl) supplemented with 0.1% of Tween-20 (TBS-T 0.1%). 10 μίοί the original phage library (corresponding to 2xl0ⁿ different phages) were added to each well and the plates were incubated for 1 hour at room temperature with gentle agitation. The phage solutions were then discarded and the plates were washed three time with (TBS-T 0.1%) . The stringency of selection was increased by using 0.5% Tween-20 in TBS for the three last rounds of panning to reduce the frequency of non-specific phage binding. The remaining phages bound to the plates were eluted with 0.2M Glycine-HCl (pH 2,2) supplemented with lmg/ml BSA.

[0137] For phage titration, a single colony of ER2738 was inoculated in lOmL of LB and incubated with shaking until mid-log phase (OD600 ^¾ 0.5) . A 10-fold serial dilution of eluted phages were prepared in LB, in a range of 10⁸-10¹¹ for amplified phages or ΙΟ^ΙΟ⁴ for crude panning eluate. 10 μΐ of each dilution were added to 200 μΐ of mid-log phase bacteria and incubated at room temperature for 5 min. Infected cells were transferred to a culture tube containing pre-warmed agarose top (45°C) , vortexed quickly, and poured onto a pre-warmed LB/IPTG /Xgal plate. Plates were incubated overnight at 37°C and plates containing approximately 100 lysis plaques were counted for titration. For amplification of the selected phages, an overnight culture of ER2738 was diluted 1 : 100 in LB and inoculated with blue plaques from plates having 10 to≡ 100 plaques. Inoculated tubes were incubated at 37°C with shaking for 4-5 hours. After incubation, cultures were centrifuged 30 seconds and supernatants were transferred to a fresh tube and centrifuged again. Using a pipet, the upper 80% of supernatants were transferred to clean tubes and amplified phages were stored at 4°C until next processing. For phages sequencing, the QIAprep® spin Ml 3 DNA extraction kit (Qiagen, Mississauga, On., Canada) was used according to themanufacturer's instructions. 10 clones of the third and the last panning procedures (5 consecutive rounds of panning were carried out) were DNA sequenced using the following primer 5 GTATGGGATTTTGTAATACATCA 3' [SEQ ID NO:3] .

Results:

[0138] NP was used as the bait for the selection of high affinity peptides by phage display. After five rounds of panning of the phages toward NP, 10 clones were sequenced. The peptide FHEFWPT [SEQ ID NO:4] was found in half of the clones sequenced, the peptide FHENWPT [SEQ ID NO:5] was found 3 times out of 10 sequenced clones, and finally, the peptides KVWQIPH [SEQ ID NO:6] and LPTPPWQ [SEQ ID NO: 7] were found in one out of 10 sequenced clones (Fig IB). The peptides FHEFWPT [ANP1 , SEQ ID NO:4] and KVWQIPH [ANP2, SEQ ID NO:6] were selected for cloning at the surface of the PapMV VLP.

EXAMPLE 3: PREPARATION OF HIGH AVIDITY PAPMV VLP (PAPMV HAV ANP)

Cloning and engineering of PapMV HAV.

[0139] The PapMV-CP (coat protein) clone was generated as described previously [28] . The nucleotide and amino acid sequences of this coat protein are shown in Figure 9. To prepare the PapMV HAV-ANP constructs containing the PapMV coat protein attached to the high affinity peptides, oligonucleotides containing sequences corresponding to selected peptides for PapMV- ANP 1 (5 '-CTA-GTT-TTC-ATG-AAT-TCT-GGC-CGA-CCA-3' [SEQ ID NO: 17] and 5 ' -CGC-GTG-GTC-GGC-C AG-AAT-TCA-TGA- AAA-3 ' [SEQ ID NO:8]) and for PapMV-ANP2 (5 '-CTA-GTA-AAG-TGT- GGC-AGA-TTC-CGC-ATA-3' [SEQ ID NO:9] and 5'-CGC-GTA-TGC-GGA-ATC-TGC-CAC-ACT-TTA-3' [SEQ ID NO: 10]) were annealed together, digested with Spel and Mlul enzymes and ligated into the Spel/Mlul site located at the C-terminus of PapMV-CP cloned in an Escherichia coli expression vector pET3D (Novagen) . The integrity of the PapMV HAV-ANP clones was confirmed by DNA sequencing.

Expression and purification of PapMV HAV-ANP recombinant proteins from E. coli

[0140] Expression in E. coli and purification of PapMV-CP were performed as described previously [31] , with some minor modifications. Briefly, the E. coli expression strain BL21 (DE3) RIL was transformed with the plasmid pET-3d containing PapMV-CP constructs, and maintained in 2xYT medium containing ampicillin (50 μg·mL·l). Bacterial cells were grown at 37 °C to an optical density of 0.6 ± 0.1 at 600 nm and protein expression was induced with 1 mM isopropyl^-d-thiogalactopyranoside (IPTG) . Induction was continued for 16 h at 22°C. Bacteria were harvested by centrifugation for 15 min at 8,983g. The pellet was resuspended in ice-cold lysis buffer (50mm Na ^PC^ (pH 8.0), 300mM NaCl, lOmM imidazol, 20 μΜ phenylmethanesulfonyl fluoride, 1 mg'mL-l lyso zyme) and the bacteria were lysed by one passage through a French press at 750 PSIG. The lysate was submitted to DNase (10 OOOU/ml) treatment with 60mM MgCl₂ for 15 min. at room temperature and was centrifuged twice for 30 min at 20442g to eliminate cellular debris. The supernatant was incubated overnight with 2 mL Ni-NTA under gentle agitation at 4°C. Lysates were loaded onto a column and the beads were washed with 2 x 30 mL washing buffer (50mM NaF^PC^ (pH 8.0) , 300mM NaCl) containing increasing concentrations of imidazole (20mm and 50mm) .Two washing steps to remove lipopolysaccharide contaminants from the preparations were included: the first one with 15 ml of (lOmM Tris-HCl (pH 8), 50mM imidazole and 0.5% Triton X- 100) , and the second one with 5mL of (lOmM Tris-HCl (pH 8) , 50mM imidazole and 1% Zwittergent) with a 30 min. incubation period at 4°C. At the end of each of these two additional washing steps, the beads were washed with 40 mL working buffer (lOmM Tris-HCl (pH 8) and 50mM imidazole) . Proteins were eluted in a working buffer containing 1M imidazole. The eluted proteins were subjected to high-speed ultracentrifugation (100,000 x g) for 45 min in a Beckman 50.2 Ti rotor. VLP pellets were resuspended in endotoxin-free phosphate-buffered saline (PBS) and finally, the protein solutions were filtered with 0.45-μιη filters. The purity, concentration and LPS content in the protein samples were evaluated as described in Example 1, and only samples containing below 5 endotoxin units/mg of protein was used. EXAMPLE 4 : MORHPOLOGICAL EVALUATION OF PAPMV HAV-ANPs

[0141] The morphology of PapMV HAV-ANP 1 and PapMV HAV-ANP2, prepared in Example 3, was evaluated by electron microscopy. For morphological evaluation by electron microscopy, PapMV-ANP HAV proteins were diluted in water to a concentration of 20 for PapMV VLPs and 40 ng/ml for NP protein, and mixed at 1 : 1 ratio with 3% uranyl acetate solution and incubated in darkness for 7 min. Following uranyl acetate staining, the VLPs were absorbed for 5 min on carbon-coated formvar grids and then observed on a JEOL - 1010 (Tokyo, Japan) transmission electron microscope. Images were acquired with a Bioscan Camera from Gatan (Warrendale, PA, USA) and analysed with the Gatan Digital Micrograph acquisition software. The length of the VLPs was measured with Metamorph software version 6.2r2 (Molecular Devices, Synnyvale, CA, USA) as described in the instruction manual from the manufacturers.

Results

[0142] In order to increase the protein- protein interaction between the adjuvant (PapMV VLP) and the antigen (NP) , the two affinity peptide candidates identified as described in Example 2 were fused to the surface of the PapMV VLP. It has been previously demonstrated that the fusion of a low affinity peptide at the surface of an highly ordered structure like the PapMV VLPs can generate a VLP that shows a high avidity to its target that is comparable to the binding of an antibody [37] . The fusion of the affinity peptides FHEFWPT (ANP 1) and KVWQIPH (ANP2) to the C-terminus of the PapMV CP was shown to be tolerated and expressed at high levels and generated newly engineered PapMV VLPs that harboured the affinity peptide at their surface. These newly engineered PapMV VLPs are referred to respectively as high avidity PapMV HAV-ANP 1 and PapMV HAV-ANP2 (Fig 2 A) . Morphologic evaluation by electron microscopy revealed that the engineered PapMV VLPs are similar in length (average of 60nm) , shape and in structure to the WT PapMV VLP (Fig 2B) .

EXAMPLE 5 : MEASUREMENT OF AVIDITY OF PAPMV HAV-ANP1 AND

PAPMV HAV-ANP2 TO NP BY ELISA [0143] NP protein at ^g/ml, was diluted in 0.1M NaHC0₃ buffer (pH 9.6) and 100 L well of diluted antigens were coated overnight at 4°C. Plates coated with buffer only were used as controls. Plates were blocked with PBS/0.1% Tween-20/2% BSA (150 IJwell) for 1 h at 37°C. After washing three times with PBS/0.1% Tween-20, PapMV, PapMV HAV-ANP l and PapMV HAV-ANP2 proteins were added in 2-fold serial dilutions starting from l ug/ml. The plates were incubated for lh. at room temperature, washed six times and then incubated for 1 hour with 100 of rabbit polyclonal antibodies generated against purified PapMV virus (Tremblay et al. 2006) at a dilution of 1 :5000 in PBS/0.1% Tween-20/2% BSA. Plates were then washed four times and incubated for lh. with 100 peroxidase-conjugated goat anti-rabbit IgG (Jackson Immunoresearch, Baltimore, PA), at a dilution of 1 : 10,000 in PBS/0.1% Tween-20/2% BSA for 1 h at 37 °C. After four washes, the presence of IgG was detected with ΙΟΟμΙ of TMB-S (Ultra-TMB-S, Research Diagnostics, Flanders, NJ) according to the manufacturer's instructions. The reaction was stopped by adding ΙΟΟμί of 0.18M H2SO4. The OD was read at 450 nm. Results are expressed as a ratio of NP coated/Buffer coated OD at 450 nm.

Measurement of avidity by silicon nano-porous biosensor analysis.

[0144] A Ski Pro system from Silicon kinetics was used to measure the avidity of PapMV HAV-ANP proteins to NP protein (Latterich and Corbeil 2008; Proteome Sci 2008;6:31) . The analysis was performed with a porous carboxy chip PEG 2000. First, COOH groups were modified to sulfo-succinimide esters with activation buffer (200mM EDC(l-ethyl-3-(3- dimethylaminopropyl)carbodimidehydrochloride) ; 50 mM sulfo-NHS(N-

Hydroxysulfosuccinimide) ; 100 mM MES; 150 mM NaCl at pH 6.0). The chip was activated for 600 sec in this solution. Next, NP protein was immobilized on the chips for 1200 sec with immobilization buffer (20 mM NaAc, 1 mM EDTA, pH 4.5) at a 5 μΜ final concentration. Then, free succinimide was deactivated with blocking buffer (1 M Ethanolamine-HCl pH 5.0) for 300 sec. The chips were equilibrated 30 min with binding buffer (HBS-EP from Biacore; 0.01M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20) before binding. For the binding step: PapMV, PapMV HAV-ANP l and PapMV HAV-ANP2 were diluted in binding buffer at 5 μΜ final concentration and bound on the chip for 200 sec. and then washed with binding buffer for 400 sec. OPD was monitored at each binding step, and depicted as a graph of OPD, nm vs.Time, in seconds. Results

[0145] Measurement of the avidity of the PapMV HAV-ANPs for their antigen

[0146] To evaluate the level of avidity of the PapMV HAV-ANPs to the antigen NP, a modified ELISA assay was first performed. In brief, the antigen NP was bound to the ELISA plate as usual, but instead of using an antibody for binding NP, the respective PapMV HAV- ANPs were used and PapMV VLPs were used as a negative control. The amount of PapMV HAV-ANP bound to NP was then revealed using an rabbit antibody directed to PapMV CP followed by a secondary goat anti rabbit antibody conjugated to peroxidase to reveal the complex. The assay showed a significant increase of the avidity of PapMV HAV-ANP2 over PapMV HAV-ANP 1 and PapMV VLPs as revealed by the five fold increase of the signal (Fig. 3A) . To confirm this result, a biosensor platform was used for monitoring direct protein- protein interaction based on the combination of a defined nano-porous silicon surface coupled to light interferometry [33] . Consistent with the ELISA analysis, the biosensor revealed a significant increase of the avidity (again by a factor of approximately 5 times) of HAV-ANP2 over HAV-ANP 1 and PapMV VLPs as seen with the increase of OPD (nm) for the PapMV HAV-ANP2 (Fig.3B) .

EXAMPLE 6: IMMUNIZATION OF MICE WITH PAPMV HAV-ANP1 AND

PAPMV HAV-ANP2

[0147] The following experiment was performed to determine whether the avidity of the PapMV HAV-ANP 1 and PapMV HAV-ANP2 affected the immune response to NP. Briefly, mice were immunized with recombinant NP protein (NP) with or without 3C^g of the PapMV, PapMV HAV-ANP 1 or PapMV HAV-ANP2 as described below. Serum from these animals was harvested two weeks after each immunization and ELISA was performed to measure IgG, IgGl and IgG2a levels in order to measure the humoral response to the NP antigen.

Immunization.

[0148] We immunized ten 6-8-week-old BALB/c mice (Charles River, Wilmington, MA) subcutaneously with 10 μg of recombinant NP protein (NP) with or without 30μg of the PapMV, PapMV HAV-ANP1 or PapMV HAV-ANP2. Primary immunization was followed by two booster doses given at 2 weeks interval. Blood samples were obtained 14 days after each shot and stored at -20 °C until analysis.

Antibody titration by ELISA.

Expression and purification of recombinant NP-GST proteins from E. coli for ELISA

[0149] The influenza NP protein was cloned as a GST fusion protein in the expression vector pGEX-2T to generate pGEX-NP. E. coli expression strain BL21 (DE3) RIL was transformed with pGEX-NP and maintained in 2xYT medium containing ampicillin (50 Jig^L Bacterial cells were grown, induced and harvested as described in Example 3 for the preparation of PapMV-CP. The bacterial cell pellet was resuspended in ice-cold lysis buffer (PBS IX) and stored at -80^°C for at least one day. Frozen pellets were thawed at 4^°C on ice and the cells lysed by one passage through a French press at 750 PSIG. The lysate was centrifuged for 45 min at 20442g to eliminate cellular debris and was loaded on glutathione separose beads from the bulk GST purification module (GE Healthcare, Little Chalfont, UK) . The beads were washed three times with 10X bed of PBS IX. GST-Proteins were eluted in 50mM Tris-HCl (pH 8.0) buffer containing lOmM reduced glutathione.

Antibody titration by ELISA

[0150] NP-GST at ^g/ml, was diluted in 0.1M NaHC0₃ buffer (pH 9.6) and ΙΟΟμΙ/well of diluted antigen was used to coat ELISA plates overnight at 4°C. Plates were blocked with PBS/0.1% Tween-20/2% BSA (150μίΛνβΠ) for 1 h at 37°C. After washing three times with PBS/0.1% Tween-20, sera were added in 2-fold serial dilutions starting from 1 :50. The plates were incubated for 90 min at 37 °C, washed four times and then incubated with 100

of peroxidase-conjugated goat anti-mouse IgG, IgGl , IgG2a, (all from Jackson Immunoresearch, Baltimore, PA), at a dilution of 1/10,000 in PBS/0.1% Tween-20/2% BSA for 1 h at 37 °C. After four washes, the presence of IgG was detected with ΙΟΟμί of TMB-S (Ultra-TMB-S, Research Diagnostics, Flanders, NJ) according to the manufacturer's instructions. The reaction was stopped by adding ΙΟΟμί of 0.18M H2SO4 . The OD was read at 450 nm. Results are expressed as an antibody endpoint titer, determined when the OD value is 3-fold greater than the background value obtained with a same dilution of serum from pre-immune mice.

ELISPOT.

[0151] The day before splenocyte isolation, 70% ethanol-treated MultiScreen-IP opaque 96- well plates (High Protein Binding Immobilon-P membrane, Millipore, Bedford, MA) were coated overnight at ^°4C with ΙΟΟμίΛνβΙΙ of capture IFN-γ antibody, diluted in DPBS as suggested in the murine interferon-gamma ELISPOT kit (Abeam, Cambridge, MA, USA) . After the overnight incubation, the plates were washed three times with 200 PBS/well and blocked with 100 nL/well of 2% skimmed dry milk in PBS for 2 h at 37°C, 5% C0₂. Two weeks after the last boost, the mice were sacrificed and their spleens were removed aseptically. Spleens were minced in culture medium and homogenates were passed through a 100-μιη cell strainer. The cells were centrifuged and red blood cells were removed by incubation for 5 min. at room temperature in ammonium chloride-potassium lysis buffer (150mM NH₄C1, lOmM KHCO₃, O. lmM Na₂EDTA (pH 7.2-7.4)) . Isolated red blood depleted spleen cells were washed twice in PBS and dilute in culture media (RPMI 1640 supplemented with 25 mM Hepes, 2mM L-glutamine, ImM sodium pyruvate, 50μΜ 2- Mercaptoethanol, 10% heat inactivated fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen, Canada). Duplicate samples at 2.5 x 10⁵ cells/well were reactivated with either culture medium alone or with 50 μg/ml of rNP and were cultured for 36h. at 37°C, 5% CO₂. At the end of incubation, the plates were washed manually, 3 times with 200 L well of PBS/0.1% Tween 20. 100 L well of biotinylated anti-mouse IFN- gamma detection antibody in PBS/1 %BSA was added and the plates were incubated for lh.30min. at 37°C, 5% CO₂. Plates were manually washed 3 times with PBS and 100 L/well of streptavidine-alkaline phosphatase conjugated secondary antibody diluted in PBS/1% BSA was added for lh. At 37°C, 5% C02. The plates were washed a final 3 times with PBS/0.1 % Tween 20. Spots were visualized by adding ΙΟΟμί of ready-to-use BCIP/NBT buffer in each well for 2-15 min. Plates were scanned and counted using the ImmunoSpot analyzer (Cellular Technology Ltd., Shaker Heights, OH, USA) to determine the number of spots/well. The images were acquired by the Image Acquisition program (version 4.5) and analysed with the ImmunoSpot program (version 3). The precursor frequency of specific T cells was determined by subtracting the background spots in media alone from the number of spots seen in wells reactivated with NP.

Results

Improvement of the adjuvant property of the PapMV VLPs through an increase in avidity to the antigen.

[0152] The structural characterisation of the PapMV VLPs, PapMV HAV-ANP 1 and PapMV HAV-ANP2 suggest that they are all comparable in size and in structure. However, their avidity for the antigen NP differs. Through immunization of mice with the different conjugates, the ability of the avidity of the adjuvant for the antigen to influence the immune response to the antigen was evaluated. Balb/C mice, 10 per groups, were immunized three times by the subcutaneous route at 2 week intervals. Two weeks after each immunization, serum was harvested and ELISA was performed to measure the humoral response to the NP antigen.

[0153] The IgGl titers appeared to be similar and comparable with each immunization regime and the VLP did not increase the amount of antibody isotype significantly (Fig. 4A). However, the PapMV HAV-ANP2 appeared to significantly improve, by 5 fold, the amount of IgG2a directed to the NP antigen as compared to the other treatments, suggesting that the closer contact of the adjuvant to the antigen demonstrated a benefit (Fig. 4B). Therefore, the ratio between the IgGl/IgG2a ( ^m) is significantly lower with the PapMV HAV- ANP2+NP treatment and shows a strong bias toward a THI response that is indicative of a higher quality of the humoral immune response and indicative of the trigger of a CTL response. To further support this data, an INF-γ ELISPOT against NP protein was performed two weeks after the last boost. Consistent with humoral response, as shown in Fig. 4D, only immunization with the PapMV HAV-ANP2-NP conjugate significantly increased (by almost 8 fold) the number of NP-specific T-cells secreting INF-γ, as compare to NP alone.

EXAMPLE 7: ABILITY OF PAPMV HAV-ANP2 TO PROTECT MICE AGAINST CHALLENGE WITH INFLUENZA VIRUS [0154] The following experiment was performed to determine the ability of PapMV HAV- ANP2 to protect mice against a challenge with influenza virus. In this experiment, mice were immunized as described in Example 6, with recombinant NP protein (NP) with or without 3(^g of the PapMV, or PapMV HAV-ANP2.

Influenza A strain.

[0155] The influenza virus A strain used in this study was A/WSN/33 (H1N1), which was derived from a mouse lung-adapted clinical isolate, A/WSN/33, obtained by serial passage in neonatal mice and brains of adult mice [Stuart-Harris Lancet 1939; 1 :497-9] . The LD50 (Lethal Dose inducing 50% mortality) of this strain was previously evaluated as being approximately 10³ plaque-forming units (pfu) [Abed Antivir Ther 2006; 1 1 (8) :971-6] . Under the experimental conditions described here, the LD50 was estimated to approximately 2.5xl0² plaque-forming units (pfu) as determined in a pilot challenge experiment (data not shown) .

Infection with influenza in mice.

[0156] Balb/C mice were infected intranasally with 50μΙ_^ containing 5 x 10² pfu (1LD50) of influenza A/WSN/33. Mice were monitored daily for clinical symptoms (loss of body weight, abnormal behaviour and ruffled fur) . Deaths were recorded over a period of 14 days. Mice were sacrificed when the total body weight loss reached more than 20% of initial weight. For the virus titration, animals were sacrificed at day 7 and lungs were removed aseptically and stored at -80 °C in 1 ml of sterile PBS.

Viral titration.

[0157] Lungs were homogenized and centrifuged at 2500 rpm/4 °C for 10 min and supernatants were titrated in MDBK cells using a standard plaque assay as described previously [ Abed et al. Antimicrob Agents Chemother 2005 Feb;49(2) :556-9] .

CD8+ T-cell depletion. [0158] For T-cell depletion experiments, mice were injected with O. lmg i.p. of monoclonal antibodies directed to CD8⁺ in vaccinated or immunized mice at day 33 and 35. After depletion, which was validated by FACS, mice were challenged as before on day 36.

Statistical analysis.

[0159] Data were analysed with parametric (or non parametric when the variance were significantly different) ANOVA test. Student's or Tukey's post tests were used to compare differences (antibody titers, ELISPOT, Weight losses, symptoms and viral titers) among groups of mice. Differences among survival curves were analysed by Kaplan-Meier survival analysis. Values of *p < 0.05, **p < 0.01 , ***p < 0.001 were considered statistically significant. Statistical analyses were performed with GraphPad PRISM 5.01.

Results:

Protective effect of the generated immune response

[0160] The improvement of the THI response to NP using the PapMV HAV-ANP2 was convincing and was expected to provide protection to an influenza challenge. To confirm this hypothesis, Balb/C mice were immunized with NP alone, NP + PapMV VLPs and NP + PapMV HAV-ANP2 using a protocol similar to that described in Example 6, and the capacity of the vaccinated animals to be protected to a challenge with the influenza mouse adapted strain A/WSN/33 H1N1 was tested. The increased immunity generated by the PapMV HAV- ANP2 adjuvant was translated in a decreased weight losses and a significant improvement of the symptoms observed on the animals vaccinated with the conjugated vaccine NP+ PapMV HAV-ANP 2 (Fig. 5A and B) seven days after the challenge. Mice were sacrificed at day 7 and the titers of WSN/33 strain were evaluated to measure the clearance of the virus in the animals. As expected and consistent with previous observations, the animals vaccinated with the NP+ PapMV HAV-ANP2 showed a significant reduction in the viral load as compared to the other treatments since more than half of the animals treated with this vaccination regimen had almost completely cleared the virus from their lungs (Fig. 5C). To confirm this result this experiment was repeated and the survival of the animals (10 per group) followed 14 days after challenge. Consistent with previous results, the IgGl titers to NP were similar with all the treatments (Fig 6A) , but the IgG2a titers was significantly improved in the group immunized with the PapMV HAV-ANP2+NP as compared to NP alone (Fig 6B). As expected, antibodies directed to PapMV CP were comparable in mice receiving the adjuvanted vaccines (Fig 6C) . Interestingly, the IgG2a titers against NP were found to be always higher in the animals vaccinated with the PapMV HAV-ANP2+NP vaccine (Fig 6D).

[0161] PapMV HAV-ANP2+NP was the best treatment of those tested and provided 40% survival as compared to non-vaccinated mice or mice immunized with NP alone that did not survive the challenge. The addition of WT PapMV VLP to NP was less efficient than the treatment PapMV HAV-ANP2+NP and showed only 20% survival (Fig. 5D) . Finally, in order to evaluate the contribution of the CD8+ mediated immune response to the observed protection, CD8+ cells were depleted using a monoclonal antibody directed to CD8 in mice that were previously immunized three times with the PapMV HAV-ANP2+NP regimen. As expected, the depletion of CD8⁺ cells erased the benefits of the vaccination with PapMV HAV-ANP2+NP suggesting that the protection that observed was caused by the CD8⁺ mediated immune response.

Discussion

[0162] The data shown in Examples 1 to 7 demonstrate the ability of the native PapMV VLP and an engineered form harbouring a high avidity peptide to NP to its surface (high avidity VLP; HAV) to improve the immune response directed to the influenza NP protein. Multimerisation of the affinity peptides for NP at the surface of HAV improves its affinity for the NP antigen which increases the efficacy of protection against an influenza challenge.

[0163] The recent circulation of the highly pathogenic H5N1 influenza virus in some human populations and the appearance of a new highly contagious HlNl of swine origin triggered a surge of interest in the development of new vaccine strategies that are not based on protective antibodies directed to HA and NA proteins that are highly variable. The structural protein NP is one of the most attractive targets for the development of a so-called universal vaccine because the amino acid sequence of this protein is highly conserved through all the strains of influenza. The antigen NP in DNA form alone [Ulmer et al. Science 1993 Mar

19;259(5102) : 1745-9; Macklin et al. J Virol 1998 Feb;72 (2) : 1491-6; Epstein et al. Emerg

Infect Dis 2002 Aug;8(8) :796-801 ; Luo et al. J Virol Methods 2008 Dec; 154(l-2) : 121-7] , in viral vectors [Andrew et al. Scand J Immunol 1987 Jan;25 (l) :21-8; Webster et al. Vaccine 1991 May;9(5) :303-8; Wesley et al. Vaccine 2004 Sep 3;22 (25-26) :3427-34; Epstein et al. Vaccine 2005 Nov 16;23(46-47) :5404-10; Altstein et al. Arch Virol 2006 May; 151 (5) :921- 31 ; Roy et al. Vaccine 2007 Sep 28;25(39-40) :6845-51 ; Barefoot et al. Clin Vaccine Immunol 2009 Apr; 16(4) :488-98] or as a soluble protein [Carragher et al. J Immunol 2008 Sep 15; 181 (6) :4168-76; Tite et al. Immunology 1990 Oct;71 (2) :202-7; Tamura et al. J Immunol 1996 May 15; 156(10) :3892-900; Guo et al. Arch Virol 2010 Jul 22, Wraith et al. J Gen Virol 1987 Feb;68 ( Pt 2) :433-40] was shown previously to confer protection in homologous and heterologous challenges. All of the studies that used soluble protein as source of NP required an adjuvant to some extent to stimulate higher immunity and confer protection. Many of these potent adjuvants, particularly LPS, cause considerable side effects such as toxicity or inflammation and are not allowed for human use [Aguilar et al. Vaccine 2007 May 10;25 (19) :3752-62] . Here, a new form of adjuvant derived from PapMV VLPs conjugated to soluble recombinant NP free of LPS contamination was tested. It is well known that VLPs like PapMV, are capable of inducing strong cellular and humoral immune response [Grgacic Methods 2006 Sep;40(l) :60-5] . Effectively, B cells are efficiently activated by repetitive structures like PapMV VLPs which lead to cross-linking of B cell receptors on the cell surface [ Denis et al. Virology 2007 Jun 20;363(l) :59-68, Bachmann et al. Science 1993 Nov 26;262(5138) : 1448-51] . Thus, it is possible to render weak target antigens more immunogenic for B cells by presenting them in a more organised and repetitive fashion. PapMV VLPs are also known to be cross-presented on MHC-I through TAP-independent pathway [29] . Thus, it is also possible to trigger cellular response by more efficient antigen presentation. However, insertion of large epitopes into immunodominant region of VLPs often interferes with their correct conformation and interferes with formation of the VLP. Previous studies have circumvented this problem by introducing linker sequences which allows covalent linkage of large antigen to VLP carrier by using chemical cross-linkers [Jegerlehner et al. Vaccine 2002 Aug 19;20(25-26) :3104-12] . Others use the high specific interaction of biotin/streptavidin protein to increase the avidity of VLP to target antigen [Chackerian et al. J Clin Invest 2001 Aug; 108(3) :415-23] . The approach described in Examples 1 to 7 was to improve the avidity of the PapMV VLP adjuvant through the fusion of an affinity peptide to the NP antigen to the surface of the VLP. The resulting molecule showed an improved avidity for NP which, consequently, improved the immune response directed to the antigen.

[0164] The HAV-ANP2 was more efficient than the PapMV VLP in increasing the IgG2a and the cellular response to the NP antigen. IgG2a is a more effective class of antibody in preventing intracellular virus replication since it is more efficient in complement activation and antibody-dependant cellular immunity [Coutelier et al. J Exp Med 1987 Jan 1 ; 165(1) :64- 9; Hocart et al. J Gen Virol 1989 Sep;70 ( Pt 9) :2439-48] . Some authors have reported that non-neutralizing antibodies against NP might have a role in protection against influenza virus [Carragher et al. J Immunol 2008 Sep 15; 181 (6) :4168-76; Zheng et al. J Immunol 2007 Nov 1 ; 179(9) :6153-9] . Here, with the CD8+ depletion experiment described in Example 7, it was confirmed as some previous reports [Epstein et al. J Immunol 1997 Feb 1 ; 158(3) : 1222-30, Stitz et al. J Gen Virol 1990 May;71 ( Pt 5) : 1169-79; Epstein et al. J Immunol 1998 Jan 1 ; 160(1) :322-7] have demonstrated before, that serum antibodies to NP do not significantly contribute to protective effect but rather the cellular response was important to the observed protection induced by the PapMV HAV-ANP2+NP vaccine.

[0165] The NP protein is an important target antigen for influenza A virus cross-reactive CTL [ Townsend et al. J Exp Med 1984 Aug 1 ; 160(2) :552-63; Yewdell et al. Proc Natl Acad Sci U S A 1985 Mar;82 (6) : 1785-9; McMichael et al. J Gen Virol 1986 Apr;67 ( Pt 4) :719-26; Chen et al. Immunity 2000 Jan; 12(1) :83-93] . The protective effect of the HAV adjuvanted NP vaccine described herein is characterized by a more rapid, reduction in viral titers, viral clearance and reduction in morbidity and mortality, all features characteristic of heterosubtypic immunity [ Epstein et al. Expert Rev Anti Infect Ther 2003 Dec; l (4) :627-38] . Moreover, the mechanism of immune protection generated by the PapMV HAV-ANP2+NP vaccine can be explained by the proliferation of CTLs specific to NP [McMichael Curr Top Microbiol Immunol 1994; 189:75-91] . Engineered PapMV VLPs fused to CTL epitopes were previously showed to be efficient in improving the loading of the MHC class I with the CTL epitope (Leclerc et al. J Virol 2007 Feb;81 (3) : 1319-26) . It is likely that the attachment of the HAV to NP also triggers, as for the fusion directly on the PapMV CP, a similar mechanism of cross presentation. [0166] It has been recently shown that intranasal administration of soluble NP protein in combination with cholera toxin B subunit adjuvant can confer protection to homologous and heterologous viruses by inducing mucosal and cell-mediated immunity [Guo et al. Arch Virol 2010 Jul 22] . Because NP is an highly conserved target through all the strains of influenza, it is likely that the PapMV HAV-ANP2+NP vaccine could also provide a benefit in protecting against heterosubtypic strains of the virus.

EXAMPLE 8: PREPARATION AND ANALYSIS OF PAPMV VLPS

Expression and purification of recombinant proteins PapMV-CP

[0167] Expression and purification of PapMV-CP in E. coli were performed as described previously (Denis et al Vaccine 2008 Jun 25;26(27-28) :3395-403). The lipopolysaccharide (LPS) levels in the purified proteins was evaluated with the Limulus test according to the manufacturer's instructions (Cambrex, Walkersville, MD) . In all cases, the LPS contamination was less than 5 endotoxin (EU) units/mg of protein.

Morphological evaluation of PapMV VLPs.

[0168] Morphological evaluation of VLPs was carried out by electron microscopy as previously described (Tremblay et al. Febs J 2006 Jan; 273(1) : 14-25). VLPs were observed on a JEOL -1010 (Tokyo, Japan) transmission electron microscope. Images were acquired with a Bioscan Camera from Gatan (Warrendale, PA, USA) and analysed with the Gatan Digital Micrograph acquisition software. VLP content of preparations was evaluated by gel filtration chromatography using Superdex 200 10/300 (GE Healthcare, Baie d'Urfe, Canada) as previously described (Denis et al Vaccine 2008 Jun 25;26(27-28) :3395-403) . The dynamic light scattering (DLS) , was also used to evaluate the homogeneity of the VLP population and its size. VLPs were diluted at 250μg/ml in PBS and size measurements were performed with a Zetasizer Nano ZS (Malvern, Worcestershire, UK) . Particle size distributions were evaluated from intensity measurements.

Results [0169] PapMV VLPs were produced using the bacterial expression vector pET-3D (Novagen) as described previously (Tremblay et al. Febs J 2006 Jan; 273(1) : 14-25,; Denis et al. Virology 2007 Jun 20;363(l) :59-68; Denis et al Vaccine 2008 Jun 25;26(27-28) :3395- 403). The purification profile is shown in (Fig. 7). The PapMV VLP preparation was homogenous as demonstrated by SDS-PAGE showing only one protein of 30kDa (Fig. 7A) that was able to self assemble as PapMV VLPs (Fig. 7B) that show an average length of 70nm as measured by dynamic light scattering (DLS) (Fig. 7C).

EXAMPLE 9: ABILITY OF PAPMV VLPs TO BE TRANSPORTED TO

SECONDARY LYMPHOID ORGANS

In Vivo fluorescent Imaging

[0170] For in vivo fluorescent imaging, 25μg of Alexa@680 (Invitrogen, Burlington, On, Canada) stained VLPs (0.34 M. of Alexa@680 by M. of PapMV VLPs) were injected in the footpad of 3 anesthetized mice. Three other mice were injected with an equivalent quantity of Alexa@680 staining as negative control. The images were gathered with an IVIS 200 imaging system (Xenogen, Alameda, CA, USA) at 24, 48 and 72 hours. The data are represented as pseudocolor images indicating fluorescence intensity (red and yellow, most intense) , which were superimposed over gray-scale reference photographs.

[0171] It has been previously reported that PapMV VLPs alone or fused to a peptide antigen are immunogenic (Denis et al. Virology 2007 Jun 20;363(l) :59-68; Denis et al Vaccine 2008 Jun 25;26(27-28) :3395-403) and taken up by dendritic cells (Lacasse et al, I Virol. 2008 Jan;82(2) :785-94. Epub 2007 Nov 7) . To illustrate the speed of capture of the PapMV VLPs by the immune cells, labelled VLPs were injected in the foot pad of mice. It was observed that the proximal propliteal lymph node became fluorescent 24 hours after injection (Fig. 8A). The signal progressively declined 48 and 72 hours suggesting that the PapMV VLPs were rapidly degraded.

EXAMPLE 10: ABILITY OF PAPMV VLPS TO INDUCE VARIOUS CYTOKINES AND CHEMOKINES Evaluation of cytokine and chemokine profile.

[0172] To evaluate the cytokines and chemokines profile generated following PapMV VLPs immunization, 2 groups of 5 BALB/c mouse were injected with 3C^g of PapMV VLPs once or twice at 2 week intervals. Two weeks after the last boost (both groups were synchronized), the mice were sacrificed and the spleens were removed aseptically. Splenocytes, 2.5 x 10⁵ cells/well were reactivated with either culture medium alone or with 100 μg/ml of PapMV VLPs were cultured for 36h at 37°C. The concentration of cytokines and chemokines were evaluated with MILLIPLEX MAP Mouse Cytokine/Chemokine - Premixed 22 Plex (Millipore, Billerica, MA, USA) for Luminex® xMAP® platform. Measurements were performed with a Luminex 100IS liquichip workstation (Qiagen, Canada).

[0173] To characterise the type of immune response induced by immunization with PapMV VLPs, the cytokine/chemokine profile secreted by spleen cells was evaluated following one or two subcutaneous injections in the back neck of the animals. Reactivation of spleen cells of mice immunized only once led to the secretion of MIP-Ια and KC (Fig. 8B) . Lower but still significant amounts of IL-6, G-CSF, TNF-a, IL-2, RANTES, MCP- 1, IL- la, 11-5, INF-γ and IL- 17 were also measured. Two immunizations led to an increase of MIP- la, KC levels followed by an abundant secretion of IL-2, 5 and 6 secretion (Fig. 8C). Lower but significant levels of IL- 13, G-CSF, GM-CSF, INF-γ, 11- 10, IL-la, RANTES, MCP-1 , IL- 17, TNF-a and 11-4 were also detected. This result suggests that PapMV VLPs are efficiently perceived by the immune system and trigger a balanced THI and TH2 cytokine profile. This result also indicates that PapMV VLP can be considered a pathogen associated molecular pattern (PAMP) that is recognized by the immune system as a danger signal. Therefore, PapMV VLPs show excellent potential as an adjuvant for improvement of the flu vaccines.

[0174] The data shown in Examples 9 and 10 demonstrate that PapMV VLPs can be used as an efficient adjuvant that is readily recognised by the immune cells that transport the molecule rapidly to secondary lymphoid organs, where it is degraded. It has been previously shown that antigen-presenting cells (APCs) are able to uptake PapMV VLPs in vivo and which leads to their maturation (Denis et al. Virology 2007 Jun 20;363(l) :59-68, Lacasse et al, J Virol. 2008 Jan;82(2) :785-94. Epub 2007 Nov 7). It has also been shown that PapMV VLPs induce an active secretion of MIP- la and KC after one or two immunizations of PapMV VLPs. MIP-la (CCL3) is a chemotactic and pro-inflammatory chemokine that is produced by macrophages, dendritic cells and lymphocytes (Maurer and Von Stebut Int J Biochem Cell Biol. 2004 Oct;36(10) : 1882-6) . This chemokine family is crucial for T-cell chemotaxis from the circulation to inflamed tissue and plays an important role in the regulation of transendothelial migration of monocytes, DCs and NK cells (Maurer and Von Stebut Int J Biochem Cell Biol. 2004 Oct;36(10) : 1882-6). In addition, CCL3 and its receptor CCR5 promote THI skewing cytokine profiles (Andres et al. J Immunol. 2000 Jun 15; 164 (12) :6303- 12, Luther and Cyster Nat Immunol. 2001 Feb;2 (2) : 102-7) . PapMV VLPs reactivated splenocytes also induced the secretion of KC (for keratinocyte chemoattractant, also designated N51 in the murine system) , a rodent a-chemokine related to the human chemokine interleukin-8 (Tani et al. J Clin Invest. 1996 Jul 15;98(2) :529-39, Bozic et al. J Immunol. 1995 Jun 1 ; 154 (1 1) :6048-57). KC stimulates chemotaxis specifically of neutrophils, which exit rapidly from the circulation to provide the first line of cellular defense against invading pathogens. The cytokine/chemokine profile shown here, stimulated after only one injection of PapMV VLPs, suggests that immune cells, presumably APCs, could induce the recruitment of lymphocytes following secretion of MIP- la and KC. It has also been suggested that following this recruitment, APCs are able to cross-present CTL epitopes and induce proliferation of specific CD8+ (Leclerc et al. J Virol 2007 Feb; 81 (3) : 1319-26). After the second immunization of PapMV VLPs, an increase in secretion of 11-6 and IL-5 (T_fK-like cyokine) and IL-2 (Tni-like cytokine) was observed, indicating an activation of a THI TH2 mixed specific T-cell response. IL-6 augments immunoglobulin production by B- cells and enhances B-cell growth and differentiation (Van Damme Eur J Biochem. 1987 Nov 2; 168(3) :543-50) and synergizes with IL- 1 in augmenting antigen presentation (Kupper et al. 1988) . IL-5 is an interleukin produced by TH2 cells and mast cells. Its acts as a growth and differentiation factor for both B cells and eosinophils (Adachi and Alam, Am J Physiol. 1998 Sep;275(3 Pt l) :C623-33). IL-5 is known to enhance several functions of murine B cells, including immunoglobulin production, growth, and differentiation (Takatsu et al. Adv Immunol. 1994;57: 145-90). This cytokine is also the main regulator of eosinopoiesis, eosinophil maturation and activation (Takatsu et al. Adv Immunol. 2009; 101 : 191-236) . Finally, IL-2 is an interleukin secreted by THI cells (Mosmann et al J Immunol. 1986 Apr 1 ; 136(7) :2348-57). Its functions are to stimulates the growth, differentiation and survival of antigen-selected cytotoxic T cells via the activation of the expression of specific genes (Malek 2008) and is necessary for the development of T cell immunologic memory. Therefore, PapMV VLPs are powerful inducers of the immune response and are recognized by the immune system as a pathogen associated molecular pattern (PAMP) as previously suggested (Lacasse et al, J Virol. 2008 Jan;82(2) :785-94. Epub 2007 Nov 7, Acosta Ramirez et al, Immunology. 2008 Jun; 124(2) : 186-97. Epub 2007 Dec 7).

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[0164] 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. An affinity-conjugated nucleoprotein-PapMV virus-like particle system comprising an influenza nucleoprotein (NP) and a virus-like particle (VLP) derived from PapMV coat protein, said PapMV coat protein modified by the addition of one or more peptides capable of specifically binding to influenza NP, wherein said system is capable of inducing an immune response in an animal.

2. The system of claim 1 , wherein the one or more peptides comprise the sequence set forth in SEQ ID NO:4.

3. The system of claim 1 , wherein the one or more peptides comprise the sequence set forth in SEQ ID NO:6.

4. The system of any one of claims 1 to 3, wherein said immune response comprises a humoral response.

5. The system of any one of claims 1 to 4 wherein said immune response comprises a cellular response.

6. The system according to any one of claims 1 to 5, wherein said animal is a mammal.

7. The system according to any one of claims 1 to 5, wherein said animal is a human.

8. The system according to any one of claims 1 to 5, wherein said animal is a non-human animal.

9. An immunogenic composition comprising the affinity-conjugated nucleoprotein-PapMV virus-like particle system according to any one of claims 1 to 8, and a pharmaceutically acceptable carrier.

10. A method of inducing an immune response to influenza nucleoprotein in an animal comprising administering to said animal an effective amount of the affinity-conjugated nucleoprotein-PapMV virus-like particle system according to any one of claims 1 to 8.

11. The method according to claim 10, wherein said immune response comprises the production of antibodies.

12. The method according to claim 10 or 11 , wherein said immune response comprises the induction of a cytotoxic T lymphocyte (CTL) response.

13. The method according to any one of claims 10 to 12, wherein said system is administered by injection.

14. The method according to any one of claims 10 to 13, wherein said animal is a mammal.

15. The method according to any one of claims 10 to 13, wherein said animal is a human.

16. The method according to any one of claims 10 to 13, wherein said animal is a non-human animal.

17. The method according to any one of claims 10 to 16 wherein said method further comprises administering to said animal a booster dose of the system.

18. A method of preventing or treating influenza in an animal, said method comprising administering to said animal an effective amount of the affinity-conjugated nucleoprotein- PapMV virus-like particle system according to any one of claims 1 to 8.

19. The method according to claim 18, wherein said system is administered by injection.

20. The method according to claim 18 or 19, wherein said animal is a mammal.

21. The method according to claim 18 or 19, wherein said animal is a human.

22. The method according to claim 18 or 19, wherein said animal is a non-human animal.

23. The method according to any one of claims 18 to 22, wherein said method further comprises administering to said animal a booster dose of the system.

24. Use of an effective amount of the affinity-conjugated nucleoprotein-PapMV virus-like particle system according to any one of claims 1 to 8, to induce an immune response to influenza nucleoprotein in an animal in need thereof.

25. The use according to claim 24, wherein said immune response comprises the production of antibodies.

26. The use according to claim 24 or 26, wherein said immune response comprises the induction of a cytotoxic T lymphocyte (CTL) response.

27. The use according to any one of claims 24 to 26, wherein said system is formulated for administration by injection.

28. The use according to any one of claims 24 to 27, wherein said animal is a mammal.

29. The use according to any one of claims 24 to 27, wherein said animal is a human.

30. The use according to any one of claims 24 to 27, wherein said animal is a non-human animal.

31. The use according to any one of claims 24 to 30, wherein said use further comprises a booster dose of the system.

32. Use of an effective amount of the affinity-conjugated nucleoprotein-PapMV virus-like particle system according to any one of claims 1 to 8, to prevent or treat influenza in an animal in need thereof.

33. The use according to claim 32, wherein said system is formulated for administration by injection.

34. The use according to claim 32 or 33, wherein said animal is a mammal.

35. The use according to claim 32 or 33, wherein said animal is a human.

36. The use according to claim 32 or 33, wherein said animal is a non-human animal.

37. The use according to any one of claims 32 to 36, wherein said use further comprises a booster dose of the system.

38. Use of the affinity-conjugated nucleoprotein-PapMV virus-like particle system of any one of claims 1 to 8, in the manufacture of a medicament.

39. A method of preparing an immunogenic composition comprising admixing influenza nucleoprotein with a papaya mosaic virus (PapMV) virus-like particle (VLP) derived from PapMV coat protein, said PapMV VLP comprising one or more peptides attached to coat proteins of said PapMV VLP, said peptides capable of specifically binding to influenza nucleoprotein.

40. The method of claim 39, wherein the one or more peptides comprise the sequence set forth in SEQ ID NO:4.

41. The method of claim 39, wherein the one or more peptides comprise the sequence set forth in SEQ ID NO:6.

42. An immunogenic composition prepared by the method according to any one of claims 39 to 41.

43. A fusion protein comprising a papaya mosaic virus (PapMV) coat protein fused to one or more peptides capable of specifically binding to influenza nucleoprotein.

44. The fusion protein of claim 43, wherein the peptide comprises the sequence set forth in SEQ ID NO:4.

45. The fusion protein of claim 43, wherein the peptide comprises the sequence set forth in SEQ ID NO:6.

46. An isolated polynucleotide encoding the fusion protein according to any one of claims 43 to 45. Use of the fusion protein according to any one of claims 43 to 45, or the polynucleotide according to claim 46, to prepare a virus-like particle.