HK1211473B - Vaccine of conferring a protective immune response to norovirus - Google Patents

Description

Vaccine conferring a protective immune response against norovirus

The present application is a divisional application of an invention application having an application date of 2008/9/18, a chinese application number of 200880116659.6, and an invention name of "vaccine for imparting protective immune response against norovirus".

Cross Reference to Related Applications

Priority of U.S. provisional application No.60/973,389 filed on 18 th 9 th 2007 and U.S. provisional application No.60/986,826 filed on 9 th 2007, are hereby incorporated by reference in their entirety.

Technical Field

The present application is in the field of vaccines, in particular vaccines against norovirus (norovirus). In addition, the invention relates to methods of preparing vaccine compositions and methods of inducing protective immune responses.

Statement regarding government support

The invention was made with government support from the U.S. army medical research and materials commander under contract number W81 XWH-05-C-0135. The government may have certain rights in the invention.

Background

Norovirus is an incurable human calicivirus that has become the single most important cause of an epidemic outbreak of nonbacterial gastroenteritis (Glass et al, 2000; Hardy et al, 1999). Prior to the development of sensitive molecular diagnostic assays, the clinical significance of norovirus was inadequately valued. The cloning of the genogroup I (genogroup I) Norwalk Virus (NV) genome and the production of virus-like particles (VLPs) from recombinant baculovirus expression systems has led to the development of assays that reveal widespread norovirus infection (Jiang et al.1990; 1992).

Norovirus is a single-stranded, positive-sense RNA virus that contains an unfragmented RNA genome. The viral genome encodes three open reading frames, the latter two of which specify the production of major capsid and minor structural proteins, respectively (Glass et al 2000). When expressed at high levels in eukaryotic expression systems, NV and some other norovirus capsid proteins self-assemble into VLPs that structurally mimic the native norovirus virions (virion). When viewed by transmission electron microscopy, VLPs are morphologically indistinguishable from infectious virions isolated from human stool samples.

The immune response against norovirus is complex and the association of protection (corrlate) is now being elucidated. Human volunteer studies conducted with native viruses demonstrated that mucosal-derived memory immune responses provide short-term protection from infection and suggest that vaccine-mediated protection is feasible (Lindesmith et al 2003; Parrio et al 1997; Wyattet al, 1974).

Although norovirus cannot be cultured in vitro due to the availability of VLPs and their ability to be produced in large quantities, considerable progress has been made in defining the antigen topology and structural topology of norovirus capsids. VLPs retain the authentic conformation of the viral capsid protein (antigenic conformation), but lack infectious genetic material. As a result, VLPs mimic the functional interaction of the virus with cellular receptors, thereby eliciting an appropriate host immune response, but lack the ability to reproduce or cause infection. In conjunction with NIH, Baylor College of Medicine studied the humoral, mucosal and cellular immune responses against NV VLPs in human volunteers in an academic, investigator-initiated phase I clinical trial. Oral administration of VLPs is safe and immunogenic in healthy adults (Ball et al 1999; tag et al 2003). Preclinical experiments in animal models have demonstrated, at other academic centers, an enhancement of the immune response against VLPs when administered intranasally with bacterial exotoxin adjuvants (Guerrero et al 2001; nicolilier-Jamot et al 2004; Periwal et al 2003; Souza et al (2007) Vaccine, doi:10.1016/j. vaccine.2007.09.040). However, no studies have reported that protective immunity against norovirus can be achieved using any norovirus vaccine.

Summary of The Invention

The present invention provides methods of inducing protective immunity against norovirus infection in a subject (particularly a human subject) comprising administering a vaccine comprising at least one norovirus antigen. In one embodiment, the antigen is a norovirus Virus Like Particle (VLP). The vaccine used in the method of the invention may further comprise one or more adjuvants. The norovirus VLPs may be selected from a genogroup I or genogroup II virus or mixtures thereof. In one embodiment, the vaccine comprises norovirus VLPs at a concentration of about 0.01% to about 80% (by weight). In another embodiment, the vaccine comprises a norovirus VLP dose of about 1 μ g to about 100mg per dose.

In some embodiments, the vaccine further comprises a delivery agent that functions to enhance antigen uptake, provide a depot effect, prolong retention time of the antigen at the delivery site, or enhance the immune response via relaxation of cellular tight junctions at the delivery site. The delivery agent may be a bioadhesive, preferably a mucoadhesive selected from the group consisting of: dermatan sulfate, chondroitin, pectin, mucins, alginates, cross-linked derivatives of polyacrylic acid, polyvinyl alcohol, polyvinyl pyrrolidone, polysaccharides, hydroxypropyl methylcellulose, lectins, pilus/fimbrial proteins, and carboxymethyl cellulose. Preferably, the mucoadhesive is a polysaccharide. Most preferably, the mucoadhesive is chitosan or a mixture containing chitosan, such as a chitosan salt or chitosan base.

In other embodiments, the vaccine comprises an adjuvant. The adjuvant may be selected from the group consisting of: toll-like receptor (TLR) agonists, monophosphoryl lipid ASynthetic lipid a, lipid a mimetics or analogues, aluminium salts, cytokines, saponins, Muramyl Dipeptide (MDP) derivatives, CpG oligomers, Lipopolysaccharides (LPS) of gram negative bacteria, polyphosphazenes, emulsions, virosomes, Cochleates, poly (lactide-co-glycolide) (PLG) microparticles, poloxamer particles, microparticles, endotoxins (e.g. bacterial endotoxins) and liposomes. Preferably, the adjuvant is toll-likeReceptor (TLR) agonists. More preferably, the adjuvant is

The methods of the invention comprise administering norovirus vaccines formulated as liquids or dry powders. The dry powder formulation may contain an average particle size of about 10 to about 500 microns in diameter. Routes suitable for administration of the vaccine include mucosal, intramuscular, intravenous, sub-epidermal (subeutaneous), intradermal (intradermal), subdermal (subdermal), or transdermal (transdermal). In particular, the route of administration may be intramuscular or mucosal, preferably mucosal routes of administration, including intranasal, oral, or vaginal routes of administration. In another embodiment, the vaccine is formulated as a nasal spray, nasal drops, or dry powder, wherein the vaccine is administered by rapid deposition within the nasal passages from a device containing the vaccine in proximity to the nasal passages. In another embodiment, the vaccine is administered to one or both nostrils.

The present invention relates to the following items.

1. A method of eliciting protective immunity to norovirus infection in a human comprising administering to the human a vaccine comprising norovirus Virus Like Particles (VLPs) and at least one adjuvant.

2. The method of item 1, wherein the norovirus VLPs are selected from the group consisting of: norovirus genogroup I and genogroup II strains.

3. The method of item 1, wherein the norovirus VLPs are monovalent VLPs.

4. The method of item 1, wherein the norovirus VLPs are multivalent VLPs.

5. The method of item 1, wherein the vaccine comprises a second type of norovirus VLPs.

6. The method of item 5, wherein said first and second norovirus VLPs are monovalent VLPs from different gene groups.

7. The method of item 6, wherein said first norovirus VLPs are norwalk virus VLPs and said second norovirus VLPs are Huston virus VLPs.

8. The method of clause 1, wherein the vaccine further comprises a delivery agent.

9. The method of clause 8, wherein the delivery agent is a bioadhesive.

10. The method of item 9, wherein said bioadhesive is a mucoadhesive.

11. The method of item 10, wherein the mucoadhesive is selected from the group consisting of: dermatan sulfate, chondroitin, pectin, mucins, alginates, cross-linked derivatives of polyacrylic acid, polyvinyl alcohol, polyvinyl pyrrolidone, polysaccharides, hydroxypropyl methylcellulose, lectins, pilus/fimbrial proteins, and carboxymethyl cellulose.

12. The method of item 11, wherein the mucoadhesive is a polysaccharide.

13. The method of item 12, wherein the polysaccharide is chitosan, a chitosan salt, or a chitosan base.

14. The method of item 1, wherein the adjuvant is selected from the group consisting of: toll-like receptor (TLR) agonists, monophosphoryl lipid a (mpl), synthetic lipid a, lipid a mimetics or analogs, aluminum salts, cytokines, saponins, Muramyl Dipeptide (MDP) derivatives, CpG oligomers, Lipopolysaccharides (LPS) of gram-negative bacteria, polyphosphazenes, emulsions, virosomes, Cochleates, poly (lactide-co-glycolide) (PLG) microparticles, poloxamer particles, microparticles, and liposomes.

15. The method of item 14, wherein the adjuvant is a toll-like receptor (TLR) agonist.

16. The method of item 14, wherein the adjuvant is MPL.

17. The method of item 1, wherein the adjuvant is not a toxin adjuvant.

18. The method of item 1, wherein the vaccine is in a powder formulation.

19. The method of item 1, wherein the vaccine is in a liquid formulation.

20. The method of item 1, wherein the vaccine is administered to a human by a route selected from the group consisting of: mucosal, intranasal, intramuscular, intravenous, subcuticular, intradermal, subdermal, and transdermal routes of administration.

21. The method of item 20, wherein the vaccine is administered intranasally.

22. The method of item 21, wherein the vaccine is administered to the nasal mucosa by rapid deposition within the nasal passages from one or more devices comprising the vaccine near the nasal passages.

23. The method of item 22, wherein the vaccine is administered to one or both nostrils.

24. The method of item 1, wherein the norovirus VLPs are present at a concentration of about 0.01% (w/w) to about 80% (w/w).

25. The method of item 1, wherein the norovirus VLPs are present in an amount of from about 1 μ g to about 100mg per dose.

26. The method of item 25, wherein the norovirus VLPs are from about 1 μ g to about 100 μ g per dose.

27. The method of item 1, wherein the vaccine confers protection from one or more symptoms of norovirus infection.

Brief Description of Drawings

Figure 1 shows that Norwalk Virus (NV) -specific IgG was elicited in rabbits immunized with dry powder VLPs. The intranasal route of administration was given 3 times to rabbits on days 1, 22 and 43 (arrows) with 50 μ g NV-VLP +50 μ g MPL. Sera from each rabbit were tested for NV-VLP specific IgG by ELISA on the indicated days. Only the rabbits vaccinated with the VLP vaccine had NV-VLP specific IgG, whereas no antigen specific antibodies were detected in the untreated and placebo treated groups (data not shown). The arithmetic mean of the responses is shown and expressed in U/mL (1U-1. mu.g). Bars indicate standard error of mean.

Figure 2 depicts the results of an ELISA assay measuring serum IgA (panel a) and IgG (panel B) levels from human volunteers immunized with a control (adjuvant/vehicle) or a vaccine formulation containing one of three doses (5,15,50 μ g) of norwalk virus VLPs. The geometric mean fold increase in anti-VLP titer at 35 days (day 56) after the second immunization is shown for each dose level. Volunteers received immunizations on day 0 and day 21.

Figure 3 shows the levels of IgA (panel a) and IgG (panel B) Antibody Secreting Cells (ASC) in human volunteers receiving a vaccine formulation containing a 50 μ g dose of norwalk virus VLPs or a control (adjuvant/excipient). Geometric mean ASC (GMN) of every 106 Peripheral Blood Mononuclear Cells (PBMCs) was plotted against the study days (day 7 or day 28), specifically seven days post-immunization. Volunteers received immunizations on day 0 and day 21.

Detailed Description

The present invention relates to methods of eliciting protective immunity to norovirus infection in a subject. In particular, the present invention provides a method of administering a vaccine comprising norovirus VLPs and at least one adjuvant to a human, wherein the vaccine confers protection from at least one symptom of norovirus infection. Additionally or alternatively, the vaccine may further comprise at least one delivery agent.

Norovirus antigens

The present invention provides compositions comprising one or more norovirus antigens. "norovirus" (NOR) and grammatical equivalents herein refer to members of the norovirus genus of the Caliciviridae family (Caliciviridae). In some embodiments, norovirus may include a group of related, plus-sense, single-stranded RNA, non-enveloped viruses, which may be infectious to human or non-human mammalian species. In some embodiments, the norovirus is capable of causing acute gastroenteritis in humans. Norovirus may also be referred to as Small Round Structure Virus (SRSV), which has a defined surface structure or rough edges when viewed by electron microscopy. Norovirus includes at least four gene groups (GI-IV) defined by nucleic acid and amino acid sequences, comprising 15 genetic clusters. The major gene groups are GI and GII. GIII and GIV have been proposed but have not been generally accepted. Representatives of GIII are the Yena (Jena) strain of cattle. GIV currently contains one virus, Alphatron. For further description of norovirus see Vinjeet al.J.Clin.Micro.41:1423-1433 (2003). "norovirus" also refers herein to recombinant norovirus virus-like particles (rNOR VLPs). In some embodiments, at least recombinant expression of the norovirus capsid protein encoded by ORF2 in a cell (e.g., using a baculovirus vector in Sf9 cells) results in spontaneous self-assembly of the capsid protein into VLPs. VLPs are structurally similar to norovirus, but lack the viral RNA genome and are therefore not infectious. Thus, "norovirus" includes virions, which can be infectious or non-infectious particles, including defective particles.

Non-limiting examples of norovirus include Norwalk virus (Norwalk virus, NV, GenBank M87661, NP)₀₅₆₈₂₁) South Ampelon virus (south Ampton virus, SHV, GenBank L07418), Saurovirus (DesertShield virus, DSV, U04469), Selenavirus (Hesse virus, HSV), Chiba virus (Chiba virus, CHV, GenBank AB042808), Hawaii virus (Hawaii virus, HV, GenBank U07611), snow mountain virus (SnowMount virus, SMV, GenBank U70059), Toronto virus (Toronto virus, TV, Leite et al, Arthrol Virol.004865: 865), Bristol virus (Bristol virus, Yak virus), Naja virus (JV virus, BV, AJ01099), Maryland virus (Marylvirus, MV, AY032605), Setarian virus (Genbank virus, Squarry virus), Securi virus (Squarry virus), Haemarrh virus (Sjo virus, Squal, Skul virus, Sjogren virus), Haemark virus (Sjor virus, Sjogren virus, Sjo virus, Sjor virus (Sjor virus, Sjor virus), Haemarlt virus, Sjor virus (Sjor virus), Sjor virus, Sjor 103, Sjor virus, Sjor virus, BUDS (AY660568), Houston virus (Houston virus, HoV, AY502023), MOH (AF 3976156), Parish Island virus (Parris Island, PiV; AY652979), VA387(AY 038600)) VA207(AY038599), and free iraq free move (Operation Iraqi free, OIF, AY 675554). All nucleic acids and corresponding amino acid sequences of each are fully included by reference. In some embodiments, the ciphertext may be used for authentication purposes, or the ciphertext may be organized: abbreviations of host species/genus/name of abbreviation of species/name of strain/country of origin of discovery for isolated virus (Green et al, Human Caliciviruses, in Fields Virology Vol.1841-874(Knipe and Howley, editors-in-chip, 4th ed., Lippincott Williams)&Wilkins 2001)). Norwalk, snow mountain, and houston viruses are preferred in some embodiments.

The norovirus antigen may be in the form of a peptide, protein, or virus-like particle (VLP). In a preferred embodiment, the norovirus antigen comprises a VLP. As used herein, "virus-like particle or VLP" refers to a virus-like particle, fragment, aggregate, or portion thereof generated from the capsid protein coding sequence of norovirus and comprising similar antigenic features as infectious norovirus particles. The norovirus antigen may also be in the form of a capsid monomer, capsid multimer, protein or peptide fragment, or aggregate or mixture thereof, of the VLP. Norovirus antigenic proteins or peptides may also be in denatured form, produced using methods known in the art.

VLPs of the invention may be formed from full-length norovirus capsid proteins such as VP1 and/or VP2 proteins, or certain VP1 or VP2 derivatives, using techniques standard in the art. Alternatively, the capsid protein used to form the VLP is a truncated capsid protein. In some embodiments, for example, at least one VLP comprises a truncated VP1 protein. In other embodiments, all VLPs comprise a truncated VP1 protein. The truncation may be an N-terminal or C-terminal truncation. Truncated capsid proteins are suitably functional capsid protein derivatives. A functional capsid protein derivative is capable of eliciting an immune response in the same manner as VLPs composed of full-length capsid proteins elicit an immune response (if necessary, when appropriate adjuvants are present).

VLPs may contain a major VP1 protein and/or a minor VP2 protein. Preferably, each VLP contains VP1 and/or VP2 proteins from only one norovirus gene panel, resulting in a monovalent VLP. As used herein, the term "monovalent" refers to antigenic proteins derived from a single norovirus gene group. For example, VLPs contain VP1 and/or VP2 from a genogroup I virus strain (e.g., VP1 and VP2 from norwalk virus). Preferably, the VLPs are predominantly composed of VP1 protein. In one embodiment of the invention, the antigen is a mixture of monovalent VLPs, wherein the composition comprises VLPs consisting of VP1 and VP2 from a single norovirus gene group mixed with VLPs consisting of VP1 and VP2 from different norovirus gene groups (e.g., norwalk virus and houston virus) taken from multiple virus strains. Purely by way of example, the composition may comprise monovalent VLPs from one or more strains of norovirus genogroup I and monovalent VLPs from one or more strains of norovirus genogroup II. Preferably, the norovirus VLP mixture is composed of multiple strains of norwalk and houston norovirus.

However, in another embodiment of the invention, the VLP may be a multivalent VLP comprising, for example, VP1 and/or VP2 proteins from one norovirus gene group mixed with VP1 and/or VP2 proteins from a second norovirus gene group, wherein the different VP1 and VP2 proteins are not chimeric VP1 and VP2 proteins but join together within the same capsid structure to form an immunogenic VLP. As used herein, the term "multivalent" refers to antigenic proteins derived from two or more norovirus gene groups or strains. The multivalent VLP may contain VLP antigens taken from two or more virus strains. Purely by way of example, the composition may comprise a multivalent VLP composed of capsid monomers or multimers from one or more strains of norovirus gene group I (e.g., norwalk virus) and capsid monomers or multimers from one or more strains of norovirus gene group II (e.g., houston virus). Preferably, the multivalent VLP contains capsid proteins from multiple strains of norwalk and houston norovirus.

The combination of monovalent or multivalent VLPs within the composition preferably does not reduce the immunogenicity of each VLP type. In particular, it is preferred that there is no interference between the norovirus VLPs in the combination of the invention, such that the combination VLP composition of the invention is capable of eliciting immunity against infection of each norovirus genotype presented in the vaccine. Suitably, the immune response against a given VLP type in combination is at least 50%, preferably 100% or substantially 100% of the immune response against the same VLP type when measured separately. The immune response may suitably be measured, for example by an antibody response, as exemplified in the examples herein.

Multivalent VLPs can be produced by expressing individual capsid proteins separately, followed by combining to form VLPs. Alternatively, multiple capsid proteins may be expressed from one or more DNA constructs within the same cell. For example, multiple DNA constructs can be transformed or transfected into a host cell, each vector encoding a different capsid protein. Alternatively, a single vector having multiple capsid genes under the control of a shared promoter or multiple separate promoters may be used. Where appropriate, the IRES element may also be incorporated into a vector. Using such expression strategies, the co-expressed capsid proteins can be co-purified for subsequent VLP formation, or multivalent VLPs can be formed spontaneously, which can then be purified.

A preferred process for the production of multivalent VLPs comprises preparing VLP capsid proteins or derivatives from different norovirus genotypes, such as VP1 protein, mixing the proteins, and assembling the proteins to produce multivalent VLPs. Prior to mixing, the VP1 protein may be in the form of a crude extract, partially purified or purified. Assembled monovalent VLPs of different gene subgroups can be disassembled, mixed together, and reassembled into multivalent VLPs. Preferably, the protein or VLP is at least partially purified prior to combining. Optionally, the multivalent VLPs may be further purified after assembly.

Suitably, the VLPs of the invention are prepared by disassembly and reassembly of the VLPs to provide homogeneous and pure VLPs. In one embodiment, multivalent VLPs may be prepared by reassembling two or more VLPs, followed by combining the reassembled VLP components at any suitable point prior to reassembly. This approach is suitable when VLPs are formed spontaneously from the expressed VP1 protein, as occurs, for example, in some yeast strains. In cases where expression of VP1 protein does not result in spontaneous VLP formation, preparations of VP1 protein or capsid may be combined prior to assembly into VLPs.

In the case of multivalent VLPs, it is preferred that the components of the VLPs are mixed in their desired proportions in the final mixed VLP. For example, a mixture of identical amounts of partially purified VP1 protein from norwalk and houston viruses (or other norovirus strains) provides multivalent VLPs containing approximately equal amounts of each protein.

Compositions comprising multivalent VLPs may be stabilized by solutions known in the art, such as WO 98/44944; WO 00/45841, incorporated herein by reference.

In addition to VP1 and VP2 proteins or derivatives, the compositions of the invention may comprise other proteins or protein fragments. Other proteins or peptides may also be co-administered with the compositions of the present invention. Optionally, the composition may also be formulated or co-administered with a non-norovirus antigen. Suitably, these antigens provide protection against other diseases.

The VP1 protein or functional protein derivative is suitably capable of forming VLPs, and VLP formation can be assessed by standard techniques, such as, for example, electron microscopy and dynamic laser light scattering.

Antigen preparation

The antigenic molecules of the invention may be prepared by isolation and purification from the organism in which they naturally occur, or they may be prepared by recombinant techniques. Preferably, the norovirus VLP antigens are prepared from insect cells such as Sf9 or H5 cells, although any suitable cell may be used, such as e.coli (e.coli) or yeast cells, e.g. saccharomyces cerevisiae/saccharomyces cerevisiae (s.cerevisiae), schizosaccharomyces pombe (s.pombe), Pichia pastoris (Pichia pastoris) or other Pichia expression systems, mammalian cells expressing e.g. CHO or HEK systems. When prepared by recombinant methods or by synthesis, one or more insertions, deletions, inversions or substitutions may be made to the amino acids constituting the peptide. Preferably, each of the above antigens is used in a substantially pure state.

Procedures for the production of norovirus VLPs in insect cell culture have been previously disclosed in U.S. patent No.6,942,865, incorporated herein by reference in its entirety. Briefly, a cDNA from the 3' end of the genome containing the viral capsid gene (ORF2) and the minor structural gene (ORF3) was cloned. Recombinant baculoviruses carrying viral capsid genes were constructed from the cloned cdnas. Norovirus VLPs are produced in Sf9 or H5 insect cell cultures.

Adjuvant

The invention further provides compositions comprising an adjuvant for use with norovirus antigens. Most adjuvants contain substances designed to protect antigens from rapid metabolism (such as aluminium hydroxide or mineral oil) and stimulators of the immune response (such as bordetella pertussis (bordetella pertussis) or mycobacterium tuberculosis (mycobacterium tuberculosis) derived proteins). Suitable adjuvants are commercially available, for example Freund's incomplete adjuvant and complete adjuvant (Pifco Laboratories, Detroit, Mich.); merck adjuvant 65(Merck and Company, inc., Rahway, n.j.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine acylated sugars; a cationically or anionically derivatized polysaccharide; polyphosphazene; biodegradable microspheres; and Quil a.

Suitable adjuvants also include, but are not limited to, toll-like receptor (TLR) agonists, monophosphoryl lipid a (mpl), synthetic lipid a, lipid a mimetics or analogs, aluminum salts, cytokines, saponins, Muramyl Dipeptide (MDP) derivatives, CpG oligomers, Lipopolysaccharides (LPS) of gram-negative bacteria, polyphosphazenes, emulsions, virosomes, Cochleates, poly (lactide-co-glycolide) (PLG) microparticles, poloxamer particles, microparticles, and liposomes. Preferably, the adjuvant is a bacterially derived exotoxin. Adjuvants that stimulate Th1 type responses are also preferred, such as 3DMPL or QS 21.

Monophosphoryl lipid a (mpl), a nontoxic derivative of lipid a from Salmonella (Salmonella), is a potent TLR-4 agonist that has been developed as a vaccine adjuvant (Evans et al 2003). In preclinical murine studies, intranasal MPL was shown to enhance secretory as well as systemic humoral responses (Balbridge et al 2000; Yang et al 2002). It has also been demonstrated in clinical studies of over 120,000 patients to be safe and effective as a vaccine adjuvant (Baldrick et al, 2002; 2004). MPL stimulates induction of innate immunity via the TLR-4 receptor and is therefore capable of eliciting non-specific immune responses against a variety of infectious pathogens, including both gram-negative and gram-positive bacteria, viruses, and parasites (Baldrick et al 2004; Persing et al 2002). The inclusion of MPL in intranasal formulations should provide rapid induction of innate responses, eliciting non-specific immune responses from viral challenges (challenge), while enhancing specific responses generated by antigenic components of the vaccine.

Thus, in one embodiment, the present invention provides a composition comprising monophosphoryl lipid aOr 3 De-O-acylated monophosphoryl lipid A(as an enhancer of adaptive and innate immunity). In the chemical aspect of the raw materials,is a mixture of 3 De-O-acylated monophosphoryl lipid A having 4, 5 or 6 acylated chains. A preferred form of 3 De-O-acylated monophosphoryl lipid A is disclosed in European patent 0689454B 1(SmithKline Beechambiologicals SA), incorporated herein by reference. In another embodiment, the invention provides a composition comprising a synthetic lipid A, lipid A mimetic, or analog, such as PET lipid A from BioMira,Or a composition of synthetic derivatives designed to function as a TLR-4 agonist.

The term "effective adjuvant amount" or "effective amount of adjuvant" is well understood by those skilled in the art and includes an amount of one or more adjuvants capable of stimulating an immune response to an administered antigen, i.e., increasing an immune response to an administered antigen composition, as measured in terms of IgA levels, serum IgG or IgM levels, or B and T cell proliferation in nasal washes. Suitable effective elevations of immunoglobulin levels include more than 5%, preferably more than 25%, in particular more than 50%, as compared to the same antigen composition without any adjuvant.

Delivery agent

The invention also provides compositions comprising a delivery agent that functions to enhance antigen uptake, provide a depot effect, or prolong the retention time of antigen at the delivery site (e.g., delay excretion of antigen). Such delivery agents may be bioadhesives. In particular, the bioadhesive may be a mucoadhesive, such as chitosan, a chitosan salt, or a chitosan base (e.g., chitosan glutamate).

Chitosan, a positively charged linear polysaccharide derived from chitin in the shell of crustaceans, is a bioadhesive for epithelial cells and their overlying mucus layer. Formulating antigens with chitosan extends their time of contact with the nasal membranes, thereby increasing uptake by virtue of the storage effect (Illum et al 2001; 2003; Davis et al 1999; Bacon et al 2000; van der Lubben et al 2001; 2001; Lim et al 2001). Chitosan has been tested as a nasal delivery system for several vaccines, both in animal models and in humans, including influenza, pertussis and diphtheria (Illum et al 2001; 2003; Bacon et al 2000; Jabbal-Gill et al 1998; Mills et al 2003; McNeelaet al 2004). In these experiments, chitosan was shown to enhance the systemic immune response to levels equivalent to parenteral vaccination. In addition, significant antigen-specific IgA levels were also measured in mucosal secretion. In this manner, chitosan can greatly enhance the efficacy of nasal vaccines. Furthermore, chitosan is particularly suitable for intranasal vaccines formulated as powders due to its physical characteristics (van der Lubben et al 2001; Mikszta et al 2005; Huang et al 2004).

Thus, in one embodiment, the invention provides an antigenic composition or vaccine composition adapted for intranasal administration, wherein the composition comprises an antigen and an effective amount of an adjuvant. In a preferred embodiment, the present invention provides a kit comprising a delivery agent (such as chitosan) and at least one adjuvant (such asAn antigenic composition or vaccine composition of a norovirus antigen (such as a norovirus VLP) in combination with CPG, imiquimod, gardiquimod, or synthetic lipid a or lipid a mimetics or analogues.

The molecular weight of the chitosan may be between 10kDa and 800kDa, preferably between 100kDa and 700kDa and more preferably between 200kDa and 600 kDa. The concentration of chitosan in the composition will generally be up to about 80% (w/w), for example 5%, 10%, 30%, 50%, 70% or 80%. The chitosan is preferably at least 75% deacetylated, such as 80-90%, more preferably 82-88% deacetylated, with specific examples being 83%, 84%, 85%, 86% and 87% deacetylated.

Vaccine and antigenic formulations

The compositions of the invention may be formulated for administration as a vaccine or antigenic formulation. As used herein, the term "vaccine" refers to a formulation containing a norovirus VLP or other norovirus antigen of the invention as described above, in a form capable of being administered to a vertebrate and inducing a protective immune response sufficient to induce immunity to prevent and/or ameliorate an infection and/or alleviate at least one symptom of an infection and/or enhance the efficacy of another dose of VLP or antigen. As used herein, the term "antigenic formulation" or "antigenic composition" refers to a preparation that induces an immune response when administered to a vertebrate (e.g., a mammal). As used herein, the term "immune response" refers to both a humoral immune response and a cell-mediated immune response. The humoral immune response involves the stimulation of B lymphocytes to produce antibodies that, for example, neutralize infectious agents, block the entry of infectious agents into cells, block the replication of said infectious agents, and/or protect host cells from infection and destruction. A cell-mediated immune response refers to an immune response exhibited by a vertebrate (e.g., a human) against an infectious agent, mediated by T lymphocytes and/or other cells (such as macrophages), that prevents or ameliorates an infection or reduces at least one symptom thereof. In particular, "protective immunity" or "protective immune response" refers to immunity exhibited by a vertebrate (e.g., a human) against an infectious agent or elicits an immune response against an infectious agent that prevents or ameliorates an infection or alleviates at least one symptom thereof. In particular, induction of a protective immune response from administration of the vaccine is manifested by elimination or reduction of the presence of one or more symptoms of gastroenteritis, or a reduction in the duration or severity of such symptoms. Clinical symptoms of gastroenteritis caused by norovirus include nausea, diarrhea, loose stools, vomiting, fever, and general malaise. A protective immune response that reduces or eliminates the symptoms of a disease will reduce or stop the spread of norovirus outbreaks in a population. Vaccine preparations are generally described in Vaccine Design ("The subabunit and adjuvant Vaccine" (edited by Powell M.F. and Newman M.J.) (1995) Plenum Press New York). The compositions of the invention may be formulated for delivery, for example, to one or more of the oral, gastrointestinal, and respiratory (e.g., nasal) mucosa.

Where the composition is intended to be delivered to the respiratory (e.g. nasal) mucosa, it is typically formulated as an aqueous solution (for administration as an aerosol or nasal drops) or as a dry powder (e.g. for rapid deposition within the nasal passages). Compositions for administration as nasal drops may contain one or more types of excipients commonly included in such compositions, such as preservatives, viscosity modifiers, tonicity modifiers, buffers, and the like. The viscosity agent may be microcrystalline cellulose, chitosan, starch, polysaccharides, and the like. Compositions for administration as a dry powder may also contain one or more excipients typically included in such compositions, such as mucoadhesives, fillers, and agents that provide suitable powder flow and size characteristics. Bulking agents and powder flow and sizing agents can include mannitol, sucrose, trehalose, and xylitol.

In one embodiment, the norovirus vaccine or antigenic formulation of the invention contains one or more norovirus gene subgroup antigens (as immunogens), adjuvants (such as) Biopolymers such as chitosan for promoting adhesion to mucosal surfaces, and bulking agents such as mannitol and sucrose. For example, norovirus vaccines can be formulated to contain one or more norovirus genogroup antigens (e.g., norwalk virus, houston virus, snow mountain virus),Adjuvant, chitosan mucoadhesive, and 10mg dry powder of mannitol and sucrose (as bulking agent and to provide appropriate flow characteristics). The formulation may comprise about 7.0mg chitosan (25-90% w/w range), about 1.5mg mannitol (0-50% w/w range), about 1.5mg sucrose (0-50% w/w range), about 25 μ g(in the range of 0.1-5% w/w), and about 100. mu.g norovirus antigen (in the range of 0.05-5% w/w).

Norovirus antigens can be present at a concentration of about 0.01% (w/w) to about 80% (w/w). In one embodiment, the norovirus antigen may be formulated at a dosage of about 5 μ g, about 15 μ g, about 25 μ g, about 50 μ g, about 100 μ g, about 200 μ g, about 500 μ g, and about 1mg per 10mg of dry powder formulation (0.05, 0.15, 0.25, 0.5, 1.0, 2.0, 5.0, and 10.0% w/w) (for administration into both nostrils, 10mg per nostril) or about 10 μ g, about 30 μ g, about 50 μ g, about 100 μ g, about 200 μ g, about 400 μ g, about 1mg, and about 2mg (0.1, 0.3, 0.5, 1.0, 2.0, 4.0, 10.0, and 20.0% w/w) per 20mg of dry powder formulation (for administration into one nostril). The formulation may be administered in one or both nostrils during each administration. There may be booster administrations 1-12 weeks after the first administration to improve the immune response. The content of each norovirus antigen in the vaccine and antigenic formulations may be in the range 1 μ g to 100mg, preferably in the range 1-1000 μ g, more preferably 5-500 μ g, most typically in the range 10-200 μ g. The total norovirus antigen administered per dose may be about 10 μ g, about 30 μ g, about 200 μ g, about 250 μ g, about 400 μ g, about 500 μ g, or about 1000 μ g. The total vaccine dose may be administered into one nostril, or may be split in half and administered to both nostrils. The dry powder is characterized in that less than 10% of the particles are smaller than 10 μm in diameter. The average particle size ranges from 10 to 500 μm in diameter.

In another embodiment, the antigenic and vaccine compositions can be formulated as a liquid for subsequent administration to a subject. A liquid formulation intended for intranasal administration will comprise a norovirus gene panel antigen, an adjuvant, and a delivery agent such as chitosan. A liquid formulation for intramuscular (i.m.) administration would contain norovirus gene group antigens, adjuvants, and buffers, without a delivery agent (e.g., chitosan).

Preferably, the antigenic and vaccine compositions described above are lyophilized and stored anhydrous until they are ready for use, at which time they are reconstituted with a diluent. Alternatively, the different components of the composition may be stored separately in the kit (any or all of the components are lyophilized). The ingredients may be kept in lyophilized form for dry or reconstituted liquid formulation and either mixed prior to use or administered separately to the patient. For dry powder administration, the vaccine or antigenic formulation may be pre-loaded into an intranasal delivery device and stored until use. Preferably, such intranasal delivery devices will protect and ensure the stability of their contents.

Lyophilization of antigenic formulations and vaccines is well known in the art. Typically, the liquid antigen is freeze-dried in the presence of a medicament that protects the antigen during lyophilization and produces a cake (cake) with the desired powder characteristics. Sugars (such as sucrose, mannitol, trehalose, or lactose), present at initial concentrations of 10-200mg/mL, are often used for cryoprotection of protein antigens and to produce lyophilized cakes with desirable powder characteristics. Lyophilizing a composition theoretically results in a more stable composition. Although the goal of most formulation processes is to minimize protein aggregation and degradation, the inventors found that the presence of aggregated antigens enhances the immune response against norovirus VLPs (see examples 3 and 4). Thus, the inventors have developed methods that can control the percentage of antigen aggregation during the lyophilization process to generate the optimal ratio of aggregated antigen to intact antigen to induce the maximum immune response.

As such, the present invention also encompasses a method of preparing a norovirus antigen formulation comprising (a) preparing a pre-lyophilization solution comprising norovirus antigen, sucrose, and chitosan, wherein the ratio of sucrose to chitosan is from about 0:1 to about 10: 1; (b) freezing the solution with liquid nitrogen; and (c) lyophilizing the frozen solution at ambient temperature for 48-72 hours, wherein the final lyophilized product contains a percentage of the norovirus antigen in aggregated form. In one embodiment, the pre-lyophilization solution further comprises a bulking agent. In another embodiment, the bulking agent is mannitol.

A suitable ratio of sucrose to chitosan that produces the desired aggregation percentage can be determined by the following guidelines. A pre-lyophilization mixture containing a sucrose to chitosan weight ratio in the range of about 2.5:1 to about 10:1 will yield more than 95% of intact norovirus antigen (i.e., less than 5% aggregated antigen; see example 13) after lyophilization. A sucrose to chitosan weight ratio range of about 1:1 to about 2.1:1 will yield about 50% to about 90% of intact norovirus antigen (i.e., about 10% to about 50% aggregated antigen). A sucrose to chitosan weight ratio of 0:1 will yield less than 30% of intact norovirus antigen. Omitting both sucrose and chitosan would result in less than 5% of the whole antigen (i.e. more than 95% of aggregated antigen). Using these guidelines, the skilled artisan can adjust the weight ratio of sucrose to chitosan in the mixture prior to lyophilization to obtain the desired amount of aggregation necessary to produce an optimal immune response.

In addition, the inclusion of sucrose and chitosan in the solution prior to lyophilization improves the stability of the intact norovirus antigen over time. When stored as a dry powder for a period of about 12 months or longer, the ratio of aggregated antigen/intact antigen in the formulation does not increase (see example 10). In this manner, this lyophilization procedure ensures a stable formulation with a predictable and controllable ratio of aggregation to intact norovirus antigen.

Method of stimulating an immune response

The amount of antigen in each antigenic or vaccine formulation dose is selected to be an amount that induces a strong immune response without significant adverse side effects. Such amounts will vary depending upon which particular antigen is employed, the route of administration, and the adjuvant used. In general, in the context of the present invention, the dose administered to a patient should be sufficient to elicit a protective immune response in the patient over time, or to induce the production of antigen-specific antibodies. As such, the compositions are administered to a patient in a sufficient amount to elicit an immune response against a particular antigen and/or to prevent, alleviate, reduce, or cure symptoms and/or complications from a disease or infection, and thus reduce or stop the spread of norovirus outbreaks in a population. An amount sufficient to achieve this is defined as a "therapeutically effective amount".

For norovirus antigens in substantially pure form, it is expected that each dose will contain from about 1 μ g to 10mg, preferably from about 15-500 μ g, of each norovirus antigen in the formulation. In a typical immunization regimen using the antigenic preparations of the invention, the formulation may be administered in several doses (e.g., 1-4 doses), each containing 1-1000 μ g of each antigen. The dosage will be determined by the immunological activity produced by the composition and the condition of the patient and the weight or surface area of the patient to be treated. The size of the dose will also be determined by the presence, nature, and extent of any adverse side effects that may accompany the administration of a particular composition in a particular patient.

Antigenic and vaccine formulations of the invention may be administered via a non-mucosal or mucosal route. These administrations may include in vivo administration via parenteral injection (e.g., intravenous, subcutaneous, and intramuscular) or other conventional direct routes, such as buccal/sublingual, rectal, oral, nasal, topical (such as transdermal and ocular), vaginal, pulmonary, intra-arterial, intraperitoneal, intraocular, or intranasal routes or directly into specific tissues. Alternatively, the vaccines of the present invention can be administered by any of a variety of routes, such as oral, topical, subcutaneous, mucosal, intravenous, intramuscular, intranasal, sublingual, transdermal, subdermal, intradermal, and suppository. Administration can be achieved simply by direct administration using a needle, catheter or related device at a single point in time or multiple points in time.

In a preferred embodiment, the antigenic and vaccine formulations of the present invention are administered by the intranasal route. Vaccination via mucosal surfaces offers numerous potential advantages over other routes of vaccination. The most obvious benefits are 1) mucosal immunization without the need for needles or trained personnel to administer, and 2) the generation of an immune response at the site of pathogen entry and systemically (Isaka et al 1999; kozlowski et al 1997; mestecky et al 1997; wuet al 1997).

In another aspect, the invention provides methods of eliciting both IgA mucosal immune responses and IgG systemic immune responses by administering (preferably intranasally) to a mucosal surface of a patient an antigenic or vaccine composition comprising one or more norovirus antigens, at least one effective adjuvant and/or at least one delivery agent.

The present invention also encompasses means for providing an intranasal formulation for dispensing a norovirus antigen as defined above and at least one adjuvant or at least one delivery agent as defined above. The dispensing means may take the form of, for example, an aerosol delivery system and may be arranged to dispense only a single dose or a plurality of doses. Such devices deliver a metered dose of vaccine or antigenic formulation to the nasal passage. Other examples of suitable devices include, but are not limited to, droppers (droppers), swabs (swabs), nebulizers (aerosolizers), insufflators (e.g., Valois Monospoder nasal administration devices, single dose Bespak Unidose DP dry powder intranasal delivery devices), nebulizers (nebulizers), and inhalers (inhales). The device may deliver the antigenic or vaccine formulation by passive means that require the subject to inhale the formulation into the nasal cavity. Alternatively, the device may actively deliver the formulation by pumping or spraying a dose into the nasal cavity. The antigenic formulation or vaccine may be delivered into one or both nostrils by one or more such devices. Administration may include two devices per subject (one device per nostril). The actual dose of active ingredient (norovirus antigen) may be about 5-1000 μ g. In a preferred embodiment, the antigenic or vaccine formulation is administered to the nasal mucosa by rapid deposition within the nasal passages from a device containing the formulation in close proximity to the nasal passages.

The present invention also provides a method of generating antibodies against one or more norovirus antigens, said method comprising administering to a subject a vaccine or antigenic formulation of the invention as described above. These antibodies can be isolated and purified by routine methods in the art. Isolated antibodies specific for norovirus antigens can be used in the development of diagnostic immunological assays. These assays can be used to detect norovirus in clinical samples and to identify the specific virus causing the infection (e.g., norwalk, houston, snow mountain, etc.). Alternatively, the isolated antibody may be administered to a subject susceptible to norovirus infection to confer passive or short-term immunity.

The present invention provides a method for eliciting protective immunity against norovirus infection in a subject comprising administering a vaccine to the subject, wherein the vaccine comprises norovirus VLPs and at least one adjuvant. In one embodiment, the subject is a human and the vaccine confers protection from one or more symptoms of norovirus infection. While others have reported methods of inducing immune responses with norovirus antigens (see U.S. patent application publication No. us 2007/0207526), no one has demonstrated that a protective immune response is induced in humans. Unlike several vaccines currently licensed in the united states, where the effectiveness of the vaccine is related to serum antibodies, studies have shown that markers of immune responses, such as elevated serum antibody titers against norwalk virus, are not related to protective immunity in humans (Johnson et al (1990) j. infection Diseases 161: 18-21). In addition, another study examining Norwalk virus challenge (challenge) in humans indicated that susceptibility to Norwalk infection is multifactorial and includes factors such as secretor status and memory mucosal immune response (Lindesmith et al (2003) Nature Medicine 9: 548-. Because norovirus cannot be cultured in vitro, no virus neutralization assay is currently available. One functional assay that serves as an alternative to neutralization assays is the hemagglutination inhibition (HAI) assay. HAI measures the ability of antibodies induced by norovirus vaccines to inhibit the agglutination of antigen-coated red blood cells by norovirus VLPs (because norovirus VLPs bind to red blood cell antigens). In this assay, a fixed amount of norovirus VLPs is mixed with a fixed amount of red blood cells and serum from an immunized subject. If the serum sample contains functional antibodies, the antibodies compete with the VLPs for binding to erythrocytes, thereby inhibiting erythrocyte agglutination.

Similar findings were observed for vaccines against other viruses, such as rotavirus. For rotaviruses, there is controversy as to whether serum antibodies are directly involved in protection or merely reflect recent infection (Jiang, 2002; Franco, 2006). Determining such associations of protection in the context of diarrheal diseases such as rotavirus or norovirus is particularly difficult, where preclinical studies conclude that protection can be enriched by contributions from mucosal immunity such as intestinal IgA, cytokine fine-tuning (elibosation), and cell-mediated immunity (multifaceted). The difficulty in measuring such immune responses during clinical development and the lack of correlation with serum antibody measurements requires that the effectiveness of vaccines against these types of viruses can only be demonstrated through human clinical trials.

As noted above, administration of the vaccine of the invention prevents and/or reduces at least one symptom of norovirus infection. Symptoms of norovirus infection are well known in the art and include nausea, vomiting, diarrhea, and gastric colic. In addition, patients with norovirus infection may have low fever, headache, chills, muscle pain, and fatigue. The present invention also encompasses methods of inducing a protective immune response in a subject experiencing a norovirus infection by administering to the subject a vaccine formulation of the invention such that at least one symptom associated with the norovirus infection is reduced and/or decreased. The reduction in symptoms can be determined subjectively or objectively, e.g., by the subject self-assessment, by a clinician assessment, or by performing a suitable assay or measurement (e.g., body temperature), including, for example, quality of life assessment, slowing of the progression of norovirus infection or other symptoms, reduction in severity of norovirus symptoms, or a suitable assay (e.g., antibody titer, RT-PCR antigen detection, and/or B-cell or T-cell activation assay). Effective responses can also be determined by direct measurement (e.g., RT-PCR) of viral load in stool samples, which reflects the amount of virus shed from the intestine. Objective assessment includes both animal and human assessment.

The stability and efficacy of the vaccines and antigenic formulations disclosed herein in animal models is reported in international application No. pct/US07/79929 (incorporated herein by reference in its entirety).

Examples

The present invention will now be illustrated in more detail by reference to specific embodiments described in the following examples. The examples are intended purely to illustrate the invention and are not intended to limit its scope in any way. Example 1: GLP toxicity studies in rabbits with norovirus vaccine formulations

The objective of this study was to evaluate the potential toxicity of norwalk virus-like particle (NV-VLP) vaccine after three intranasal administrations in rabbits. The NV-VLP vaccine contains (per 10mg dry powder) 25. mu.g gene panel I VLP, 25. mu.g MPL, 7mg chitosan glutamate, 1.475mg mannitol, and 1.475mg sucrose. The study was conducted over an eight week period. The persistence, reversibility, or delayed onset of any effect was assessed after a four week, no treatment recovery interval. Three subgroups (10 rabbits/gender/group) were randomly assigned to 60 new zealand white rabbits (30 per gender). Group 1 animals were not dosed (i.e. not treated (naive)). Group 2 animals were administered 10 mg/nostril (20 mg total) of placebo (i.e. adjuvant/vehicle: MPL, chitosan, sucrose, and mannitol). Animals in group 3 were administered 10 mg/nostril (20 mg total) of NV-VLP vaccine, which represents 25 μ g antigen/nostril (50 μ g total). Animals in groups 2 and 3 were dosed by intranasal administration using a Bespak Unidose intranasal dry powder device on study days 1, 22, and 43 (SD). Animals (5/group/sex) were subjected to a complete gross necropsy at SD 46 and 74. Parameters assessed during the study included mortality, clinical and cage-side observations, body weight changes, food consumption, body temperature, ophthalmic examinations, clinical pathology (clinical chemistry, hematology, and urinalysis), gross pathology, organ weight data, and histopathology. The study summary is summarized in table 1. The results of the study are summarized in table 2.

Table 1: study parameters for GLP toxicity studies of norwalk vaccine formulations

Species (II)	SPF New Zealand white rabbit with ear tag ID
		Animal number/sex/dose groups	10 males and 10 females/group
Total number of animals in the study	60

Group 1	Untreated control
		Group 2	Adjuvants/excipients
Subgroup 3	1x maximum human dose VLP in adjuvant/vehicle

Table 2: safety and toxicology findings of norwalk vaccine formulations

Cage-side observation revealed no significant findings. Hematological measurements (elevation of globulin and total protein) are typical for polyclonal activation of B lymphocytes and can be attributed to adjuvant effects. Histopathological findings included inflammatory infiltrates of varying degrees, either within the lamina propria of the turbinate or free within the nasal passages, and/or mild bleeding in the nasal passages of rabbits in both groups. The observed lesions are expected to occur in the immune response. The lesions in both groups were limited in nature and were completely resolved by SD 74.

Analysis of serological samples of NV-VLP-specific IgG by ELISA showed that 30% of the immunized animals had measurable titers against NV-VLP at day 10 after a single dose (see FIG. 1). The boosting treatment at days 22 and 43 increased both the number of seroconverted animals and the product-specific antibody levels, and by day 73, 90% of the immunized animals were seroconverted. None of the untreated or matrix-treated controls had quantifiable levels of NV-VLP-specific antibodies (data not shown).

The immune response was further characterized by assessing memory B cell responses in another group of rabbits immunized intranasally with the same formulation on days 1, 15, and 29. Memory B cell responses were measured as described in International application No. PCT/US07/79929 (incorporated herein by reference in its entirety). Tissues collected 156 days after the last boost showed the presence of NV-VLP specific memory B cells in peripheral blood, spleen, and (most notably) mesenteric lymph nodes. Antigen-specific memory B cells in mesenteric lymph nodes were IgA positive. In addition, the presence of NV-VLP specific antibodies in the bone marrow secretes long-lived plasma cells.

Example 2: dose-escalation safety study of Norwalk vaccine formulations in humans

A double-blind, controlled, phase 1 dose-escalation safety and immunogenicity study of norovirus genogroup 1 vaccines was performed. The vaccine consists of lyophilized norwalk virus-like particles (VLPs) in a dry powder matrix designed for intranasal administration. Vaccinees included healthy adult volunteers of type H1 antigen secretors. The rationale for the inclusion of type H1 antigen secretors is that type H1 antigen secretors are susceptible to Norwalk virus infection and not to secretors. As a control, 2 additional volunteers at each dose level received matrix only. The dry powder matrix comprises 25 μ gAdjuvant, 7mg chitosan, 1.5mg mannitol, and 1.5mg sucrose. Volunteers were dosed on days 0 and 21 and were asked to take a 7 day symptom diary after each dose. Blood (for serology, Antibody Secreting Cells (ASC)), and stool and saliva samples (for mucosal antibody assessment) were collected.

The components of the norwalk VLP vaccine are listed in table 3. The vaccine is packaged into an intranasal delivery device. A single administration of the norwalk VLP vaccine was packaged into a single dose Bespak (Milton Keynes, UK) unite dose DP dry powder intranasal delivery device. Each device delivered 10mg of dry powder vaccine formulation. Each dose of vaccine consisted of two delivery devices, one in each nostril. The total vaccine dose was 20mg of dry powder. The formulation of the adjuvant/excipient is the same as the norwalk VLP vaccine, except that norwalk VLP antigen is not included in the formulation. The formulation of adjuvants/excipients (also referred to as dry powder matrix) is summarized in table 4.

Table 3: norwalk VLP vaccine compositions

Table 4: adjuvants/excipients (Dry powder base)

Composition (I)	Molecular categories	The amount of each 10mg of the dry powder	% final formulation
				Monophosphoryl lipid A	Phospholipids	25μg	0.25％
Chitosan	Polysaccharides	7.0mg	70％
				Mannitol	Candy	1.5mg	15％
Sucrose	Candy	1.5mg	15％

Specifically, the dose escalation of the vaccine was performed as follows: after proper screening for good health, a group of 3 volunteers was randomly assigned to receive either 5 μ g of norwalk VLP vaccine plus dry powder matrix (n-2) or dry powder matrix alone (n-1) by the intranasal route. The 3 volunteers were followed for 21 days of Safety and their Safety data were reviewed by an Independent Safety Monitor (ISM). After ISM approval, these individuals received their second dose of vaccine or matrix on day 21 and were randomly assigned to receive 5 μ g of VLP protein plus dry powder matrix (n-3) or matrix alone (n-1) by the intranasal route to 4 additional volunteers. The ISM reviews safety data from this second group and following ISM approval, an intranasal second dose is administered 21 days after the first dose. Volunteers were logged for 7 days after each dose. After ISM decided that scaling up to the next higher dose was acceptable, another group of 7 volunteers was randomly assigned to receive either a norwalk VLP vaccine containing 15 μ g of VLP proteins (n-5) or a separate dry powder matrix (n-2) by intranasal route on day 0 and day 21. Again, a 7-day symptom diary was recorded and reviewed by the ISM prior to the second dose on day 21. Finally, after reviewing the safety data from the first two dose groups, ISM decided that dose escalation was acceptable, and the last group of 7 volunteers was randomly assigned to receive either norwalk VLP vaccine containing 50 μ g VLP protein (n-5) or dry powder matrix alone (n-2) by intranasal route on days 0 and 21. Seven day symptom diaries and other safety data were reviewed by the ISM again before the second dose on day 21.

Volunteers were logged with a diary of symptoms (including local symptoms such as nasal discharge, nasal pain/discomfort, nasal congestion, rhinorrhea, nasal itching, epistaxis, headache, and systemic symptoms such as daily oral temperature, myalgia, nausea, vomiting, abdominal cramps, diarrhea, and anorexia) 7 days after receiving either the Norwalk VLP vaccine or the dry powder matrix alone. Obtaining an intra-term (intercerim) medical history at each follow-up visit (day 7 ± 1, day 21 ± 2, day 28 ± 2, day 56 ± 2 and day 180 ± 14); the volunteers were asked for disease, medication, and doctor visit during the session. Volunteers were asked to report all serious or severe adverse events, including unsolicited (solicited) events during follow-up visits. Volunteers were evaluated for CBC and serum creatinine, glucose, AST, and ALT on days 7 and 28 (7 days after each immunization) and, if abnormal, the abnormal laboratory tests were followed until the tests became normal or stable.

Blind data suggest that of the volunteers receiving low dose (n-5) or matrix (n-2), 4 of 7 reported some or all of the following within the first 24 hours after vaccination: nasal discharge, nasal pain, stuffiness, itching, sneezing, headache, and/or sore throat. One subject reported a slight epistaxis on both day 1 and day 6. Of the subjects receiving the medium dose (n-5) or the base (n-2), 5 of 7 reported mild nasal discharge, stuffiness, itching, sneezing, and/or headache during the first 24 hours. Symptoms generally resolved within the first 72 hours, but stuffiness persisted to day 7 in one volunteer. Table 5 below presents a summary of findings on non-blinded data, which also included adverse events reported in high doses. These findings indicate that intranasal norovirus VLP vaccines are associated with local, usually mild, short-term symptoms that appear to be independent of VLP concentration. No differences were seen between the adjuvant/vehicle (or matrix) control group and the norwalk VLP vaccine group with respect to adverse events, hematology, blood chemistry and/or physical examination results.

Table 5: number of volunteers with adverse events against norwalk VLP vaccine or adjuvant/excipient

One subject in cohort 3 did not receive the second dose.

Blood was collected prior to immunization and at days 7 ± 1, 21 ± 2, 28 ± 2, 56 ± 2, and 180 ± 14 to measure serum antibodies to the norwalk VLP vaccine by enzyme-linked immunosorbent assay (ELISA). Peripheral blood lymphocytes were collected before and on day 7 after administration of each dose of vaccine or dry powder matrix alone to detect antibody secreting cells by ELISPOT assay. Whole blood was obtained before and after vaccination on days 21 ± 2, 56 ± 2, and 180 ± 14 to isolate cells and frozen for future studies of cell-mediated immunity, including cytokine production in response to norwalk VLP antigens, and lymphoproliferation. Whole stool samples were collected for anti-Norwalk VLP sIgA screening prior to immunization and at days 7+ -1, 21+ -2, 28+ -2, 56+ -2, and 180+ -14. Saliva was collected with a commercial device (Salivette, Sarstedt, Newton, N.C.) before immunization and at days 7+ -1, 21+ -2, 28+ -2, 56+ -2, and if mucosal antibodies were positive at day 56, samples were collected at day 180+ -14 and screened for anti-Norwalk VLPsIgA. Finally, blood from volunteers receiving the highest dose of norwalk VLPs (50 μ g, third cohort described above) was screened for memory B cells on days 0, 21, 56 and 180.

Blood, stool, and saliva samples collected from immunized individuals or individuals receiving the dry powder matrix alone were analyzed using the following methods:

A. measurement of serum antibodies by ELISA

20mL of blood was collected prior to vaccination and at various time points after vaccination for measurement of antibodies against norwalk virus by ELISA, wherein the encoded specimens were screened using purified recombinant norwalk VLPs as target antigen. Briefly, microtiter plates were coated with norwalk VLPs in carbonate coating buffer ph 9.6. The coated plates were washed, blocked, and incubated with serial two-fold dilutions of test serum, followed by washing and incubation with enzyme-conjugated secondary antibody reagents specific for human IgG, IgM, and IgA. Appropriate substrate solutions were added, developed, plates read, and IgG, IgM, and IgA end points titers were determined, comparing each antibody class to a reference standard curve. A positive response is defined as a 4-fold increase in titer after vaccination. Figure 2 shows the serum titers at day 56 (35 days after the second immunization) for each vaccine dose. The results show a dose-dependent increase in serum titers for IgG and IgA. Significant serum titers of IgG and IgA were observed in volunteers receiving vaccines containing 50 μ g norovirus antigen.

B. Antibody secreting cell assay

PBMCs were collected from heparinized blood (30mL fraction 1 and 2, 25mL fraction 3) for use in ASC assays to detect cells secreting antibodies to norwalk VLPs. These assays were performed on days 0, 7 ± 1, 21 ± 2, and 28 ± 2 after administration of either the norwalk VLP vaccine or the dry powder matrix alone. The response rate per time point and per 10 th dose are reported⁶Mean of ASC number of individual PBMC. Positive responses were defined as every 10 post-vaccination⁶The ASC counts of individual PBMCs exceeded the mean of all subjects pre-vaccination counts by at least 3 Standard Deviations (SD) (in a logarithmic scale) and at least 8 ASC spots, which corresponds to the mean of medium-stimulated negative control wells (2 spots) plus 3 SD, as determined in a similar assay.

Figure 3 depicts the results of an ASC assay of 50 μ g dose norwalk VLPs. Circulating IgG and IgA antibody secreting cells were observed seven days after prime and boost vaccination, suggesting that the vaccine was immunogenic.

C. Measuring functional antibody responses

Sera collected as described in paragraph B above were further analyzed to determine the functional properties of anti-norwalk virus antibodies. Test sera at serial two-fold dilutions were analyzed for their ability to inhibit hemagglutination of erythrocytes by norwalk VLPs (a functional assay indicative of a protective immune response). A positive response is defined as a 4-fold increase in titer after vaccination. Table 6 shows serum titers and hemagglutination inhibition titers at day 56 (35 days post-boost) for five subjects receiving a 50 μ g dose of norwalk VLP vaccine. The results show that seventy-five percent (75%) of individuals exhibiting seroconversion responses (as measured by serum IgG titers) also develop functional antibody responses capable of blocking binding receptors on human red blood cells (as measured by hemagglutination inhibition).

Table 6: serum IgG and hemagglutination inhibition (HAI) (functional) titers from five human volunteers at day 0 and day 35 post-reinforcement (day 35 PB).

D. Measurement of Norwalk virus-specific memory B cells

Heparinized blood was collected from cohort 3(30mL day 0 and 21, 50mL day 56 and 180) at days 0, 21, 56 and 180 post-vaccination to measure memory B cells using the ELISpot assay, prior to in vitro antigen stimulation. A similar assay was successfully used to measure the frequency of memory B cells elicited by norwalk VLP formulations in rabbits (see international application No. pct/US07/79929, incorporated herein by reference). Mononuclear cells from peripheral blood (5X 10)⁶cells/mL, 1 mL/well, 24-well plate) were incubated with norwalk VLP antigen (2-10 μ g/mL) for 4 days to allow clonal expansion and differentiation of antigen-specific memory B cells into antibody-secreting cells. Controls include cells incubated in the same conditions without the antigen and/or cells incubated with an unrelated antigen. After stimulation, cells were washedCounted and transferred to norwalk VLP-coated ELISpot plates. To determine the frequency of virus-specific memory B cells/total Ig secreting B lymphocytes, expanded B cells were also added to wells coated with anti-human IgG and anti-human IgA antibodies. Bound antibody was revealed with HRP-labeled anti-human IgG or anti-human IgA, followed by True Blue substrate. Conjugates to IgA and IgG subclasses (IgA1, IgA2 and IgG1-4) can also be used to determine antigen-specific subclass responses, which may be associated with different effector mechanisms and immune priming sites. Spots were counted using an ELISpot reader. The expanded cell population was examined by flow cytometry for each volunteer to confirm their memory B cell phenotype, i.e., CD19+, CD27+, IgG +, IgM +, CD38+, IgD-.

E. Cellular immune response

Heparinized blood (50mL fraction 1 and 2, 25mL fraction 3) was collected as coded specimens, Peripheral Blood Mononuclear Cells (PBMCs) were isolated and cryopreserved in liquid nitrogen for possible future assessment of CMI responses against norwalk VLP antigens. Assays that can be performed include PBMC proliferation and cytokine responses to norwalk VLP antigens, and can be determined by measuring Interferon (IFN) - γ and Interleukin (IL) -4 levels according to established techniques.

F. Stool and saliva collection for anti-Norwalk VLP sIgA

Anti-recombinant norwalk virus IgA was measured in stool and saliva samples. Saliva samples were treated with protease inhibitors (i.e., AEBSF, leupeptin, bestatin, and aprotinin) (Sigma, St. Louis, Mo.), stored at-70 ℃ and assayed using a modified version of the previously described assay (Millset al. (2003) feed. Immun.71: 726-. Stool was collected on multiple days post vaccination and specimens were stored at-70 ℃ until analysis. The specimens were thawed and protease inhibitor buffer was added to prepare a 10% w/v stool suspension. Recombinant norwalk virus (rNV) -specific mucosal IgA was assayed on stool supernatants by ELISA, as described below.

Approximately 2-3mL of whole saliva was collected before vaccination and at various time points after vaccination. Saliva was collected by a commercial device (Salivette, Sarstedt, Newton, NC) in which a Salivette swab was chewed or placed under the tongue for 30-45 seconds until the swab was saturated with saliva. Saliva was collected from the swab by centrifugation.

G. Measurement of anti-Norwalk VLPs in stool and saliva

ELISA was performed using plates coated with anti-human IgA antibody reagents or target rNV VLP antigen coatings to determine total IgA and titer specific anti-VLP IgA responses for each specimen. Total or specific IgA was revealed with HRP-labeled anti-human IgA as described above. An internal total IgA standard curve was included to quantify IgA content. Response was defined as a 4-fold increase in specific antibody.

Example 3: safety and immunogenicity studies in humans of two doses of intranasal Norwalk VLP vaccine

A randomized, double-blind study was conducted in healthy adults to compare the safety and immunogenicity of two dose levels of norwalk virus-like particle (VLP) vaccine with adjuvant/vehicle and placebo controls (empty devices). The vaccine consisted of norwalk Virus Like Particles (VLPs) in a dry powder matrix designed for intranasal administration as described in example 2. Vaccinees included healthy adult volunteers of type H1 antigen secretors. Human volunteers were randomly assigned to one of four groups, and each group received one of the following treatments: one dose of 50 μ g norwalk VLP vaccine, one dose of 100 μ g norwalk VLP vaccine, adjuvant/vehicle, or placebo. Volunteers were dosed on days 0 and 21 and were asked to take a 7 day symptom diary after each dose. Blood (for serology, Antibody Secreting Cells (ASC)), and stool and saliva samples (for mucosal antibody assessment) were collected.

Example 2 the components of the vaccine are listed in table 3. The vaccine is packaged into an intranasal delivery device. Norwalk with single administrationThe VLP vaccine was packaged into a single dose Bespak (Milton Keynes, UK) unite dose DP dry powder intranasal delivery device. Each device delivered 10mg of dry powder vaccine formulation. Each dose of vaccine consisted of two delivery devices, one in each nostril. The total vaccine dose was 20mg of dry powder. Thus, a 50 μ g vaccine dose consists of two devices, each delivering 10mg of dry powder formulation, wherein each 10mg of dry powder formulation consists of 25 μ g of norwalk VLPs, 25 μ gAdjuvant, 7mg chitosan, 1.5mg mannitol, and 1.5mg sucrose. Similarly, a 100 μ g vaccine dose consisted of two devices, each delivering 10mg of dry powder formulation, where each 10mg of dry powder formulation consists of 50 μ g of Norwalk VLP, 25 μ gAdjuvant, 7mg chitosan, 1.5mg mannitol, and 1.5mg sucrose. The formulation of the adjuvant/excipient is the same as the norwalk VLP vaccine, except that norwalk VLP antigen is not included in the formulation. The formulation of adjuvants/excipients (also referred to as dry powder matrix) is summarized in example 2, table 4. The placebo group received two empty devices.

Volunteers were logged daily for symptoms (including local symptoms such as nasal discharge, nasal pain/discomfort, nasal congestion, rhinorrhea, nasal itching, epistaxis, headache, and systemic symptoms such as daily oral temperature, myalgia, nausea, vomiting, abdominal cramps, diarrhea, and loss of appetite) 7 days after receiving either dose of the two doses of the Norwalk VLP vaccine, dry powder matrix alone, or placebo. Acquiring an interim medical history at each follow-up visit (day 7+1, day 21+2, day 28+2, day 56+2, and day 180+ 14); the volunteers were asked for disease, medication, and doctor visit during the session. Volunteers were asked to report all serious or severe adverse events, including events not requested during follow-up visits. Volunteers were evaluated for CBC and serum creatinine, glucose, AST, and ALT on days 7 and 28 (7 days after each immunization) and, if abnormal, the abnormal laboratory tests were followed until the tests became normal or stable.

Blood was collected prior to immunization and at days 7+1, 21+2, 28+2, 56+2, and 180+14 to measure serum antibodies to the norwalk VLP vaccine by enzyme-linked immunosorbent assay (ELISA). Peripheral blood lymphocytes were collected to detect antibody secreting cells by ELISPOT assay prior to and on day 7 after administration of each dose of vaccine, dry powder matrix alone, or placebo. Whole blood was obtained prior to vaccination and at days 21+2, 56+2 and 180+14 post vaccination to isolate cells and frozen for future studies of cell-mediated immunity, including cytokine production in response to norwalk VLP antigens, and lymphoproliferation. Whole stool samples were collected for anti-norwalk VLP sIgA screening prior to immunization and at days 7+1, 21+2, 28+2, 56+2, and 180+ 14. Saliva was collected with a commercial device (Salivette, Sarstedt, Newton, NC) before immunization and on days 7+1, 21+2, 28+2, 56+2, and if mucosal antibodies were positive on day 56, samples were collected on day 180+14 and screened for anti-norwalk VLP sIgA. Blood was also screened for memory B cells on days 0, 21, 56 and 180.

Methods for analyzing blood, stool, and saliva samples collected from immunized individuals or individuals receiving either matrix dry powder or placebo alone are described in detail in example 2.

Example 4: norwalk virus challenge study in humans immunized with Norwalk virus VLP vaccine formulation

A multi-site, randomized, double-blind, placebo-controlled phase 1-2 challenge study was performed in 80 human volunteers immunized with the norwalk VLP vaccine described in example 2 above. Eligible subjects included those aged 18-50 years, good health, expressing type H-oligosaccharide 1 (as measured by positive salivary secretor status) and outside of type B or AB blood. Subjects who were not type H-1 secretors or who had type B or AB blood reported to be more resistant to norwalk virus infection were excluded from the study. Based on these two criteria, at least 80% of the volunteers were expected to be eligible.

After screening, qualified volunteers who met all acceptance criteria were randomly (1:1) divided into one of two equal-sized groups of approximately 40 volunteers in each group. Group 1 was immunized with norwalk VLPs and group 2 received placebo. Volunteers were immunized with 10mg of norwalk VLP vaccine (20 mg dry powder total) or placebo in each nostril. Each 10mg of Norwalk VLP vaccine contains 50 μ g Norwalk VLP, 7mg chitosan, 25 μ g Norwalk VLP1.5mg sucrose and about 1.5mg mannitol. Thus, each volunteer in cohort 1 received a total dose of 100 μ g norwalk VLP antigen at each immunization. Volunteers received vaccine or placebo on days 0 and 21.

The safety of norwalk virus VLP vaccine was assessed compared to placebo. Volunteers were diary to record the severity and duration of adverse events 7 days after each immunization with vaccine or placebo. The occurrence of Severe Adverse Events (SAE) and any major new medical condition was followed 6 months after the last dose of vaccine or placebo and 4 months after challenge with infectious virus.

All volunteers were challenged with infectious norwalk virus between 21-42 days after the second vaccine or placebo (between study days 42-56). Each volunteer received a dose of greater than or equal to 50% human infectious dose (HID50), i.e., the amount of infectious virus expected to cause disease in at least 50% of the volunteers in the placebo group. HID50 is between about 48 and about 480 virus equivalents norwalk virus. Norwalk virus is mixed with sterile water and administered orally. 500mg of sodium bicarbonate in water was taken prior to inoculation to prevent the virus from being broken down by gastric acid and pepsin. A second intake of sodium bicarbonate solution (500 mg of sodium bicarbonate in water) was performed 5 minutes after oral inoculation with infectious virus. Volunteers were left at the test facility for at least 4 days and at least 18 hours after the symptoms/signs of acute gastroenteritis (vomiting, diarrhea, loose stools, abdominal pain, nausea, and fever) disappeared.

Several metrics were tested to determine the efficacy of the norwalk VLP vaccine in preventing or alleviating the symptoms/signs of acute gastroenteritis induced by viral challenge. Clinical symptoms of acute gastroenteritis were recorded in all volunteers and documented by the investigator at the study site. Disease symptoms/signs from cohort 1 receiving the vaccine were compared to cohort 2 placebo recipients.

Serum and stool samples were collected from all volunteers before immunization with vaccine or placebo and routinely after challenge. Serum samples were analyzed for IgA and IgG, titers against novok VLPs by ELISA. Norwalk antigen and norwalk RNA were tested in stool samples by ELISA and PCR, respectively, indicating the presence of virus, the amount of virus shed from the intestine, and the duration of virus shed. Additional laboratory studies, including serum chemistry, BUN, creatinine, and liver function tests were performed on subjects with illness after challenge until symptoms/signs were resolved.

The results from the vaccine group (group 1) and the placebo group (group 2) were compared to assess the protective efficacy of the vaccine against norovirus disease in general (primary endpoint) and/or its efficacy in ameliorating symptoms/signs (severity and days of disease) and/or reduction in the presence, amount and/or duration of viral shedding (secondary endpoint).

The invention is not to be limited in scope by the specific embodiments described, which are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and compositions are within the scope of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings using no more than routine experimentation. Such modifications and equivalents are intended to fall within the scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference.

Citation or discussion of a reference herein shall not be construed as an admission that it is prior art with respect to the present invention.

Reference to the literature

1.Glass,RI,JS Noel,T Ando,RL Fankhauser,G Belloit,A Mounts,UDParasher,JS Bresee and SS Monroe.The Epidemiology of Enteric Calicivirusesfrom Human:A Reassessment Using New Diagnostics.J Infect Dis 2000；181(Sup 2):S254-S261.

2.Hardy,ME.Norwalk and“Norwalk-like Viruses”in EpidemicGastroenteritis.Clin Lab Med 1999；19(3):675-90.

3.Jiang,X,DY Graham,KN Wang,and MK Estes.Noralk Virus Genome Cloningand Characterization.Science 1990；250:1580-1583.

4.Jiang,X,M Want,DY Graham,and MK Estes.Expression,Self-Assembly,andAntigenicity of the Norwalk Virus Capsid Protein.J Virol 1992；66:6527-6532.

5.Glass,P,LJ White,JM Ball,I Leparc-Goffart,ME Hardy,and MKEstes.Norwalk Virus Open Reading Frame 3 Encodes a Minor Structural Protein.JVirol 2000；74:6581-6591.

6.Lindesmith,L,C Moe,S Marionneau,N Ruvoen,X Jiang,L Lindblad,PStewart,J LePendu,and R Baric.Human Susceptiblity and Resistance to NorwalkVirus Infection.Nat Med 2003；9:548-553.

7.Parrino,TA,DS Schreiber,JS Trier,AZ Kapikian,and NRBlacklow.Clinical Immunity in Acute Gastroenteritis Caused by Norwalk Agent.NEngl J Med 1977；297:86-89.

8.Wyatt,RG,R Dolin,NR Blacklow,HL DuPont,RF Buscho,TS Thornhill,AZKapikian,and RM Chanock.Comparison of Three Agents of Acute InfectiousNonbacterial Gastroenteritis by Cross-challenge in Volunteers.J Infect Dis1974；129:709.

9.Ball,JM,DY Graham,AR Opekum,MA Gilger,RA Guerrero,and MKEstes.Recombinant Norwalk Virus-like Particles Given Orally to Volunteers:Phase I Study.Gastroenterology 1999；117:40-48.

10.Tacket,CO,MB Sztein,GA Losonky,SS Wasserman,and MK Estes.Humoral,Mucosal,and Cellular Immune Responses to Oral Nowalk Virus-like Particles inVolunteers.Clin Immunol 2003；108:241.

11.Guerrero,RA,JM Ball,SS Krater,SE Pacheco,JD Clements,and MKEstes.Recombinant Norwalk Virus-like Particles Administered Intranasally toMice Induce Systemic and Mucosal(Fecal and Vaginal)Immune Responses.J Virol2001；75:9713.

12.Nicollier-Jamot,B,A Ogier,L Piroth,P Pothier,and EKohli.Recombinant Virus-like Particles of a Norovirus(Genogroup II Strain)Administered Intranasally and Orally with Mucosal Adjuvants LT and LT(R192G)in BALB/c Mice Induce Specific Humoral and Cellular Th1/Th2-like ImmuneResponses.Vaccine 2004；22:1079-1086.

13.Periwal,SB,KR Kourie,N Ramachandaran,SJ Blakeney,S DeBruin,D Zhu,TJ Zamb,L Smith,S Udem,JH Eldridge,KE Shroff,and PA Reilly.A Modified CholeraHolotoxin CT-E29H Enhances Systemic and Mucosal Immune Responses toRecombinant Norwalk Virus-like Particle Vaccine.Vaccine 2003；21:376-385.

14.Isaka,M,Y Yasuda,S Kozuka,T Taniguchi,K Matano,J Maeyama,T Komiya,K Ohkuma,N Goto,and K Tochikubo.Induction of systemic and mucosal antibodyresponses in mice immunized intranasally with aluminium-non-adsorbeddiphtheria toxoid together with recombinant cholera toxin B subunit as anadjuvant.Vaccine 1999；18:743-751.

15.Kozlowski,PA,S Cu-Uvin,MR Neutra,and TP Flanigan.Comparison of theoral,rectal,and vaginal immunization routes for induction of antibodies inrectal and genital tract secretions of women.Infect Immun 1997；65:1387-1394.

16.Mestecky,J,SM Michalek,Z Moldoveanu,and MW Russell.Routes ofimmunization and antigen delivery systems for optimal mucosal immuneresponses in humans.Behring Inst Mitt 1997；33-43.

17.Wu,HY,and MW Russell.Nasal lymphoid tissue,intranasalimmunization,and compartmentalization of the common mucosal immunesystem.Immunol Res 1997；16:187-201.

18.Evans,JT,CW Cluff,DA Johnson,MJ Lacy,DH Persing,and JRBaldridge.Enhancement of antigen-specific immunity via the TLR4 ligands MPLadjuvant and Ribi 529.Expert Rev Vaccines 2003；2:219-229.

19.Baldridge,JR,Y Yorgensen,JR Ward,and JT Ulrich.Monophosphoryllipid A enhances mucosal and systemic immunity to vaccine antigens followingintranasal administration[In Process Citation].Vaccine 2000；18:2416-2425.

20.Yang,QB,M Martin,SM Michalek,and J Katz.Mechanisms ofmonophosphoryl lipid A augmentation of host responses to recombinant HagBfrom Porphyromonas gingivalis.Infect Immun 2002；70:3557-3565.

21.Baldrick,P,D Richardson,G Elliott,and AW Wheeler.Safety evaluationof monophosphoryl lipid A(MPL):an immunostimulatory adjuvant.Regul ToxicolPharmacol 2002；35:398-413.

22.Baldridge,JR,P McGowan,JT Evans,C Cluff,S Mossman,D Johnson,and DPersing.Taking a toll on human disease:Toll-like receptor 4 agonists asvaccine adjuvants and monotherapeutic agents.Expert Opin Biol Ther2004；4:1129-1138.

23.Persing,DH,RN Coler,MJ Lacy,DA Johnson,JR Baldridge,RM Hershberg,and SG Reed.Taking toll:lipid A mimetics as adjuvants andimmunomodulators.Trends Microbiol 2002；10:S32-37.

24.Illum,L.Nasal drug delivery--possibilities,problems andsolutions.J Control Release 2003；87:187-198.

25.Illum,L,I Jabbal-Gill,M Hinchcliffe,AN Fisher,and SSDavis.Chitosan as a novel nasal delivery system for vaccines.Adv Drug DelivRev 2001；51:81-96.

26.Davis,SS.Delivery of peptide and non-peptide drugs through therespiratory tract.Pharm Sci Technol Today 1999；2:450-456.

27.Bacon,A,J Makin,P J Sizer,I Jabbal-Gill,M Hinchcliffe,L Illum,SChatfield,and M Roberts.Carbohydrate biopolymers enhance antibody responsesto mucosally delivered vaccine antigens.Infect Immun 2000；68:5764-5770.

28.van der Lubben,IM,JC Verhoef,G Borchard,and HE Junginger.Chitosanfor mucosal vaccination.Adv Drug Deliv Rev 2001；52:139-144.

29.vander Lubben,IM,JC Verhoef,G Borchard,and HE Junginger.Chitosanand its derivatives in mucosal drug and vaccine delivery.Eur J Pharm Sci2001；14:201-207.

30.Lim,S T,B Forbes,GP Martin,and MB Brown.In vivo and in vitrocharacterization of novel microparticulates based on hyaluronan and chitosanhydroglutamate.AAPS Pharm Sci Tech 2001；2:20.

31.Jabbal-Gill,I,AN Fisher,R Rappuoli,SS Davis,and LIllum.Stimulation of mucosal and systemic antibody responses againstBordetella pertussis filamentous haemagglutinin and recombinant pertussistoxin after nasal administration with chitosan in mice.Vaccine 1998；16:2039-2046.

32.Mills,KH,C Cosgrove,EA McNeela,A Sexton,R Giemza,I Jabbal-Gill,AChurch,W Lin,L Illum,A Podda,R Rappuoli,M Pizza,GE Griffin,and DJLewis.Protective levels of diphtheria-neutralizing antibody induced inhealthy volunteers by unilateral priming-boosting intranasal immunizationassociated with restricted ipsilateral mucosal secretory immunoglobulin.AInfect Immun 2003；71:726-732.

33.McNeela,EA.,I Jabbal-Gill,L Illum,M Pizza,R Rappuoli,A Podda,DJLewis,and KH Mills.Intranasal immunization with genetically detoxifieddiphtheria toxin induces T cell responses in humans:enhancement ofTh2responses and toxin-neutralizing antibodies by formulation withchitosan.Vaccine 2004；22:909-914.

34.Mikszta,JA.,VJ Sullivan,C Dean,AM Waterston,JB Alarcon,JP Dekker,3rd,JM Brittingham,J Huang,CR Hwang,M Ferriter,G Jiang,K Mar,KU Saikh,BGStiles,CJ Roy,RG Ulrich,and NG Harvey.Protective immunization againstinhalational anthrax:a comparison of minimally invasive delivery platforms.JInfect Dis 2005；191:278-288.

35.Huang,J,RJ Garmise,TM Crowder,K Mar,CR Hwang,AJ Hickey,JA Mikszta,and VJ Sullivan.A novel dry powder influenza vaccine and intranasal deliverytechnology:induction of systemic and mucosal immune responses in rats.Vaccine2004；23:794-801.

36.GSK Press Room.www.gsk.com/media/archive.htm

37.Corixa Press Room.www.corixa.com/default.asp？pid＝release_detail& id＝248&year＝2004

38.BioMira Web Site.http://www.biomira.com/business/outLicensing/

Claims (18)

1. Use of a vaccine composition comprising norovirus Virus Like Particles (VLPs) from at least one norovirus gene group in the manufacture of a medicament for eliciting protective immunity against norovirus infection in a human, wherein each type of norovirus VLP is present in an amount of from 1 μ g to 200 μ g.

2. The use of claim 1, wherein the norovirus VLPs are selected from the group consisting of: norovirus genogroup I and genogroup II strains.

3. The use of claim 1, wherein the norovirus VLPs are monovalent VLPs.

4. The use of claim 1, wherein the norovirus VLPs are multivalent VLPs.

5. The use of claim 2, wherein the vaccine further comprises an adjuvant selected from the group consisting of: toll-like receptor (TLR) agonists, monophosphoryl lipid a (mpl), synthetic lipid a, lipid a mimetics or analogs, aluminum salts, cytokines, saponins, Muramyl Dipeptide (MDP) derivatives, CpG oligomers, Lipopolysaccharides (LPS) of gram-negative bacteria, polyphosphazenes, emulsions, virosomes, Cochleates, poly (lactide-co-glycolide) (PLG) microparticles, poloxamer particles, and liposomes.

6. The use of claim 1, wherein the vaccine is in a powder formulation.

7. The use of claim 1, wherein the vaccine is in a liquid formulation.

8. The use of claim 1, wherein the vaccine is formulated for administration to a human by a route selected from the group consisting of: mucosal, intranasal, intramuscular, intravenous, subcuticular, intradermal, subdermal, and transdermal routes of administration.

9. The use of claim 1, wherein each type of norovirus VLP is present in an amount of from 1 μ g to 100 μ g.

10. The use of claim 1, wherein the composition comprises at least one VLP from a norovirus genogroup I virus strain and at least one VLP from a norovirus genogroup II virus strain.

11. The use of claim 10, wherein the composition comprises norwalk virus VLPs and houston virus VLPs.

12. The use of claim 1, wherein the composition further comprises a delivery agent.

13. The use of claim 12, wherein the delivery agent is a bioadhesive.

14. The use of claim 13, wherein the bioadhesive is a mucoadhesive.

15. The use of claim 14, wherein the mucoadhesive is selected from the group consisting of: dermatan sulfate, chondroitin, pectin, mucins, alginates, cross-linked derivatives of polyacrylic acid, polyvinyl alcohol, polyvinyl pyrrolidone, polysaccharides, hydroxypropyl methylcellulose, lectins, pilus/fimbrial proteins, and carboxymethyl cellulose.

16. The use of claim 15, wherein the mucoadhesive agent is a polysaccharide.

17. The use of claim 16, wherein the polysaccharide is chitosan, a chitosan salt, or a chitosan base.

18. The use of claim 1, wherein the composition confers protection from one or more symptoms of norovirus infection.