Influenza vaccine comprising whole virus, split virus or virus like particles with partially glycosylated surface viral glycoprotein

Abstract

The present invention provides a vaccine composition comprising a therapeutically effective amount of whole influenza virus, split influenza virus, virus like particle (VLP) or a combination thereof wherein one or more surface viral glycoproteins of the whole influenza virus, split influenza virus or VLP is partially glycosylated; and wherein the whole influenza virus and the split influenza virus has been attenuated or inactivated. The present invention also provides a method of preparation of the vaccine comprising the steps of injecting working virus seed with an inhibitor of the mannosidase into embryonated chicken egg to inoculate the embryonated chicken egg; incubating the inoculated embryonated chicken egg to propagate the working vims; harvesting allantonic fluids from the inoculated embryonated chicken egg; concentrating and clarifying the harvested allantonic fluids; purifying the concentrated and clarified allantonic fluids using sucrose density centrifugation; applying deglycosylation enzyme to the purified allantonic fluid to partially glycosylate viral surface glycoprotein of the propagated vims within the purified allantonic fluid; removing deglycosylation enzyme from whole vims with partially glycosylated viral surface glycoprotein; and inactivating the vims. The present invention further provides a method of treatment for prevention of influenza infection comprising the step of administering any embodiment of the vaccine of the present invention to a subject.

Description

Influenza Vaccine ComprisingWhole Virus, Split Virus or Virus Like Particles with Partially Glycosylated Surface Viral Glycoprotein

[0001] Background of the Invention

[0002] Influenza pandemics have played a devasting role in history. Despite the availability of vaccines and antiviral therapeutics, influenza viruses cause annual epidemics and pose the threat of a deadly global pandemic that burdens health systems through rising hospitalizations and result in 290,000 to 650,000 deaths worldwide each year (2). Vaccination has remained the best approach for prevention and control of influenza infection. However, current influenza vaccines are only effective against closely matched circulating strains such that, if mismatch occurs between the vaccine strains and circulating strains, reduced efficacy of influenza vaccine can cause poor protection (doi: 10.1080 / 21645515.2015.1086047.) As such, influenza vaccines must be updated and administered every year (3). To improve upon the shortcomings caused by the mismatch between the vaccine and circulating strains, there is a need for influenza vaccines capable of inducing broad immunity effective against multiple strains of influenza that can be manufactured efficiently in largescale.

[0003] Previous studies have shown that partially glycosylated recombinant viral surface glycoproteins such as hemagglutinin (HA) provides substantially better and broader immunity than fully glycosylated viral surface glycoproteins. However, no prior art has explored whether whole viruses or split viruses with partially glycosylated virial surface glycoproteins would provide even better and broader immunity than partially glycosylated recombinant viral surface glycoproteins while safe for humans. In addition, no prior art has provided an efficient method of preparation of whole or split viruses with partially glycosylated virial surface glycoproteins at a large scale that clearly demonstrates large scale commercial production capability.

[0004]

[0005] Summary of the Invention

[0006] A vaccine composition comprising a therapeutically effective amount of whole influenza virus, split influenza virus, virus like particle (VLP) or a combination thereof wherein one or more surface viral glycoproteins of the whole influenza virus, split influenza virus or VLP is partially glycosylated and wherein the whole influenza virus and the split influenza virus has been attenuated or inactivated.

[0007] A method of preparation of the vaccine of the present invention, comprising the steps of injecting working virus seed with an inhibitor of the mannosidase into embryonated chicken egg to inoculate the embryonated chicken egg; incubating the inoculated embryonated chicken egg to propagate the working virus; harvesting allantonic fluids from the inoculated embryonated chicken egg; concentrating and clarifying the harvested allantonic fluids; purifying the concentrated and clarified allantonic fluids using sucrose density centrifugation; applying deglycosylation enzyme to the purified allantonic fluid to partially glycosylate viral surface glycoprotein of the propagated virus within the purified allantonic fluid; removing deglycosylation enzyme from whole virus with partially glycosylated viral surface glycoprotein; and inactivating and / or attenuating the virus.

[0008] A method of treatment for prevention of influenza infection comprising the step of administering any embodiment of the vaccine of the present invention to a subject.

[0009]

[0010] Brief Description of the Drawings

[0011] FIG. 1 illustrates embodiments of partially glycosylated viral surface glycoproteins of whole influenza virus, split influenza virus or influenza virus like particle (VLP) vaccine of the present invention.

[0012] FIG. 2 is a flow chart depicting an embodiment of the method of preparation of the whole influenza virus, split influenza virus or influenza VLP vaccine with partially glycosylated viral surface glycoproteins of the present invention.

[0013] FIG. 3A illustrates GMT HA Inhibition (HAI) Titre of monoglycosylated whole virus vaccine, monoglycosylated split virus vaccine and monoglycosylated recombinant HA protein vaccine each with 5ug of HA. FIG. 3B illustrates GMT microneutralization (MN) Titre of monoglycosylated whole virus vaccine, monoglycosylated split virus vaccine and monoglycosylated recombinant HA protein vaccine each with 5pg of HA. FIGs. 3A and 3B show that monoglycosylated whole virus vaccine and monoglycosylated split virus vaccine each induced substantially higher HAI and microneutralization than monoglycosylated recombinant HA protein.

[0014] FIG. 4 illustrates stability of monoglycosylated whole influenza virus vaccine, monoglycosylated split influenza virus vaccine and monoglycosylated recombinant HA protein vaccine for up to 1100 days plotting hemagglutination activity (log2) (HAU) on the y axis and number of days on the x axis. As shown in the figure, monoglycosylated whole influenza virus vaccine and monoglycosylated influenza split virus vaccine are each substantially more stable than monoglycosylated recombinant HA protein vaccine.

[0015] FIG. 5A illustrates inhibition of IVR-190 NA activity of monoglycosylated whole influenza virus vaccine, monoglycosylated split influenza virus vaccine and monoglycosylated recombinant HA protein vaccine each with 5 pg of HA plotting NAI titre on the y axis. FIG. 5 A shows that monoglycosylated whole influenza virus vaccine and monoglycosylated split influenza influenza virus vaccine each induced substantially higher NAI than monoglycosylated recombinant HA protein. FIG. 5B illustrates inhibition of IVR- 190 M2e activity of monoglycosylated whole influenza vims vaccine, monoglycosylated split influenza vims vaccine and monoglycosylated recombinant HA protein vaccine each with 5pg of HA plotting OD450 on the y axis. FIG. 5B shows that monoglycosylated whole influenza virus vaccine and monoglycosylated split influenza vims vaccine each induced substantially higher M2e inhibition than monoglycosylated recombinant HA protein. In addition, FIG. 5B shows that monoglycosylated whole influenza vims vaccine induced substantially higher M2e inhibition than monoglycosylated split influenza vims vaccine.

[0016] FIG. 6 A quantifies IgA induced by monoglycosylated whole influenza vims vaccine, monoglycosylated split influenza vims vaccine and monoglycosylated recombinant HA protein vaccine each with 5pg of HA plotting end-point IgA Titre on the y axis. As shown in FIG. 6A, monoglycosylated whole influenza vims vaccine induces substantially higher IgA than monoglycosylated split influenza vims vaccine, and monoglycosylated split influenza vims vaccine, in turn, induces substantially higher IgA than monoglycosylated recombinant HA protein. FIG. 6B illustrates vaccine protection against IVR-127 vims challenges of mice vaccinated with monoglycosylated whole influenza vims vaccine, monoglycosylated split influenza vims vaccine and monoglycosylated recombinant HA protein vaccine each with 5pg of HA and each based upon IVR-190. As shown in FIG. 6B, monoglycosylated whole influenza vims vaccine provides substantially higher protection than both monoglycosylated split influenza vims vaccine and monoglycosylated recombinant HA protein when mice challenged with IVR-217.

[0017] FIG. 7A, 7B and 7C illustrates CD4+ / CD8 + T cell immune response induced by monoglycosylated whole influenza vims vaccine, monoglycosylated split influenza vims vaccine and monoglycosylated recombinant HA protein vaccine each with 5 pg of HA. As shown in FIG. 7A, 7B, and 7B, monoglycosylated whole influenza vims vaccine induces more CD4+ / CD8 + T cell immune response than monoglycosylated split influenza vims vaccine and monoglycosylated recombinant HA protein vaccine.

[0018] FIG. 8 A illustrates HA inhibition (HAI) of 2pg HA / dose of monoglycosylated split influenza vims vaccine with JR300 adjuvant, 2pg HA / dose of monoglycosylated splitinfluenza virus vaccine without JR 300 adjuvant and JR300 only. As shown in FIG. 8A, monoglycosylated split influenza virus vaccine with JR300 adjuvant resulted in substantially higher HAI than monoglycosylated split influenza virus vaccine without JR 300 adjuvant and JR300 adjuvant alone. FIG. 8B illustrates microneutralization (MN) of 2pg HA / dose of monoglycosylated split influenza virus vaccine with JR300 adjuvant, 2pg HA / dose of monoglycosylated split influenza virus vaccine without JR 300 adjuvant and JR300 only. As shown in FIG. 8B, monoglycosylated split influenza virus vaccine with JR300 adjuvant resulted in substantially higher MN than monoglycosylated split influenza virus vaccine without JR 300 adjuvant and JR300 adjuvant alone.

[0019] FIG. 9 illustrates vaccine protection from virus challenges of 2pg HA / dose of monoglycosylated split influenza virus vaccine with JR300 adjuvant, 2pg HA / dose of monoglycosylated split influenza virus vaccine without JR 300 adjuvant and JR300 only each vaccine. As shown in FIG. 9, monoglycosylated split influenza virus vaccine with JR300 adjuvant provides substantially higher protection than both monoglycosylated split influenza virus vaccine without JR 300 adjuvant and JR 300 adjuvant alone when mice challenged with IVR-217.

[0020] FIG. 10 illustrates optimization of upstream inoculation parameters for monoglycosylated IVR-190 virus (Virusmg) production in embryonated chicken eggs. FIG. 10A illustrates determination of the optimal infectious dose at 103, 104or 105dilution for virus inoculation and incubation time of 48 and 72 hours for virus production. The virus yield from each test group was evaluated by hemagglutination assay and the data are presented as HA unit titers. FIG. 10B illustrates evaluation of the effect of kifunensine concentration on Virusmgproduction from kifunensine concentration of 0 up to 400 pg / mL plotted on the x axis and total HA unit titres plotted on the y axis. FIG. 10C illustrates examination of the minimal sufficient concentration of kifunensine for Virusmgproduction. Western blot analysis of the viral HAs in the purified virus pretreated with various concentrations of kifunensine following Endo H digestion. Arrowheads indicate viral HA with high-mannose-type glycans (HAlhm / HA2hm) and monoglycosylated viral HA (HAlmg / HA2mg).

[0021] FIG. 11 illustrates monoglycosylated IVR-190 split influenza virus vaccine (IVR- 190mg) production process development. FIG. 11 A provides a schematic overview of three embodiments of the processes designed for IVR-190mg production of the present invention adding Endo H at three different stages including allantoic fluid (AF) harvest (Method I), AF concentration (Method II), and ultracentrifugation (Method III). The different glycosylation states of viral HA are indicated. HAhm, viral HA with high mannose-type N-glycans; HAmg,viral HA with GlcNAc at its N-glycosylation sites. The outcome of each process stage is labeled and illustrated. FIG. 1 IB illustrates measurement of kifunensine content in each stage of the three processes. The residue of kifunensine in each step is presented as a percentage (%). FIG. 11C illustrates comparative measurements of Endo H contents between the initial5 addition step and final vaccine bulk from the three designed processes. The residue of Endo H in each step is presented as a percentage (%). FIG. 1 ID illustrates comparison of IVR- 190mgyield from the three processes. The recovery yield (presented as a percentage) was used to measure the total protein in the final vaccine bulks produced by the three methods determined by microBCA assay. FIG. HE illustrates SDS-PAGE analysis of the final0 vaccine bulks produced from the three methods and kifunensine pretreated virus bulk (HAhm). Arrowheads indicate the viral NA hm, HAs (HAlhm, HA2hm, HAlmg, and HA2mg), NP (nucleoprotein), and Ml (matrix-1) proteins.

[0022] FIG. 12 illustrates purification, characterization, and long-term stability of IVR- 190mg. FIG. 12A illustrates purification of IVR-190fgand IVR-190mgby zonal sucrose density5 gradient ultracentrifugation and fractionation analysis by western blotting against HA1 proteins. FIG. 12B illustrates the profiles of sucrose content (Brix%), total protein content (TP; pg / mL), and HA activity were plotted for the indicated fractions. FIG. 12C are representative electron micrographs of IVR-190fgor IVR-190mgvirions observed by transmission electron microscopy. FIG. 12D illustrates statistical analysis of the spike lengths of the IVR-190fgand IVR-190mg0 virions. FIG. 12E illustrates measurements of HA activity of the IVR-190fgand IVR-190mgvaccine bulks after inactivation. FIG. 12F illustrates evaluation of the long-term stability of HA potency from the IVR-190fgand IVR-190mgvaccine bulks by single radial immunodiffusion (SRD) assay. FIG. 12G illustrates determination of the long-term stability of viral protein content from the IVR-190fg and IVR-190mgvaccine bulks by microBCA. FIG. 12H illustrates5 analysis of the particle size distribution of the IVR-190fgand IVR-190mgvaccine bulks by dynamic light scattering (DLS). Comparative study of the agglutination abilities of the IVR- 190fg and IVR-190mg samples to chicken RBCs. FIG. 121 illustrates microscopic observations of hemagglutination between RBCs and vaccine samples. Vaccine (100 pg / ml) was serially diluted 2-fold and mixed with a 0.5% RBC suspension in a 1 : 1 ratio. The mixture was 0 incubated for 30 mins at room temperature, samples were applied to a glass slide, and hemadsorption was observed under a microscope 1, 24, or 120 h postincubation. Pictures were taken with a DP80 digital camera (Olympus). All negative control samples did not exhibit any visible agglutination. The red blocks in the pictures indicate obvious RBC agglutination with vaccine samples. FIG. 12J illustrates time-course determination of hemagglutination unit titers(HAU) resulting from treatment with vaccine samples in the microtiter plate. The relative agglutination ability of each vaccine sample with RBCs over time was calculated and the data are presented as a percentage.

[0023] 5

[0024] Detailed Description of the Invention

[0025] The compositions and methods of the present invention can comprise, consist of, or consist essentially of the essential elements and limitations of the invention described herein, as well as any of the additional or optional ingredients, components, or limitations described herein. 0

[0026] Unless defined otherwise, 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. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), provides one skilled in the art with a general guide to many of the terms used in the present application. 5

[0027] As used in the specification and claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a” cell includes a plurality of cells, including mixtures thereof.

[0028] “About” in the context of amount values refers to an average deviation of maximum of ±20%, ±10% or ±5% based on the indicated value. For example, an amount of about 300 mol % anionic lipid refers to 30 mol %±6 mol %, 30 mol %±3 mol % or 30 mol %±1.5 mol % anionic lipid with respect to the total lipid / amphiphile molarity.

[0001] An “effective amount” or “therapeutically effective amount” is an amount sufficient to effect beneficial or desired results. The exact amount required will vary from subject to subject, depending on the species, age, general condition of the subject, the severity of the5 disease, the particular vaccine, its mode of administration, the desired outcome, and the like. In certain embodiments of the present invention, a “therapeutically effective amount” of a compound or pharmaceutical composition is that amount effective for preventing influenza infection in a subject or a biological sample (e.g., in cells). In certain embodiments, infection is prevented in about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about0 70%, about 80%, about 90%, about 95%, about 99% or about 100% of the subjects receiving a therapeutically effective amount of the vaccine of the present invention. An effective amount can be administered in one or more administrations, applications or dosages.

[0029] The term “polypeptide” is used in its conventional meaning, i.e., as a sequence of amino acids. The polypeptides are not limited to a specific length of the product. Peptides,oligopeptides, and proteins are included within the definition of polypeptide, and such terms may be used interchangeably herein unless specifically indicated otherwise. This term also does not refer to or exclude post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. A polypeptide may be an entire protein, or a subsequence thereof. Particular polypeptides of interest in the context of this invention are amino acid subsequences comprising CDRs and being capable of binding an antigen or influenza virus-infected cell.

[0030] A “subject,” “individual” or “patient” is used interchangeably herein, which refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, pets, laboratory test animals (e.g., mouse, rabbit, rat, guinea pig. hamster), captive wild animals (e.g., fox. deer), and any other organisms who can benefit from the vaccines of the present disclosure. There is no limitation on the type of animal that could benefit from the presently described vaccines. A subject regardless of whether it is a human or non-human organism may be referred to as a patient, individual, animal, host, or recipient.

[0031] Influenza viruses are segmented negative- strand RNA viruses and belong to the Orthomyxoviridae family. There are three main types of influenza virus: A, B and C. Type A strains of influenza virus can cause severe illness and are the only type to have caused human pandemics. The H5N1 strain is a type A influenza virus. Type B strains cause sporadic human cases and small-scale outbreaks. Type C strains only rarely cause human infection and have not caused large outbreaks. Of the influenza A viruses, only subtypes Hl, H2 and H3 have been transmitted easily between humans.

[0032] Influenza A virus comprises 9 structural proteins and codes additionally for one nonstructural NS1 protein with regulatory functions. The non-structural NS1 protein is synthesized in large quantities during the reproduction cycle and is localized in the cytosol and nucleus of the infected cells. The segmented nature of the viral genome allows the mechanism of genetic reassortment (exchange of genome segments) to take place during mixed infection of a cell with different viral strains. The 9 structural proteins include hemagglutinin (HA) and neuraminidase (NA) viral surface glycoproteins which are used to classify the influenza A virus into various subtypes.

[0033] The HA of the influenza virus is a homotrimeric transmembrane protein with an ectodomain composed of a globular head and a stem region. Both regions carry N-linked oligosaccharides, the biosynthesis of which follows the general pathways of N glycosylation.The HA is the most abundant protein in influenza viruses responsible for binding to sialic acid on the surface of target cells. Viral transmission begins with a critical interaction between the HA glycoprotein and sialic acid (SA) containing glycans on the host cell surface. It is an important antigen of the influenza virus because the HA induces neutralizing antibodies against the influenza virus infection (4, 5). However, changes in the amino acid sequence of HA of the influenza virus can often affect effectiveness of vaccination from previous influenza seasons. (6, 7). The functional properties of HA are affected by glycosylation at specific sites. The glycans around the antigenic peptide epitopes interfere with the access of antibodies which likely causes antigenic drift of influenza viruses. Previous studies on HAs revealed that the peptide sequences with glycosylation are highly conserved, and the HA receptor binding specificity was affected by the absence of a complex glycan chain near the receptor binding site. In addition, the proteolytic activation of HA was also modulated by the glycans near the cleavage site, influencing infectivity of influenza viruses. The extensive variations in structure and number of glycosylation sites on the head region have been shown among different subtypes of the influenza A viruses, whereas the stem oligosaccharides were more conserved and required for fusion activity. These findings indicate the importance of HA glycosylation on its activity.

[0034] The NA is a homotetramer and the second most abundant influenza surface glycoprotein after the HA. The NA plays an integral role in the viral replication cycle during which the NA cleaves sialic acid residues from newly formed virions as they are being released from infected cells (8, 9). Serologic studies showed that mice treated with mAbs that bind to a conserved antigenic domain of N1 were protected against challenges comprising lethal doses of seasonal and pandemic viruses (10). Furthermore, purified NA proteins have been shown to induce robust NA-based immunity and protect against influenza virus infection (11, 12), indicating that the NA protein is immunogenic, and, therefore, the NA protein is also considered a candidate for the development of universal influenza vaccines.

[0035] There are 16 known HA subtypes and 9 known NA subtypes such as, for example, Hl, H3, and H5 and N1 and N2, respectively. As such, a subtype may be referred to by both H and N numbers, such as, for example, H1N1, H5N1, and H5N2. The terms specifically include all strains (including extinct strains) within each subtype, which usually result from mutations and show different pathogenic profiles. Such strains will also be referred to as various “isolates” of a viral subtype, including all past, present and future isolates. Accordingly, in this context, the terms “strain” and “isolate” are used interchangeably. Subtypes contain antigens based upon an influenza A virus. The antigens may be based upona hemagglutinin viral surface protein and can be designated as “HA antigen.” In some instances, such antigens are based on the protein of a particular subtype, such as, for example, an Hl subtype and an H5 subtype, which may be designated an Hl antigen and an H5 antigen, respectively.

[0036] The Matrix-2 (M2) glycoprotein is an integral membrane protein of 97 amino acids that is expressed at the surface of infected cells with an extracellular N-terminal domain of 18 to 23 amino acid residues, an internal hydrophobic domain of approximately 19 residues, and a C-terminal cytoplasmic domain of 54 residues. (Zebedee S L, et al. J. Virol. 1988 August; 62(8):2762-2772).

[0037] Other viral surface glycoproteins comprise polymerase protein PB 1 , polymerase protein PB2, polymerase protein PA, nucleoprotein (NP), matrix (Ml), nuclear export protein (NEP).

[0038] Glycosylation of viral surface glycoprotein such as HA, NA and M2 is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. A “sequon” is a sequence of three consecutive amino acids in a protein that can serve as the attachment site to a polysaccharide (sugar) called an N-linked- Glycan. This is a polysaccharide linked to the protein via the nitrogen atom in the side chain of asparagine (Asn). A sequon is either Asn-Xaa-Ser or Asn-Xaa-Thr, where Xaa is any amino acid except proline. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5 -hydroxyproline or 5- hydroxylysine may also be used. While the sequon Asn-X-Ser / Thr is absolutely required for the attachment of N-linked oligosaccharides to a glycoprotein (Marshall R D, Biochemical Society Symposia 40, 17-26 1974), its presence does not always result in glycosylation and some sequons in glycoproteins can remain unglycosylated. (Curling E M, et al., Biochemical Journal 272, 333-337 1990).

[0039] In eukaryotes, the O-linked glycosylations are synthesized in the Golgi or rough Endoplasmic Reticulum (ER) from nucleotide sugars. The N-linked glycosylations are synthesized from a common precursor, and subsequently processed. It is known that addition of N-linked carbohydrate chains is important for stabilization of folding, prevention of degradation in the endoplasmic reticulum, oligomerization, biological activity, and transport of glycoproteins. The addition of N-linked oligosaccharides to specific Asn residues plays an important role in regulating the activity, stability or antigenicity of mature proteins of viruses(Opdenakker G. et al FASEB Journal 7, 1330-1337 1993). The viral surface glycoproteins also provide protection against neutralizing antibodies as a “glycan shield.” As a result, strong host-specific selection is frequently associated with codon positions of potential N- linked glycosylation. Consequently N-linked glycosylation sites tend to be conserved across strains and clades.

[0040] As used in the present disclosure, the terms “deglycosylated” or “partially glycosylated” glycoprotein such as HA, NA or M2 denotes a glycoprotein that has one or more sugars removed from the glycan structure of a fully glycosylated instance of the glycoprotein. A fully glycosylated instance of the glycoprotein means the glycosylation state at the glycosylation site of the antigen glycoprotein when expressed in a host or a subject’s cell. The terms “deglycosylated” and “partially glycosylated” glycoprotein may be used interchangeably. In an embodiment, the partially glycosylated glycoprotein substantially retains its native conformation / folding. In an embodiment, a “partially glycosylated” glycoprotein is one in which a deglycosylation process leaves a monoglycosylation, a diglycosylation or a triglycosylation at one or more glycosylation sites present on the glycoprotein. In an embodiment, a partial glycosylation site contains a smaller glycan structure (containing fewer sugar units) as compared to the site in a fully glycosylated instance of the glycoprotein, but the partially glycosylated protein substantially retains its native conformation / folding. A “partially glycosylated” protein may alternatively be generated by introducing glycosylation at an unglycosylated site of a protein such that the added glycosylation sequence is smaller than the glycan structure at that same site in a fully glycosylated instance of the glycoprotein. A “partially glycosylated” protein may also be generated by synthesizing a viral glycoprotein sequence, or fragment thereof, introducing glycosylated amino acid units (e.g., GlcNAc- Arginine moieties) at glycosylation sites of the sequence, such that the added glycan structure is smaller than the glycan structure at that site in a fully glycosylated instance of the glycoprotein.

[0041]

[0042] Whole Virus, Split Virus and Virus-Like Particles (VLP) with Partially Glycosylated Viral Surface Glycoproteins as Basis for Vaccines

[0043] Viral surface glycoproteins such as HA, NA, M2 etc. . . are important targets for vaccine development. Importantly, certain viral protein sequences at these glycosylation sites are often highly conserved. However, these highly conserved regions are not readily accessible by the host's immune system due in part to the glycosylation covering or blocking those regions from the host’s immune system. As a result, the immune system can onlyusually target highly variable regions but not the highly conserved regions, reducing the subject’s immunity against subsequent viral infections as the variable regions mutate.However, if the immune system is somehow able to access these highly conserved regions, then antibodies directed to these highly conserved sequences are capable of providing a route towards inoculation against viruses that either have variable regions that mutate often or have conserved regions that are so thickly glycosylated as to be substantially inaccessible to the immune system to be able to raise these antibodies that target the conserved regions.

[0044] One way of rendering these highly conserved regions accessible to the immune system is to partially remove glycosylation so as to expose conserved regions of the viral surface glycoprotein. Importantly, as discussed above, complete removal of the sugars from the glycoprotein has been shown to cause the protein to denature; in many viral surface glycoproteins, glycosylation is a key component to tertiary structure of the glycoprotein. For example, in the 0- and N-linked glycoproteins, the first sugar (N-acetylglucosamine for N- glycoprotein and N-acetylglucosamine or N-acetylgalactosamine for O-glycoproteins) is essential to preserve the tertiary structure of the glycoprotein while the rest of the sugars are not important. Treatment of N-glycoproteins with deglycosylation enzymes such as endoglycosidase (endoH) will remove the sugar chain and keep the N-acetyl glucosamine attached to the protein. Mannosidases may also be used to cleave N-glycoproteins to di- or triglycans, which are expressly contemplated herein as possible vaccines due to the ability of the immune system to access the conserved glycosylation sites on the proteins even with di-, tri-, and larger deglycosylated proteins. Because the highly conserved glycosylation regions are now partially glycosylated and thereby exposed to the immune system, antibodies are generated against the highly conserved regions.

[0045] Whole and split influenza viruses with monoglycosylated viral surface glycoproteins of the present invention each provides substantially higher immunogenicity in comparison to monoglycosylated recombinant HA polypeptide as shown in the Examples in connection with FIGs. 3A, 3B, 5A, 5B, 6A, 7 A, 7B, 7C, 8A and 8B. Notably, the monoglycosylated whole vims vaccine even elicited substantially higher immunogenicity than the monoglycosylated split vims vaccine as shown in Examples in connection with FIGs. 5B, 6A, 7B, 7C, 8A and 8B. The whole and split vimses with monoglycosylated viral surface glycoproteins of the present invention also each provides substantially broader immunogenicity in comparison to partially glycosylated recombinant HA as shown in Examples in connection with FIG. 6B and 9. Furthermore, the whole vims with monoglycosylated viral surface glycoproteins of the present invention also provides substantially better protection in comparison to split vimswith monoglycosylated viral surface glycoproteins and partially glycosylated recombinant HA with and without adjuvant as shown in Examples in connection with FIG. 6B and 9. Importantly, the whole and split viruses with monoglycosylated viral surface glycoproteins of the present invention also provides substantially higher stability in comparison to partially glycosylated recombinant HA as shown in Examples in connection with FIG. 4.

[0046] Without being limited by theory, the antibodies raised by the whole or split viruses with partial glycosylated viral surface glycoproteins of the present invention are able to attack both the less or non-glycosylated region(s) that are more likely to mutate as well as the glycosylated region which is highly conserved. Such antibodies that target the glycosylated region of a glycoprotein are capable of pushing glycan chain away to bind to the highly conserved regions around the glycosylation sites since such antibodies have thermodynamically substantially higher affinity for the protein than the carbohydrate. In addition, the whole or split virus and, by extension, virus like particles (VLP) with partially glycosylated viral surface glycoprotein of the present invention provides substantially better and broader protection than the recombinant partially glycosylated surface glycoprotein vaccine because partially glycosylated viral surface glycoproteins of the whole, split virus and VLP have substantially better conformation than the recombinant partially glycosylated viral surface glycoproteins. Furthermore, without being limited by theory, partially glycosylating most of (over 80%, 90%, 95%, 97%, 99%) of at least 5 glycosylation sites of viral surface glycoproteins that usually exist on an influenza virus may expose important antigen or combination of antigens not previously discovered that play important role in vaccine efficacy.

[0047] Therefore, the present invention provides a vaccine composition comprising a therapeutically effective amount of whole influenza virus wherein one or more surface viral glycoproteins of the whole influenza virus is partially glycosylated and wherein the whole influenza virus has been attenuated or inactivated. In an embodiment, the partially glycosylated viral surface glycoproteins are tri-, di- or monoglycosylated. In an embodiment, the glycoprotein comprises HA, NA, M2, PB1, PB2, PA, NP, Ml, NEP or a combination thereof. In an embodiment, the glycoprotein comprises any viral surface glycoprotein or a combination thereof of an influenza virus of a particular strain and / or variant. In an embodiment, at least about 50% to at least about 99% of the glycosylate sites of the viral surface glycoproteins of the whole virus is tri-, di- or monoglycosylated such as at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97% about 99% including any percentages and percentageranges falling within these percentage values. In an embodiment, at least about 50% to at least about 99% of all the glycosylate sites of the HA, NA or M2 viral surface glycoproteins of the whole virus is tri-, di- or monoglycosylated such as at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97% about 99% including any percentages and percentage ranges falling within these percentage values. In an embodiment, at least five glycosylate sites of the HA, NA or M2 viral surface glycoproteins of the whole virus is at least about 50% to at least about 99% of tri-, di- or monoglycosylated such as at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97% about 99% including any percentages and percentage ranges falling within these percentage values. In an embodiment, the whole virus comprises any strain and variant of the influenza virus.

[0048] The present invention also provides a vaccine composition comprising a therapeutically effective amount of split influenza virus wherein one or more surface viral glycoproteins of the split influenza virus is partially glycosylated and wherein the split influenza virus has been attenuated or inactivated. In an embodiment, the partially glycosylated viral surface glycoproteins is tri-, di- or monoglycosylated. In an embodiment, the glycoprotein comprises HA, NA, M2, PB1, PB2, PA, NP, Ml, NEP or a combination thereof. In an embodiment, the glycoprotein comprises any viral surface glycoprotein or a combination thereof of an influenza virus of a particular a strain and / or variant. In an embodiment, at least about 50% to about 100% of the viral surface glycoproteins of the split virus is tri-, di- or monoglycosylated such as at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97% about 99% including any percentages and percentage ranges falling within these percentage values. In an embodiment, at least about 50% to about 100% of the HA, NA or M2 viral surface glycoproteins of the split virus is tri-, di- or monoglycosylated such as at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97% about 99% including any percentages and percentage ranges falling within these percentage values. In an embodiment, the whole virus comprises any strain and variant of the influenza virus.

[0049] The present invention also provides a vaccine composition comprising a therapeutically effective amount of virus like particle (VLP) wherein one or more surface viral glycoproteins of the VLP is partially glycosylated and wherein the VLP has been attenuated or inactivated. In an embodiment, the partially glycosylated viral surfaceglycoproteins is tri-, di- or monoglycosylated. In an embodiment, the glycoprotein comprises HA, NA, M2, PB1, PB2, PA, NP, Ml, NEP or a combination thereof. In an embodiment, the glycoprotein comprises any viral surface glycoprotein or a combination thereof of an influenza virus of a particular a strain and / or variant. In an embodiment, about 50% to about 100% of the viral surface glycoproteins of the VLP is tri-, di- or monoglycosylated such as about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% including any percentages and percentage ranges falling within these percentage values. In an embodiment, the whole virus comprises any strain and variant of the influenza virus.

[0050] In an embodiment, an embodiment of the vaccine of the present invention does not comprise a therapeutically effective amount of the partially glycosylated whole virus. In an embodiment, an embodiment of the vaccine of the present invention does not comprise a therapeutically effective amount of the partially glycosylated split virus. In an embodiment, an embodiment of the vaccine of the present invention does not comprise a therapeutically effective amount of the partially glycosylated VLP. In an embodiment, an embodiment of the vaccine of the present invention does not comprise a therapeutically effective amount of partially glycosylated recombinant viral surface glycoprotein peptide or polypeptide. In an embodiment, an embodiment of the vaccine of the present invention does not comprise a therapeutically effective amount of partially glycosylated recombinant HA peptide or polypeptide and / or a therapeutically effective amount of partially glycosylated recombinant NA peptide or polypeptide.

[0051] In an embodiment, an embodiment of the vaccine of the present invention is free of or does not comprise any of the partially glycosylated whole virus. In an embodiment, an embodiment of the vaccine of the present invention is free of or does not comprise any of the partially glycosylated split virus. In an embodiment, an embodiment of the vaccine of the present invention is free of or does not comprise any of the partially glycosylated VLP. In an embodiment, an embodiment of the vaccine of the present invention is free of or does not comprise any partially glycosylated recombinant viral surface glycoprotein peptide or polypeptide. In an embodiment, an embodiment of the vaccine of the present invention is free of or does not comprise any partially glycosylated recombinant HA peptide or polypeptide and / or a therapeutically effective amount of partially glycosylated recombinant NA peptide or polypeptide.

[0052] Method of Preparation

[0053] The present invention further provides a method of preparation of any embodiment of the vaccine composition of the present invention comprising whole virus, split virus or VLP with partially glycosylated viral surface glycoprotein.

[0054] An embodiment of the method of preparation of the present invention is illustrated in FIG. 2. As shown in FIG. 2, the method of preparation 1000 of any embodiment of the vaccine of the present invention begins with step 1010 of injecting working virus seed with an inhibitor of mannosidase such as kifunensine into the embryonated chicken egg. In an embodiment, the virus seed comprises any strain or variant of the influenza virus. In an embodiment, the virus seed comprising working virus has concentration of about 108'5EID50 / 0.1 mL diluted to about 103to 107such as about 103, 104, 105, 106or 107including any number and number ranges falling within these values. As shown in Examples in connection with FIG. 10 A, 108'5EID50 / 0. 1 mL diluted to about 105provided the highest HA unit titer. Therefore, in an embodiment, the virus seed comprising working virus has concentration of about 108'5EID50 / 0.1 mL diluted to about 105.

[0055] With regards to the amount of kifunensine to be used in step 1010, as shown in theExamples in connection with FIG. 10B, with 200 pg / ml or higher concentrations of kifunensine, we found that the virus hemagglutination titers (HAU) showed no significant difference compared with the kifunensine-untreated group. Therefore, in an embodiment, the concentration of kifunensine may be about lOOpg / mL to about 500 pg / mL such as about 100 pg / mL, about 150 pg / mL, about 200 pg / mL, about 250 pg / mL, about 300 pg / mL, about 350 pg / mL, about 400 pg / mL, about 450 pg / mL or about 500 pg / mL including any concentration and concentration ranges falling within these values. Since kifunensine and deglycosylation enzyme such as Endo H treatments are required for monoglycosylated vaccine production, eliminating these extra materials from a vaccine is a challenge regarding vaccine quality and safety issues, and, thus, using the minimum amount of kifunensine is desirable. Examples in connection with FIG. 10C shows that 300 pg / mL kifunensine in combination with Endo H treatment of step 1060 yields the highest amount of propagated virus. Therefore, in an embodiment, the concentration of kifunensine may be about 200pg / mL to about 300 pg / mL.

[0056] In an embodiment, the embryonated chick egg may be about 5 to about 20 days old at the time of inoculation such as about 5, about 7, about 10, about 12, about 14, about 16, about18 or about 20 including any days and day ranges falling within these values. In an embodiment, the working virus injection is performed with the embryonated chicken eggkept at about 30-38°C such as about 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C or 38°C including any temperature or temperature ranges falling within these values.

[0057] Next, in step 1020, the working virus injected embryonated chicken egg is incubated at about 30-38°C such as about 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C or 38°C including any temperature or temperature ranges falling within these values for about 24 hours to about 90 hours to propagate the viruses such as about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 54 hours, about 60 hours, about 66 hours, about 72 hours, about 78 hours, about 84 hours, or about 90 hours including any hours or hour ranges falling within these values.

[0058] In step 1030, allantonic fluids are harvested from the inoculated embryonated chicken egg. In step 1040, the harvested allantonic fluids are concentrated for clarification. In an embodiment, concentration and clarification is performed by filtering the allantonic fluid for the viruses. In an embodiment, each filtering process reduces the volume of the allantonic fluid by a factor of about 10 to result in not only a more clarified solution but also a lOx higher concentration of the propagated viruses. In an embodiment, concentration adjusts HAU titres to be between about 211to about 26such as about 211, 210, 29, 28, 27or 26including any HAU titres and number HAU titre ranges falling within these values.

[0059] Next, in Step 1050, the concentrated and clarified virus containing allantonic fluid is purified using sucrose density centrifugation. In an embodiment, concentration of the sucrose gradient is 5%-to-55% (w / v) such as about 5%, about 10%, about 15%, about 20%, about25%, about 30%, about 35%, about 40%, about 45%, about 50% or about 55% including any concentration percentage and concentration percentage ranges falling within these values. In an embodiment, the sucrose density centrifugation is performed for about 10 to about 20 hours such as about 10, about 12, about 14, about 15, about 16, about 18, or about 20 hours including any hours and hour ranges falling within these values. In an embodiment the sucrose density centrifugation is performed at about 20,000 rpm to about 40,000 rpm such as about 20,000 rpm, 25,000 rpm, 30,000 rpm, 35,000 rpm or 40,000 rpm including any rpm or rpm ranges falling within these values. In an embodiment, the sucrose density centrifugation is performed at about 2°C to 6 °C such as about 2 °C, 3 °C, 4 °C, 5 °C or 6 °C including any temperature and temperature ranges falling within these values. In an embodiment, concentration of total glycoprotein in the resulting purified virus fluid volume is about 5000 to 7000 pg / mL such as about 5000 pg / mL, about 5500 pg / mL, about 6000 pg / mL, about 6500 pg / mL or about 7000 pg / mL including any concentration or concentration rangesfalling within these values. In an embodiment, other types of centrifugation known in the art may also be used.

[0060] In step 1060, partial glycosylation treatment is performed to deglycosylate viral surface glycoproteins. In an embodiment, deglycosylation enzyme is applied to the viruses to deglycosylate viral surface glycoproteins of the viruses. In an embodiment, the deglycosylation enzyme comprises any deglycosylation enzymes known in the art. In an embodiment, the deglycosylation enzyme comprises P-specific galactosidase, a- neuraminidase, N-acetylglucosaminidase (PNGase F) or a combination thereof. In an embodiment, the p-specific galactosidase comprises p(l-4)-galactosidase, 0(1-6)- galactosidase or P(l-3)-galactosidase. In an embodiment, the a-neuraminidase comprises Endo-a-N-acetylgalactosaminidase H (Endo H), a(l-4)-Galactosidase or a(l-6)- Galactosidase. In an embodiment, the deglycosylation enzyme comprise Endo H. In an embodiment, Endo H treatment comprises adding Endo H to the purified virus fluid wherein the ratio of total amount of Endo H enzymes to the total amount of viral surface proteins by weight is about 1: 1 to about 1 :20 such as about 1 :1, about 1 :2, about 1:5, about 1:7.5, about 1:10, about 1 :12.5 about 1:15, about 1 :15.5 or about 20 including any ratio or ratio range falling within these values.

[0061] Next in step 1070, the deglycosylation enzyme is removed from the whole virus with partially glycosylated surface viral glycoproteins.

[0062] The method described in FIG. 2 1000 comprises Method III. Alternatively, two other methods of preparations are possible, Method I and Method II, by performing the application and removal of deglycosylation enzyme related steps 1060 and 1070 earlier in the process.For example, in Method I, the application of deglycosylation enzyme related steps 1060 and 1070 may be performed after harvest step of 1030 and before the concentration and clarification step 1040. In Method II, the application of deglycosylation enzyme related steps 1060 and 1070 may be performed after the concentration and clarification step 1040 and before the sucrose purifying step 1050.

[0063] Comparison of the Methods I, II and III are discussed in further detail in the Examples in connection with FIGs. 1 IB, 11C, 1 ID, 1 IE. As shown in FIG. 1 IB, over 96% of kifunensine could be easily removed after the TFF concentration step and was undetectable in the following de-sucrose, De-Endo H, and split vaccine bulk steps for all three methods.With regards to Endo H removal, FIG. 11C shows that that the production of IVR-190mg by Method III could reduce 98.8% of Endo H consumption compared with Method I, which was set as 100%. Moreover, Endo H can be effectively removed through the De-Endo H TFFprocess from Method III, retaining only 0.003% Endo H in the vaccine bulk. With regards to influenza virus yield, FIG. 12 D shows that, using single radial immunodiffusion (SRD) assay, the HA yield obtained from Method III was the highest and was set as 100% recovery whereas Method I and Method II merely exhibited 35% and 51% retention of HA activity compared with Method III. The results show that a comparable quality of monoglycosylated viruses can be achieved by Methods I, II, and III. However, Method III uses less Endo H in the process, retains less Endo H in the vaccine, and recovers more IVR-190mg than the other methods, thus supporting the possibility of pilot-scale production.

[0064] In step 1080, to produce vaccine comprising whole virus with partially glycosylated viral surface glycoprotein, whole virus with partially glycosylated viral surface glycoprotein is attenuated or inactivated. In an embodiment, the attenuation or inactivation step comprises application of heat, formalin, Triton X-100, 0-propiolactone and / or formaldehyde treatments. In an embodiment, the concentration of formalin for inactivation of the virus is about 0.2% to about 0.001% such as about 0.2%, 0.15%, 0.1%, 0.05%, 0.01%, 0.005% or 0.001% including any percentage concentration or percentage concentration range falling within these values.

[0065] Alternatively, in Step 1085, the whole virus is subjected to splitting to produce split virus with partially glycosylated viral surface glycoprotein. In step 1090, vaccine comprising inactivated split virus with partially glycosylated viral surface glycoprotein was prepared by ether treatment, followed by 0.01% formalin inactivation, and verified for the absence of viral infectivity by serial passage assay in eggs.

[0066] In an embodiment, the method of preparation of the present invention is capable of producing at least 50, at least 100, at least 150 at least 200 doses of any embodiment of the vaccine of the present invention wherein each dose of the vaccine comprises at least 5 pg of HA, 10 pg of HA or 15 pg of HA.

[0067]

[0068] Pharmaceutical Compositions

[0069] According to another aspect, any embodiment of the partially glycosylated whole, split virus or VLP of the present invention can be included in a pharmaceutical composition or formulation together with additional active agents, carriers, vehicles, adjuvants, excipients, or auxiliary agents identifiable by a person skilled in the art upon reading of the present disclosure.

[0070] In an embodiment, any embodiment of the vaccine comprising whole virus, split virus or VLP with partially glycosylated viral surface glycoprotein of the present invention further comprises an adjuvant, wherein the adjuvant may comprise squalene-based emulsionadjuvant or any aluminum-based vaccine adjuvant. In an embodiment, the squalene -based emulsion adjuvant of the present invention comprises squalene and sorbitan trioleate. In an embodiment, the squalene-based emulsion adjuvant of the present invention comprises about 20 to 50 mg / mL squalene oil such as about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, or about 50 mg / mL including all concentration and concentration ranges falling within these values. In an embodiment, the squalene-based emulsion adjuvant of the present invention comprises about 3 to 6 mg / mL sorbitan trioleate such as about 3 mg / mL, about 3.5 mg / mL, about 4 mg / mL, about 4.5 mg / mL, about 5 mg / mL, about 5.5 mg / mL, or about 6 mg / mL including all concentration and concentration ranges falling within these values.

[0071] In addition, suitable adjuvants for inclusion in the vaccine of the present disclosure include those that are well known in the art, such as complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IF A), squalene, squalane, alum, and various oils. In an embodiment, the squalene-based emulsion adjuvant comprises a commercially available adjuvant such as MF59®.

[0072] The pharmaceutical compositions preferably comprise at least one pharmaceutically acceptable carrier. In such pharmaceutical compositions, the whole virus, split virus or VLP with partially glycosylated virial surface glycol protein forms the “active agent.” Supplementary active compounds can also be incorporated into the compositions. A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, intravenous, intradermal, subcutaneous and intranasal. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: freeze-dry powder reconstituted with sterile saline or water before injection, a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol, or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediamine tetraacetic acid; buffers such as acetates, citrates, or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0073] Suitable pharmaceutically acceptable carriers for the compositions of the present invention are described in the standard pharmaceutical texts. See, e.g., “Remington's Pharmaceutical Sciences”, 18th Ed., Mack Publishing Company, Easton, Pa. (1990). Specificnon-limiting examples of suitable pharmaceutically acceptable carriers include water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the composition can further contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents that enhance the antiviral effectiveness of the composition.

[0074] Any embodiment of the partially glycosylated whole, split virus or VLP of the present disclosure may also advantageously comprise an adjuvant peptide in an effective adjuvant amount. As will be apparent to one skilled in the art, the optimal concentration of the adjuvant peptide or peptides will necessarily depend upon the specific peptide(s) used, the characteristics of the patient, the immunogen used, and the nature of the viral infection for which the treatment or prophylaxis is sought. These factors can be determined by those of skill in the medical and pharmaceutical arts in view of the present disclosure. In general, the adjuvant peptides are most desirably administered at a concentration level that will generally afford adjuvant activity without causing any harmful or deleterious side effects. Generally, an effective adjuvant amount is desired. An effective adjuvant amount refers to an amount of an adjuvant peptide which is capable of stimulating an immune response to an administered immunogen.

[0075]

[0076] Method of Treatment

[0077] The present invention also provides a method of treatment for the prevention of influenza infection comprising the step of administration of any embodiment of the whole virus influenza vaccine, split virus influenza vaccine or VLP vaccine with partially glycosylated viral surface glycoprotein of the present invention to a subject. The step of administration may comprise parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation) and intranasal.

[0078] For parenteral administration, any embodiment of the vaccine of the present invention may be administered by intranasal, intravenous, subcutaneous, intramuscular, intraperitoneal, or intradermal injection, alone or in compositions further comprising pharmaceutically accepted carriers. For administration by injection, it is preferred to use any embodiment of the whole virus influenza vaccine, split virus influenza vaccine or VLP vaccine with partially glycosylated viral surface glycoprotein of the present invention in a solution in a sterile aqueous vehicle which may also contain other solutes such as buffers or preservatives as well as sufficient quantities of pharmaceutically acceptable salts or of glucose to make the solution isotonic.

[0079] Pharmaceutical compositions suitable for an injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany. N.J.) or phosphate buffered saline (PBS). In all cases, the composition should be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.

[0080] The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0081] Sterile injectable solutions can be prepared by incorporating the active composition in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and freeze-drying, which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0082] Because the whole or split virus with partially glycosylated virial surface glycoprotein vaccines of the present invention have shown activity against respiratory viruses, they can also be delivered locally to the respiratory system, for example to the nose, sinus cavities, sinus membranes or lungs. The inactivate whole or split viruses, vaccines, or pharmaceutical compositions containing inactivate whole or split viruses or vaccines, can be delivered to the respiratory system in any suitable manner, such as by inhalation via the mouth or intranasally.The present compositions can be dispensed as a powdered or liquid nasal spray, suspension, nose drops, a gel or ointment, through a tube or catheter, by syringe, or by submucosal infusion. The vaccines of the present invention may be conveniently delivered in the form of an aerosol spray using a pressurized pack or a nebulizer and a suitable propellant, e.g., without limitation, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be controlled by providing a valve to deliver a metered amount. Inhaler can be a powder inhaler. Examples of intranasal formulations and methods of administration can be found in PCT publications WO 01 / 41782, WO 00 / 33813, and U.S. Pat. Nos. 6,180,603; 6,313,093; and 5,624,898. The latter-cited U.S. patents are incorporated herein by reference and for all purposes. A propellant for an aerosol formulation may include compressed air, nitrogen, carbon dioxide, or a hydrocarbon based low boiling solvent. The vaccines of the present disclosure can be conveniently delivered in the form of an aerosol spray presentation from a nebulizer or the like. In some aspects, the active ingredients are suitably micronised so as to permit inhalation of substantially all of the active ingredients into the lungs upon administration of the dry powder formulation, thus the active ingredients will have a particle size of less than 100 microns, desirably less than 20 microns, and preferably in the range 1 to 10 microns. In one embodiment, any embodiment of the vaccine of the present invention are packaged into a device that can deliver a predetermined, and generally effective, amount via inhalation, for example a nasal spray or inhaler.

[0083] It is advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0084] It will be further appreciated that the amount of a whole virus, split virus or VLP with partially glycosylated viral glycoprotein of the present disclosure that is useful in treatment or prevention of influenza will vary not only with the route of administration, the nature of the condition being treated, and the age and condition of the patient, and will ultimately be at the discretion of the attendant physician or veterinarian. In general however, a suitable dose for parenternal administration may be in the range of from about 10-50pg / dose such as about 10 pg / dose, about 15 pg / dose, about 20 pg / dose, about 25 pg / dose, about 30 pg / dose, about 35 pg / dose, about 40 pg / dose, about 45 pg / dose or about 50 pg / dose including any weight per dose or weight range per dose falling within these values. A suitable dose for intranasaladministration may be in the range of from about l-20ijg / dose such as about 1 |ig / dose, about 5 |ig / dose, about 10 |ig / dose, about 15 pg / dose or about 20 |ig / dose including any weight per dose or weight range per dose falling within these values.

[0085] Toxicity and therapeutic efficacy of such compositions may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected location to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[0086] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the disclosure, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0087] The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health or age of the subject, and other diseases present.

[0088] It is to be understood that both the foregoing general description and detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. In general, the terms used in the disclosure should not be construed to limit the technology to the specific embodiments disclosed in the specification, unless the above detailed description explicitly defines such terms. Accordingly, the actual scope of the technology encompasses the disclosed embodiments and all equivalent ways of practicing or implementing the technology.

[0089]

[0090] Examples

[0091] Materials and Experimental Method

[0092] Ethics statement

[0093] All animal experiments were conducted in accordance with guidelines of theLaboratory Animal Center of the Infectious Disease Core Facility (BioTReC), Taiwan. The animal-use protocols were reviewed and approved by the BioTReC Institutional Animal Care and Use Committee (protocol No. BioTReC-109-D-02). To perform virus challenge, mice are placed in an anesthetic inhalator chamber containing isoflurane (Initial phase: 5% Maintained phase: 1.5%~2.5%) for 1 minute before intranasally infected with influenza virus. All the tested animals were euthanized by 100% CO2 inhalation for 5 min followed by cervical dislocation to minimize the animals’ suffering after completion of the experimental protocol or when weight loss of 30% occurred (according to the guidelines of approved animal-use protocols; protocol No. BioTReC- 109-D-02).

[0094] Cell, viruses, and antibody

[0095] The vaccine strain of H 1 N1 influenza virus A / Brisbane / 02 / 2018 IVR- 190 (IVR- 190) was obtained from the Adimmune Corporation through the material transfer agreement. A / Shenzhen / 227 / 1995(HlNl) (SZ / 1995), A / Califomia / 7 / 2009(HlNl) (CA / 2009), A / Victoria / 1 / 2020(H1N1) (VIC / 2020), A / Guangdong Maonan / SWL1536 / 2019 (CNIC- 19O9)(H1N1) (GD / 2019), standard reagents of A / Brisbane / 02 / 2018 IVR-190 antigen and antiserum were purchased from the UK National Institute for Biological Standards and Control. All viruses were inoculated into the allantoic cavities of 10-day-old specific pathogen-free embryonated chicken eggs at 34°C for 48 h. Harvested allantoic fluid was aliquoted and stored at -80 °C. The 50% tissue culture infective doses of viruses in Madin- Darby canine kidney (MDCK, Bioresource Collection and Research Center, Hsinchu, Taiwan) cells and LD50 of virus in BALB / c mice were determined before experiments. The antibody used in this study was polyclonal: N1 (ab21305) from Abeam.

[0096]

[0097] Glycopeptide analysis with liquid chromatography-MS / MSa

[0098] The glycan compositions of viral HA and NA with different glycosylations were analyzed by mass spectrometry. Before loading onto SDS-PAGE, the purified virus was mixed with nonreducing sample buffer and heated to 95 °C for 10 min. After staining with Coomassie blue, the bands of viral HA and NA were cut into small pieces for in-gel digestion with trypsin and chymotrypsin (PROMEGA) overnight at 37 °C. The digested viral HA andNA peptide samples were extracted and analyzed by liquid chromatography-MS / MS (Agilent Technologies; Thermo Scientific) (31).

[0099] [000100] Hemagglutination inhibition assay (HAD and Microneutralization (MN) [000101] HAI and MN were performed according to the protocol developed in our previous study with minor modification as described below (35). Briefly, to determine HAI titers, mice sera were treated with a receptor-destroying enzyme (RDE) overnight and heat-inactivated for 40 min. The sera were tested in 2-fold dilutions starting with an initial dilution of 1 :20, and then admixed with 4 HA units of individual H1N1 viruses. After incubation at room temperature for 1 h, the fresh prepared 0.5% suspension of chicken red blood cells (Jianrong) (Tseng et al., 2019) was added and hemagglutination was assessed by observation after 1 h. HAI titer is defined as the reciprocal of the highest dilution that showed > 50% inhibition of hemagglutination. The detection of vaccine-induced neutralizing antibody titers against influenza viruses were performed with a World Health Organization recommended protocol as described previously. After tracing with HRP-conjugated secondary antibody and developed with TMB substrate, the absorbance was measured at 450 nm with a Microplate Reader (SpectraMax iD3, molecular devices). Untreated virus control (VC), uninfected cell control (CC), and back titration of virus infectivity are included on each plate. The neutralization (%) was calculated as { 1-(OD of each well - average OD of CC wells) / (average OD of VC wells - average OD of CC wells) } xl00%.[000102] Neuraminidase Inhibition Titer (NAI Titer)[000103] The NAI titer was determined according to the protocol developed from minimization and optimization of the conventional assay (24).[000104] [000105] Endpoint ELISA titer of Neuraminidase (NA), stem HA, and IgG isotype.[000106] ELISA plates were coated with I pg / well N1 glycoprotein (A / Idaho / 01 / 2017), purchased from Sino Biological, at 4°C overnight, and blocked with 1% BSA in PBST. NA- coated wells were incubated with serum samples of indicated dilution for 1 h at 37°C, traced with HRP-conjugated secondary Ab, and developed with TMB substrate. They were washed with PBST three times between each step of ELISA. Finally, for the ELISA readout, the absorbance of each well at 450 nm was measured using a microplate reader (SpectraMax iD3). The endpoint ELISA titer is defined as the reciprocal of the highest analyte dilution that gives a reading two times above the mean background reading. To quantify the anti-stem HAantibodies, sera from immunized mice were added to stem HA (#4900)-coated 96-well plates with two-fold serial dilution. The remaining experimental processes are the same as the ELISA assay described above. To measure the quantity of IgG isotype, sera from immunized mice were added to HA-coated 96-well plates with two-fold serial dilution. The remaining experimental processes are the same as the ELISA assay described above except that the bound antisera from mice were detected by HRP-conjugated secondary Ab of IgG, IgGl, IgG2a, IgG2b, and IgG3.[000107][000108] Competition ELISA [000109] To investigate the vaccine-induced antibody response directed against the HA head domain with conserved epitopes or HA stem, competition ELISA assay was performed as described previously (13). The specific anti-HA stem monoclonal antibodies of F10 and FI6 were expressed using HEK293F cells and purified using Protein A agarose. The monoclonal antibodies of FluA-20 and 5J8 against the conserved epitope of HA head domain were purchased from Leinco Technologies. 50 L of sera from immunized mice were added to HA- coated 96-well microplates with two-fold serial dilution and incubated for 1 h at RT. Further, 50pL of monoclonal antibodies (mAb) F10, FI6, 5J8, or FluA-20 were individually added into each well for another 1 h. After washing out unbound antibodies with lx PBST, the F10, 5J8, or FluA-20 were detected using goat-anti-human IgG-HRP (Bethyl). The absorbance (OD 450 nm) of the wells were read using Microplate Reader (SpectraMax iD3, molecular devices). The percentage of competition was calculated as follows: % competition = (A- B) / (A-C) xl00%, where A is the average OD value of adding F10, FI6, 5J8, or FluA-20 only, B is the OD value of ewell-addingding sera first and then F10, FI6, 5J8, or FluA-20 individually, and C is the OD value of wells without sera and F10, FI6, 5J8, or FluA-20. [000110][000111] Depletion of viral-specific neutralizing antibodies[000112] The purified ectodomain of HA and HA stem protein (50 pg / well) were separately coated on tissue culture plates (12-well) overnight at 4°C. After blocking with 3% BSA and washing with PBS, sera from immunized mice were incubated with the precoated antigens for 1 h at 25°C to deplete the HA-specific neutralizing antibodies against ectodomain of HA or stem HA. The same antisera were depleted in parallel with BSA as a control. Repeated depletion cycles were performed four times and the antisera with / without depletion of HA- specific antibodies were further applied for microneutralization.[000113][000114] Antibody-dependent cell-mediated cytotoxicity (ADCC) reporter assay[000115] ADCC measurement was performed as previously described with some modifications (17). MDCK cells were seeded on 96-well polystyrene cell culture microplates (white, Greiner) at a density of 1.2 x 104 cells per well. After incubation for 20 h, MDCK cells were infected with influenza viruses at multiplicity of infection (MOI) of 0.01 for 18 h. The virus infected target cell was replaced with RPMI 1640 medium containing 4% low IgG FBS followed by the addition of serial dilutions of antisera from vaccinated mice for 30 min incubation at 37 °C. Jurkat effector cells expressing mouse FcyRI II (Promega) were further added into each well at an effector / target ratio of 5:1. After 6 hr incubation in a humidified incubator at 37°C with 5% CO2, Bio-Gio Luciferase Assay Reagent (Promega) was added to the plates and luminescence (RLU) was quantified using a plate reader (GloMax® Navigator System). Fold induction was calculated by dividing the RLU of the wells of interest by the mean of control wells containing infected target cells and Jurkat effector cells with no serum.[000116] [000117] IL-4, IFN-y, and Granzyme B ELISpot Assays[000118] Spleens were harvested (Day 35) from mock-treated and vaccinated mice and splenocytes were isolated for ELISpot assay according to the manufacturer's instructions (R & D Systems, Minneapolis, MN, USA). Briefly, cells were depleted of erythrocytes by treatment with lx RBC lysis buffer (BioLegend, San Diego, CA). Following extensive wash with PBS, cells were resuspended in RPMI medium with 10% fetal bovine serum (Gibco, San Diego, CA) and 50 LIM 2-mercaptoethanol. Cell viability was determined by trypan blue staining. The cell suspensions (lxl06 / well) from the spleen were incubated in pre-coated anti-mouse IFN-y, IL-4, or Granzyme B (GrzB) plates and stimulated (20 h) with inactivated virus (5 pg / mL). An included positive control or additional wells of cells were mock stimulated as negative control for ELISpot assays. The plates were incubated overnight in a humidified incubator at 37°C with 5% CO2. After four times washing with PBS-0.05% Tween, IFN-y, IL-4, or GrzB spots were detected by biotinylated IFN-y, IL-4, or GrzB detection antibodies followed by addition of streptavidin-alkaline phosphatase and development with BCIP / NBT substrate solution. Spots were scanned and counted using AID ELISpot Reader System and EliSpot 5.0 iSpot image analyzer (AID, Strassberg, Germany). The results were expressed as the number of spot / lxl06 spleen cells.[000119][000120] IVR-190mg toxicology study design[000121] Three- to four-month-old male and female SPF New Zealand White (NZW) rabbits (obtained from Oriental Yeast Co., Ltd., Japan) were randomly assigned to three groups (6 animals / sex / group) before intramuscular dosing, and 4 / 6 animals / sex / group were designated as the main study animals to receive three vaccinations and sacrificed on Day 32 (dosing phase end sacrifice); the other 2 / 6 animals / sex / group were designated as recovery animals to be administered three vaccinations and then maintained for 2 weeks postvaccination before sacrifice on Day 43 (recovery end sacrifice). The detailed immunization protocol is described in Fig. 4A, and all blood sampling was conducted by the CRO. The dosing regimen was designed based on a previous clinical study of the seasonal influenza virus vaccine, as described below.[000122]Dose Level Dose Volume No. of AnimalsGroup(pg HA / animal / injection) '“uw““““•‘v Main Recovery1 4 Males + 2 Males +0 0.5(Vehicle control*) 4 Females 2 Females2 4 Males + 2 Males +15 0.5(Low dose) 4 Females 2 Females3 4 Males + 2 Males +75 0.5(High dose) 4 Females 2 Females[000123][000124] Detection of residual kifunensine[000125] The kifunensine content in vaccine bulk or process intermediates was detected by HPLC analysis. Vaccine or processed protein samples containing kifunensine were pretreated with acetonitrile to precipitate the proteins. The recovered kifunensine in the supernatant at more than 80% of the nominal concentration was determined by HPLC analysis. A stock solution of kifunensine was prepared by dissolving the compound in DMSO to a final concentration of 5 mg / ml and stored at -20 °C. Chromatographic separation was carried out on a Hypercarb column. The mobile phase was composed of potassium phosphate buffer (mobile phase A) and methanol (mobile phase B), and gradient elution was carried out at a flow rate of 0.6 mL / min. The column temperature was maintained at 50 °C. The wavelength of UV detection was set at 229 nm. The calibration curves were plotted with a series of concentrations of kifunensine standard solutions. The working linear range of the developedHPLC method was 78-2,500 ng / mL with a detection limit of 40.4 ng / ml and good linearity was shown by a regression coefficient (R2) of over 0.99.[000126] Detection of residual Endo H[000127] A sandwich ELISA method was established to evaluate the specific Endo H protein content in vaccine bulk and process intermediates. Rabbit and mouse anti-Endo H were prepared in-house, and the optimized dilution for detection was determined. Briefly, 96-well microplates were coated with rabbit anti-Endo H diluted 10,000-fold in carbonate coating buffer (pH 9.4) at 4 °C overnight. After blocking, 100 u L of protein sample was 2-fold serially diluted in blocking buffer, and the Endo H standards were added to each well for incubation at 37 °C for 1 h. After washing 3 times with PBST, the detection antibody (mouse anti-Endo H diluted 10,000-fold) was added for incubation at 37 °C for 1 h. Following washing with PBST 3 times, the bound detection antibody was traced by anti-mouse IgG conjugated-HRP and developed with TMB substrate. The absorbance (OD 450 nm) of the wells was measured by a microplate reader (SpectraMax iD3, Molecular Devices). For quantitative detection, a standard curve and quality control Endo H sample with a known concentration (as intra- or interassay control) were included in each microplate. All data were log2 transformed, and the standard curves were plotted by linear regression. The Endo H concentration in each sample was calculated from the corresponding standard curves. The working linear range of the developed sandwich ELISA method was 0.097-12.5 ng / mL with a detection limit of 0.049 ng / ml and good linearity was shown by a regression coefficient (R2) of over 0.99.[000128] Electron microscopy of ViruSfo and Virus^[000129] Purified virus (1 pg) was placed on a 200- mesh copper grid (Electron Microscopy Sciences) and stained with 2% methylamine tungstate (TED PELLA INC.). Micrographs were obtained by electron microscopy (FEG-TEM, FEI Tecnai G2 TF20 S-TWIN). Typically, the micrographs captured between 20-50 particles per image, providing enough particles to determine the morphology and numerical statistics for each sample. Approximately 10 TEM fields from each sample were randomly selected to calculate the length of the HA spike. The statistical data of the spike lengths of the virions are presented in a column plot. [000130] Statistical analysis[000131] The significance in differences between vaccine groups was statistically computed applying t-test using GraphPad Prism software, Version 6.0. Data were presented as mean ± SEM and differences were considered significant at *P < 0.05; **P <0.01; ***P < 0.001.[000132][000133] Example 1 Preparation of Vaccines[000134] Preparation of Monoglycosylated Whole Virus Influenza Vaccine and Monoglycosylated Split Virus Influenza Vaccine[000135] Working virus seeds with 200 pg / mL kifunensine were propagated in 10-day-old embryonated chicken eggs at 34 °C. The infected allantoic fluids were harvested at 48 h postinoculation and concentrated for clarification. The viruses were purified by sucrose density centrifugation in a 5%-to-55% (w / v) sucrose gradient for 15 h at 30,000 rpm at 4 °C. After the optimal Endo H treatment / removal processes, half amount of the monoglycosylated whole virus was preserved to prepare the whole virus vaccine, and the rest part of the monoglycosylated whole virus was subjected to splitting by ether treatment. Finally, the monoglycosylated whole virus and split virus vaccines were prepared by 0.01% formalin inactivation and verified for the absence of viral infectivity by serial passage assay in eggs. The HA content of inactivated virus vaccines was determined by SRID assay. The recombinant HA protein vaccine (rHA) was prepared as follows. The ectodomain of HA (amino acids 1—520) derived from the IVR-190 virus was expressed using HEK293F cells (Thermo Fisher) and purified by a Ni-NTA column (Cytiva). The protein concentration was quantified via microBCA assay for vaccination. The molecular weight of the rHA protein was verified by size exclusion chromatography analysis (Superdex 200 Increase 10 / 300).[000136] To find the optimal infectious dose for virus production, the results showed that the o r c IVR-190 seed virus (10 EID50 / 0.1 mL) prepared in a 10 -fold dilution for inoculation and48 h of incubation in eggs were the best conditions for virus productivity (Fig. 10A). Next, we investigated the minimum sufficient concentration of kifunensine to retain the N- glycosylation progress with high-mannose type glycans while completely maintaining production with the egg-based procedure. With 200 pg / ml or higher concentrations of kifunensine, we found that the virus hemagglutination titers (HAU) showed no significant difference compared with the kifunensine-untreated group (Fig. 10B). Following Endo H treatment to reduce the amount of high-mannose type glycans, the molecular weights of HA1 and HA2 of the virus gradually decreased when the kifunensine concentration increased (Fig.10C). In summary, the complete downregulation of viral HA indicated that the manufacturing process utilizing treatment with 300 pg / ml kifunensine combined with Endo H digestion is sufficient to obtain the highest amount of Virusmgfor further pilot-scale production. An ideal process to manufacture the monoglycosylated virus vaccine would use a minimal amount of essential Endo H, which would make the purification process easier and recovery as high aspossible. To investigate the optimized conditions for adding Endo H during the process, we produced 1.2 grams of Endo H with a certified E. coli strain (SHuffle® T7) without phage contamination based on pharmacopeia compliance regulations, and the specifications of this Endo H fulfilled the criteria of biologicals for human use. To explore the optimal Endo H treatment process, the harvested AF from inoculation of 1,000 eggs was separated into three smaller batches and individually tested to evaluate Endo H treatment by the following steps: AF harvest (Method I), AF concentration (Method II), and after density gradient ultracentrifugation (Method III) (Fig. 11 A).[000137] Since kifunensine and Endo H treatments are required for monoglycosylated vaccine production, eliminating these extra materials from a vaccine is a challenge regarding vaccine quality and safety issues. To understand whether kifunensine and Endo H can be removed step-by-step following the downstream processes, specific quantification methods were developed to validate their removal efficiency and residual amount in the final vaccine bulk. The results showed that over 96% of kifunensine could be easily removed after the TFF concentration step and was undetectable in the following de-sucrose, De-Endo H, and split vaccine bulk steps (FIG. 1 IB). The Endo H residue data showed that the production of IVR- 190mg by Method III could reduce 98.8% of Endo H consumption compared with Method I, which was set as 100% (FIG. 11C). Moreover, Endo H can be effectively removed through the De-Endo H TFF process from Method III, retaining only 0.003% Endo H in the vaccine bulk (FIG. 11C). As listed in Table 1, the HA contents of IVR-190mg (total HA amount / total protein amount) were found to be 25.3, 24.2, and 25.8% in Lots #1, 2, and 3, respectively, which is close to those observed from the traditional egg-based seasonal influenza vaccines.The overall recoveries based on HA potency were 18.3, 22.5, and 20.7% for Lots #1, 2, and 3, respectively. Over 96% of the added kifunensine can be efficiently removed by performing a TFF concentration step and further reach an undetectable level in the following SDG (ultracentrifugation), Endo H removal, and splitting / inactivation steps. The Endo H residues in three lots of the vaccine bulk were detected to be 23.3, 12.2, and 36.0 ng / dose. Excluding the uncertified kifunensine and Endo H residues, the IVR-190mg from the three engineering batch runs meets the acceptance criteria for human use as specified by the World Health Organization (WHO) and European Pharmacopeia (EP) (Kon et al., 2016).[000138] To confirm whether the glycan at each glycosylation site of HAmgand NAmgis indeed a single GlcNAc residue, site-specific glycan analysis was performed by liquid chromatography-MS / MS. Glycopeptide analysis results showed that the HAmgcontains seven glycosylation sites in which the monoglycosylation process successfully resulted in singleGlcNAc at > 99% for five glycosylation sites and > 97.9% for the two remaining glycosylation sites (Table 1). Of the eight NAmgglycosylation sites, glycopeptide analysis results showed that the monoglycosylation process successfully resulted in single GlcNAc at > 99% for five glycosylation sites and > 97.3% for the remaining three glycosylation sites (Table 2). These results suggest that the monoglycosylated influenza virus vaccine platform can be applied to the new vaccine strain IVR-190 with large-scale production by using the traditional egg-based virus production method with minimal modification procedure and which may practically be adapted to industrial vaccine production.[000139] The long-term stability of vaccine bulk is one of the key parameters that support a novel vaccine candidate to become a vaccine product. The frequency of test points for longterm studies should be sufficient to establish the stability profile over a given period (normally every 3 months over the first year), including the initial and final time points. To evaluate the long-term stability of IVR-190mg, the hemagglutination activity, HA potency, total protein content, and particle size distribution were determined by conventional methods for human vaccine characteristic analysis (Kon et al., 2016). Over 15 months of study, the collected data indicated that the biological and physical properties of IVR-190mg in terms of HA potency, total protein content, and particle size distribution, were as stable as those of IVR-190fg (Fig. 12F-H). Interestingly, the hemagglutination titer of IVR-190mg was lower than that of the fully glycosylated split vaccine (IVR-190fg), but IVR-190mg displayed strong and lasting agglutination ability against chicken RBCs for several days (Fig. 12E and I- J).Decreasing the glycans from the IVR-190 virus could lead to a change in cell binding affinity between HA and NA rather than loss of biological function, which has not yet been clearly investigated. In summary, these results demonstrated that IVR-190mg has sufficient stability for long-term storage at 2-8 °C for at least 15 months compared with IVR-190fg. [000140] Preparation of recombinant HA proteins[000141] The ectodomain of HA (amino acids 1-520) and head domain (amino acids 62-286) derived from the IVR-190 and CA / 2009 Influenza viruses and stem HA (16) of A / Brisbane / 59 / 2007 (BNE / 2007) were expressed using HEK293F cells (Thermo Fisher) and purified by a Ni-NTA column (Cytiva). The protein concentrations were quantified via microBCA assay for neutralizing antibody depletion, ELISA binding, and competition ELISA trials. The molecular weights of purified proteins were verified by size exclusion chromatography analysis (Superdex 200 Increase 10 / 300).[000142][000143] Example 2 Mouse immunization and virus challenge[000144] Vaccines without Adjuvant[000145] Female BALB / c mice (6- to 8-week-old; n=9) were immunized with the inactivated monoglycosylated whole virus (intranasally), monoglycosylated split virus (intramuscularly), or monoglycosylated rHA (intramuscularly) vaccines (based on an HA content of 5 pg / dose and diluted in 50 pL of PBS) without adjuvants at weeks 0 and 2. The three vaccines were prepared according to Example 1.[000146] Mouse antisera and nasal wash were collected at week 4, and virus challenge was performed at week 5. The immunized mice were challenged intranasally with a lethal dose (10 x LD50) of the H1N1 influenza virus (3.9 x 104 TCID50 of the IVR-217 diluted in 50 pL of PBS) and monitored in 2-day interval for 14 days for survival and weight loss. Results are shown in FIG. 6B. As shown in FIG. 6B, the monoglycosylated whole virus IVR-190 vaccine provided substantially higher protection against the IVR-217 virus than the other two vaccines as 100% of the mice treated with monoglycosylated IVR 190 whole virus vaccine survived as compared with only 30% survival of mice treated with the monoglycosylated split virus vaccine and 0% survival of mice treated with monoglycosylated rHA vaccine. The results indicate that the monoglycosylated whole virus vaccine provides higher as well as broader protection than both monoglycosylated split virus as well as rHA vaccines.[000147] Adjuvanted Vaccines[000148] Female BALB / c mice (6- to 8-week-old; n=10) were immunized with monoglycosylated whole virus vaccine administered intranasally with JR300 with 2 pg HA / dose of HA, monoglycosylated split virus vaccine with 2 pg HA / dose of HA administered intramuscularly with the JR300 adjuvant or JR300 adjuvant only at weeks 0 and 2. Mouse antisera were collected at week 4. At week 5, the immunized mice were challenged intranasally with a lethal dose (10 x LD50) of the H1N1 influenza virus (3.9 x 104 TCID50 of the IVR-217 diluted in 50 pL of PBS) and monitored in 2-day interval for 14 days for survival and weight loss. The results are shown in FIG. 9. As shown in FIG. 9, addition of the JR300 adjuvant substantially increased effectiveness of the monoglycosylated split virus vaccine, but the monoglycosylated whole virus vaccine still provided substantially higher protection as 100% of the mice vaccinated with the adjuvanted whole virus vaccine survived. [000149] The higher and broader protection provided by the monoglycosylated whole virus vaccine may stem from immunogenicity from partial glycosylation of glycoproteins other than HA and NA such as M2 as shown in FIG. 5B in connection with Example 5 as well as whole virus vaccine’s ability to illicit substantially higher T cell activity than themonoglycosylated split virus vaccine as shown in Example 6 in connection with FIG. 7 A, 7B and 7C below.[000150][000151] Example 3 Hemagglutination activity (HAU), Hemagglutination inhibition assay (HAI), and microneutralization (MN)[000152] To assess the hemagglutination activity, for each of the three vaccines, monoglycosylated whole virus vaccine, monoglycosylated split virus vaccine and monoglycosylated rHA vaccine, a vaccine sample with 100 pg / mE total protein and its serial 2-fold diluted samples were mixed with a 0.5% suspension of chicken red blood cells (Jianrong) (Tseng et al., 2019) in 96-well U-bottom plates. Plates were incubated for 30 min, and the endpoint titer value was considered the highest dilution yielding visible agglutination with the naked eye. HAI and MN were performed according to the protocol developed in our previous study with minor modification as described below (41). Briefly, to determine HAI titers, mice sera were treated with a receptor-destroying enzyme overnight and heat- inactivated for 40 min. The sera were tested in 2-fold dilutions starting with an initial dilution of 1 :20, and then admixed with 4 HA units of IVR-190 virus. After incubation at room temperature for 1 h, freshly prepared 0.5% suspension of chicken red blood cells was added and hemagglutination was assessed by observation after 1 h. HAI titer is defined as the reciprocal of the highest dilution that showed > 50% inhibition of hemagglutination. The detection of vaccine-induced neutralizing antibody titers against influenza viruses were performed with a World Health Organization recommended protocol as described previously. After tracing with HRP-conjugated secondary antibody and developed with TMB substrate, the absorbance was measured at 450 nm with a microplate reader (SpectraMax iD3, Molecular Devices). Untreated virus control (VC), uninfected cell control (CC), and back titration of virus infectivity are included on each plate. Half-cell infection was calculated by the following equation: X = (average OD of VC wells - average OD of CC wells) / 2 + (average OD of CC wells). Microneutralization titer is expressed as the reciprocal of the highest serum dilution that showed <50% of the cells are infected.[000153] Results of HAI and MN are shown in FIG. 3 A and 3B, respectively. As shown in FIG. 3A both monoglycosylated whole virus vaccine and monoglycosylated split virus vaccine resulted in substantially higher HAI than 5 pg of the monoglycosylated rHA vaccine. Similarly, as shown in FIG. 3B, both the monoglycosylated whole virus and the monoglycosylated split virus vaccines also resulted in substantially higher MN than 5 pg of rHA. The two results indicate much higher immunogenicity indicated higher inhibition andneutralization capability by the monoglycosylated whole virus and the monoglycosylated split virus vaccines.[000154] HAI and MN experiments were done with the monoglycosylated whole virus vaccine and the monoglycosylated split virus vaccine each administered with the JR300 adjuvant. The result is shown in FIG. 8 A and 8B. As shown in the figures, addition of the JR300 adjuvant substantially improved the performance of the monoglycosylated whole virus vaccine over the two other vaccines.[000155] Example 4 Neuraminidase Inhibition Titer (NAI Titer)[000156] The NAI titer was determined according to the protocol developed from minimization and optimization of the conventional assay (24). Results are shown in FIG. 5A. As shown in FIG. 5 A, both the monoglycosylated whole virus and split virus vaccine each with 5 pg of HA provided substantially higher NA inhibition than 5pg of monoglycosylated rHA.[000157] Example 5 Detection of anti-M2e and nasal wash IgA antibodies by ELISA assay [000158] To evaluate the anti-M2e response, mice sera was examined by ELISA against synthetic M2e peptide (2 pg / mL) coating in a 96-well plate (Wu et al., 2010). Compared to a commercial anti-M2e antibody (ab5416) used as a positive control, significant differences in response against an M2e peptide were detected in mice sera from monoglycosylated whole virus vaccine, monoglycosylated split virus vaccine, or monoglycosylated rHA vaccinations. M2e-coated wells were incubated with serum samples at 1,000-fold dilution for 1 h at 37 °C, traced with HRP-conjugated secondary Ab, and developed with TMB substrate. They were washed with PBST three times between each ELISA step. Finally, for the ELISA readout, the absorbance of each well at 450 nm was measured using a microplate reader. To measure the quantity of IgA antibody against IVR-190 HA antigen, nasal wash collected from immunized mice receiving two doses of vaccines with a two-fold serial dilution were added to HA antigen-coated ELISA plates (2 pg / well). HRP-labeled goat anti-mouse IgA (ab97235) was used to detect the bound antibodies from immune sera. The remaining experimental processes are the same as ELISA assay described above. Endpoint ELISA titer is defined as the reciprocal of the highest analyte dilution that gives a reading two times above the mean background reading. [000159] Results are illustrated in FIG. 5B. As shown in FIG. 5B, both monoglycosylated whole virus and monoglycosylated split virus each with 5 pg of HA resulted in substantially higher M2e inhibition than 5 pg of monoglycosylated rHA. Interestingly, monoglycosylated whole virus vaccine resulted in substantially higher M2e inhibition than split virus vaccine. The results indicate that monoglycosylated whole virus without the glycan shield and withconformation than the monoglycosylated split virus better presents the monoglycoyslated M2e for inducing effective antibodies.[000160] Example 6 IL-4, IFN-y, and granzyme B (GrzB) ELISpot assays[000161] Spleens were harvested (week 4) from vaccinated mice, and splenocytes were isolated for the ELISpot assay according to the manufacturer's instructions (R & D Systems, Minneapolis, MN, USA). Briefly, cells were depleted of erythrocytes by treatment with lx RBC lysis buffer (BioLegend, San Diego, CA). Following extensive washing with PBS, cells were resuspended in RPMI medium with 10% fetal bovine serum (Gibco, San Diego, CA) and 50 pM 2-mercaptoethanol. Cell viability was determined by trypan blue staining. The cell suspensions (3xl05 / well) from the spleen were incubated in precoated anti-mouse IL-4, IFN-y, or GrzB plates and stimulated (20 h) with inactivated virus or rHA (10 pg / mL). An included positive control or additional wells of cells were mock stimulated as a negative control for ELISpot assays. The plates were incubated overnight in a humidified incubator at 37 °C with 5% CO2. After four washes with PBS-0.05% Tween, IL-4, IFN-y, or GrzB spots were detected by biotinylated IL-4, IFN-y, or GrzB detection antibodies followed by the addition of streptavidin-alkaline phosphatase and development with BCIP / NBT substrate solution. Spots were scanned and counted using the AID ELISpot Reader System and EliSpot 5.0 iSpot image analyzer (AID, Strassberg, Germany). The results were expressed as the number of spot / 3xl05 spleen cells. [000162] Results are shown in FIG. 7A, 7B and 7C. As shown in the figures, both the monoglycosylated whole virus and the monoglycosylated split virus each with 5 pg of HA resulted in substantially higher T cell response than 5 pg of monoglycosylated rHA. However, the monoglycosylated whole virus elicited substantially higher IFN-y and GrzB response than split virus vaccine as shown in FIGs. 7B and 7C. [000163] Example 7 Stability[000164] The long-term stability of vaccine bulk is one of the key parameters that support a novel vaccine candidate to become a vaccine product. The frequency of test points for longterm studies should be sufficient to establish the stability profile over a given period (normally every 3 months over the first year), including the initial and final time points. To evaluate the long-term stability of IVR-190mg, the hemagglutination activity, HA potency, total protein content, and particle size distribution were determined by conventional methods for human vaccine characteristic analysis (Kon et al., 2016). Over 15 months of study, the collected data indicated that the biological and physical properties of IVR-190mg in terms of HA potency, total protein content, and particle size distribution, were as stable as those ofIVR-190fg (Fig. 12F-H). Interestingly, the hemagglutination titer of IVR-190mg was lower than that of the fully glycosylated split vaccine (IVR-190fg), but IVR-190mg displayed strong and lasting agglutination ability against chicken RBCs for several days (Fig. 12E and I- J). Decreasing the glycans from the IVR-190 virus could lead to a change in cell binding affinity between HA and NA rather than loss of biological function, which has not yet been clearly investigated. In summary, these results demonstrated that IVR-190mg has sufficient stability for long-term storage at 2-8 °C for at least 15 months compared with IVR-190fg.[000165] Results are shown in FIG. 4. As shown in FIG. 4, both the monoglycosylated whole virus and split virus vaccines prepared according to Example 1 substantially outperformed rHA vaccine in terms of stability. The higher stability of whole and split virus is an indication that natural gene sequence of the virus has been well developed through evolution resulting in powerful and extremely stable glycoprotein to allow the virus to replicate effectively.[000166] In contrast, although the recombinant proteins are designed according to the protein sequence of the virus, production cells such as sf9, 293, or CHO are not usually naturally infected by flu viruses and, therefore do not present natural environments in which the virus replicates and evolves, resulting in glycoproteins with inferior conformations. For this reason, the genetic sequences of HA must be modified to improve yields and stability to rHA. But no matter how well we modify it, it cannot compare with the protein derived from the natural genetic sequence of the virus.[000167] It can be appreciated by those skilled in the art that changes could be made to the examples described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular examples disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the appended claims.References which are hereby each incorporate in its entirety1. Asthagiri Arunkumar, G., loannou, A., Wohlbold, T.J., Meade, P., Aslam, S., Amanat, F., Ayllon, J., Garcia-Sastre, A., Krammer, F., 2019. Broadly Cross-Reactive, Nonneutralizing Antibodies against Influenza B Virus Hemagglutinin DemonstrateEffector Function-Dependent Protection against Lethal Viral Challenge in Mice. J Virol 93.2. 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Claims

What is claimed is:

1. A vaccine comprising a therapeutically effective amount of whole influenza viruses, split influenza viruses, influenza virus like particles (VLPs) or a combination thereof a. wherein one or more surface viral glycoproteins of the whole influenza viruses, split influenza viruses or influenza VLPs are partially glycosylated; and b. wherein the whole influenza viruses and the split influenza viruses have been attenuated or inactivated.

2. The vaccine of claim 1, wherein the one or more partially glycosylated viral surface glycoproteins are tri-, di- or monoglycosylated.

3. The vaccine of claim 1, wherein the one or more partially glycosylated viral surface glycoproteins comprise hemagglutinin (HA), neuraminidase (NA), matrix-2 (M2), polymerase protein PB 1, polymerase protein PB2, polymerase protein PA, nucleoprotein (NP), matrix (Ml), nuclear export protein (NEP) or a combination thereof.

4. The vaccine of claim 1, wherein the one or more partially glycosylated viral surface glycoproteins comprise any viral surface glycoprotein of an influenza virus of a particular strain and / or variant.

5. The vaccine of claim 1, wherein each glycosylation site of the one or more viral surface glycoproteins of the whole influenza viruses, split influenza viruses or influenza VLPs comprising the vaccine is at least about 50% to about 99% tri-, di- or monoglycosylated.

6. The vaccine of claim 3, wherein each glycosylation site of all of the HA, NA or M2 virial surface glycoproteins of the whole influenza viruses, split influenza viruses or influenza VLPs comprising the vaccine is at least about 90% to about 99% tri-, di- or monoglycosylated.

7. The vaccine of claim 3, wherein at least five glycosylation sites of all of the HA, NA or M2 viral surface glycoproteins of the whole influenza viruses, split influenza viruses or influenza VLPs comprising the vaccine are each at least about 97% to about 99% tri-, di- or monoglycosylated.

8. The vaccine of claim 3, wherein the HA virial surface glycoproteins each comprises at least 6 glycosylation sites.

9. The vaccine of claim 1 , wherein the vaccine does not comprise a therapeutically effective amount of the whole influenza viruses with the one or more partially glycosylated viral surface glycoprotein.

10. The vaccine of claim 1, wherein the vaccine does not comprise a therapeutically effective amount of the split influenza viruses with the one or more partially glycosylated viral surface glycoprotein.

11. The vaccine of claim 1, wherein the vaccine does not comprise a therapeutically effective amount of the influenza VLPs with the one or more partially glycosylated viral surface glycoprotein.

12. The vaccine of claim 1, wherein the vaccine does not comprise a therapeutically effective amount of partially glycosylated recombinant viral surface glycoprotein polypeptides.

13. The vaccine of claim 1, wherein the vaccine does not comprise a therapeutically effective amount of partially glycosylated recombinant HA polypeptide or a therapeutically effective amount of partially glycosylated recombinant NA polypeptides.

14. The vaccine of claim 1, further comprises an adjuvant, wherein the adjuvant comprises squalene-based emulsion adjuvant or aluminum-based vaccine adjuvant.

15. The vaccine of claim 14, wherein the squalene-based emulsion adjuvant of the present invention comprises squalene and sorbitan trioleate.

16. The vaccine of claim 14, wherein the squalene based emulsion adjuvant of the present invention comprises about 20 to 50 mg / mL squalene oil.

17. The vaccine of claim 14, wherein the squalene-based emulsion adjuvant of the present invention comprises about 3 to 6 mg / mL sorbitan trioleate.

18. The vaccine of claim 1, wherein the whole influenza viruses, split influenza viruses, or influenza VLPs comprise type A, type B or type C influenza virus.

19. The vaccine of claim 1, wherein the whole influenza viruses, split influenza viruses or influenza VLPs comprise avian influenza viruses, animal influenza viruses or human influenza viruses.

20. The vaccine of claim 1, wherein the whole influenza viruses, split influenza viruses or influenza VLPs comprise Hl, H3 or H5 virial surface glycoproteins.

21. A method of preparation of the vaccine of claim 1, comprising the steps ofinjecting working influenza virus seed with an inhibitor of mannosidase into cmbryonatcd chicken egg to inoculate the embryonated chicken egg; incubating the inoculated embryonated chicken egg to propagate the working virus; harvesting allantonic fluids from the inoculated embryonated chicken egg; concentrating and clarifying the harvested allantonic fluids; purifying the concentrated and clarified allantonic fluids using centrifugation; applying deglycosylation enzyme to the purified allantonic fluid to partially glycosylate viral surface glycoprotein of the propagated viruses within the purified allantonic fluid; removing the deglycosylation enzyme from whole influenza viruses with partially glycosylated viral surface glycoprotein; and inactivating and / or attenuating the viruses.

22. The method of preparation of claim 21, further comprising the step of splitting the whole influenza viruses to obtain split influenza viruses before the step of inactivating and / or attenuating the split influenza viruses and after the step of removing the deglycosylation enzyme.

23. The method of preparation of claim 21, wherein the deglycosylation enzyme comprises P-specific galactosidase, a-neuraminidase, N-acetylglucosaminidase (PNGase F) or a combination thereof.

24. The method of preparation of claim 23 wherein the P-specific galactosidase comprises P(l-4)-galactosidase, P(l-6)-galactosidase or P(l-3)-galactosidase and a-neuraminidase comprises Endo-a-N-acetylgalactosaminidase H (Endo H), a(l-4)-Galactosidase or a(l- 6)-Galactosidase.

25. The method of preparation of claim 24, wherein, for the step of applying deglycosylation enzyme to deglycosylate the viral surface protein, the ratio of total amount of Endo H enzymes to the total amount of viral surface proteins by weight is about 1:20 to about 1: 1.

26. The method of preparation of claim 24, wherein, for the step of applying deglycosylation enzyme to deglycosylate the viral surface protein, the ratio of total amount of Endo H enzymes to the total amount of viral surface proteins by weight is about 1: 12 to about 1:8.

27. The method of preparation of claim 21 wherein the centrifugation comprises sucrose density centrifugation in the purifying step.

28. The method of preparation of claim 21, wherein the working influenza virus seed comprises working influenza virus at concentration of about 1085EID50 / 0.1 mL diluted to about 103-107concentration.

29. The method of preparation of claim 21, wherein the working influenza virus seed comprises working influenza virus at concentration of about 1085EID50 / 0.1 mL diluted to about 104-106concentration.

30. The method of preparation of claim 21, wherein the inhibitor of mannosidase comprises kifunesine.

31. The method of preparation of claim 30, wherein the concentration of kifunensine is about lOOpg / mL to about 500 pg / mL in the injecting working virus seed step.

32. The method of preparation of claim 30, wherein the concentration of kifunensine is about 200ug / mL to about 300 pg / mL in the injecting working virus seed step.

33. The method of preparation of claim 21, wherein the inoculated embryonated chicken egg is incubated for about 24 to about 90 hours.

34. The method of preparation of claim 21, wherein, in the purifying the concentrated and clarified allantonic fluids using centrifugation step, the centrifugation comprises sucrose density centrifugation.

35. The method of preparation of claim 34, wherein concentration of the sucrose gradient is about 5% to about 55% (w / v).

36. The method of preparation of claim 21, wherein the steps of applying and removing deglycosylation enzyme is performed after the step of harvesting allantonic fluids but before the step of concentrating and clarifying.

37. The method of preparation of claim 21, wherein the steps of applying and removing deglycosylation enzyme is performed after the step of concentrating and clarifying but before the step of purifying the concentrated and clarified allantonic fluid.

38. The method of preparation of claim 21, wherein the method of preparation is capable of producing at least 100 doses of the vaccine wherein each dose of the vaccine comprises at least 10 micrograms of HA.

39. The vaccine of claim 21 , wherein each glycosylation site of all of the HA, NA or M2 virial surface glycoproteins of the whole influenza virus or split influenza virus comprising the vaccine is at least about 90% to about 99% tri-, di- or monoglycosylated.

40. The vaccine of claim 21, wherein at least five glycosylation sites of all of the HA, NA or M2 viral surface glycoprotein of the whole influenza virus or split influenza virus comprising the vaccine are each at least about 97% to about 99% tri-, di- or monoglycosylated.

41. A method of treatment for prevention of influenza infection comprising the step of administering the vaccine of claim 1 to a subject.

42. The method of claim 40, wherein the step of administering the vaccine comprises intranasal, intravenous, subcutaneous, intramuscular, intraperitoneal, or intradermal administration.

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