CATIONIC LIPID R-DOTAP AND METHODS OF USE THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority under 35 U.S. C. § 119(e) of U.S. Provisional Applications No. 63/418,381, filed October 21, 2022, and No. 63/539,066, filed September 18, 2023. The disclosure of the prior applications is considered part of and is herein incorporated by reference in the disclosure of this application in its entirety.

INCORPORATION OF SEQUENCE LISTING

[0002] The material in the accompanying sequence listing is hereby incorporated by reference into this application. The accompanying sequence listing xml file, name ST26.xml, was created on and is kb.

BACKGROUND OF THE INVENTION

BACKGROUND INFORMATION

[0003] Immunization remains one of the most effective public health measures to combat viral infections. Increased safety requirements for approval by the FDA, combined with the complexities associated with certain viral infections, have invigorated the search for new generation pep tide/ recomb inant protein-based prophylactic vaccines. However, most recombinant proteins/peptides are not immunogenic and only induce weak immune responses when administered. Hence, recombinant protein/peptide vaccines often require an adjuvant to stimulate or enhance the immune response to antigens.

[0004] There are a wide variety of adjuvants approved or under study for recombinant proteinbased vaccines with different immune modulatory properties. Many adjuvants promote strong antibody responses, e.g., alum, squalene or monophosphoryl lipid A, while others, e.g., CpG or other TLR 7, 8 or 9 agonists promote stronger Thl CD4 and CDS T cell responses. But even these distinctions are blurred somewhat because a single adjuvant can promote different responses through distinct pathways. For example, squalene-based adjuvants induce CDS T cell responses through a pathway distinct from its antibody inducing property. Thus, it is now appreciated that different adjuvants evoke distinct immunological signatures that render them more or less effective for distinct diseases, e.g., tuberculosis vs influenza. Vaccines against respiratory viruses e.g., influenza, predominantly induce antibody responses and, for influenza, a neutralizing antibody titer is the primary correlate of immunity. However, natural infection also induces potent CD4 and CDS T cell responses which are critical in forming long lasting immunity. CDS T cell responses to internal proteins are also critical in situations where antigenic drift or shift prevents antibody recognition. Thus, it is now widely recognized that next generation universal vaccines against respiratory viral pathogens should induce strong neutralizing antibody as well as strong CDS T cell immunity.

[0005] Lipid nanoparticles are some of the most promising delivery vehicles for eukaryotic cells and have been widely used since the late 1980s for the delivery of nucleic acids into cells including human gene therapy clinical trials. The novel mRNA vaccine technologies used to fight the COVID 19 pandemic also use lipid nanoparticles as delivery vehicles to deliver mRNA into cells. In recent years, cationic lipids have become attractive targets for delivering proteins or peptides for use in immunotherapy and vaccines. Importantly, cationic lipid mediated antigen uptake delivers proteins and peptides into the MHC class I and class II processing pathway. Mechanistically, cationic lipid nanoparticles efficiently bind negatively charged cell membranes in a receptor-independent manner and are rapidly internalized into endosomes in amounts exceeding receptor-mediated uptake. Once in endosomes, cationic lipids fuse with the cndosomal membrane, delivering some of their contents to the cytoplasm. Thus, specific cationic lipids are ideally suited as non-viral vectors to deliver peptides, proteins, and inactivated whole viruses intracellularly into the MHC class I and II pathways. More recent studies investigating cationic lipids as delivery agents also identified that certain cationic lipids possess immune-stimulatory properties and can activate pathways essential for effective immune responses following vaccination. However, because cationic lipids differ greatly in their immuno stimulatory properties and mechanisms of action, these characteristics are not universally exhibited by all cationic lipids. [0006] The cnantio-spccific cationic lipid l,2-diolcoyl-3-trimcthylammonium-propanc (R- DOTAP) has been shown to be particularly robust at inducing CDS T cell responses to peptide- based vaccines. Further studies revealed that R-DOTAP promoted cellular uptake and crosspresentation of CDS epitopes from long peptides and promoted the formation of polyfunctional CDS T cells. R-DOTAP alone was shown to induce type I interferons in the draining LN and type 1 interferon was required for the R-DOTAP mediated induction of antigen specific CDS T cells. While it is well established that R-DOTAP facilitates robust CDS T cell responses to peptide- based vaccines, the ability of R-DOTAP to promote CDS T cell responses to larger recombinant proteins is less clear. While R-DOTAP was also shown to induce robust antibody responses to the model antigen, OVA, there have been no studies on the ability of R-DOTAP to promote antibody responses to vaccine relevant recombinant proteins.

SUMMARY OF THE INVENTION

[0007] The present invention is based on the seminal discovery that the use of a cationic lipid as an immunomodulator enhances immunity induced by influenza recombinant protein in vaccines compositions.

[0008] In one embodiment, the present invention provides a vaccine composition including one or more non-naturally occurring recombinant influenza antigens; and a cationic lipid.

[0009] In one aspect, the one or more non-naturally occurring recombinant influenza antigens include a computationally optimized broadly reactive influenza antigen (COBRA) hemagglutinins (HA). In another aspect, the one or more non-naturally occurring recombinant influenza antigens are recombinant H1N1 and/or H3N2 hemagglutinin influenza proteins. In one aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence with at least 80%, 85% 90% or 95% sequence identity to any of SEQ ID NOs:3-22 and combinations thereof. In one aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence with at least 80%, 85% 90% or 95% sequence identity to SEQ ID NO: 3 or SEQ ID NO:4. In another aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence of any of SEQ ID NOs:3-22 and combinations thereof. In another aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4. In another aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence with at least 80%, 85% 90% or 95% sequence identity to of SEQ ID NOs:3 and SEQ ID NO:4. In one aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence of SEQ ID NO: 3 and SEQ ID NO:4. In one aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence of SEQ ID NO: 3 and SEQ ID NO:4. In another aspect, the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S- DDA, S- DOEPC, S-DOTMA, variations thereof or analogs thereof. In one aspect, the cationic lipid is R- DOTAP. In another aspect, the one or more non-naturally occurring recombinant influenza antigens are encapsulated in liposomes including cationic lipids. In another aspect, the one or more non-naturally occurring recombinant influenza antigens are mixed with preformed cationic lipid nanoparticles. In some aspects, the one or more non-naturally occurring recombinant influenza antigens and the preformed cationic lipid nanoparticles are mixed at a 1 :1 ratio. In one aspect, the vaccine composition is a universal influenza vaccine.

[0010] In another embodiment, the invention provides a method of inducing an immune response against influenza viruses in a subject including administering to the subject a vaccine composition including: a) one or more non-naturally occurring recombinant influenza antigens; and b) a cationic lipid, thereby inducing an immune response against influenza viruses in a subj ect. [0011] In one aspect, the immune response includes an induction of CD8 ⁺ effector T cells, CD4 ⁺ effector T cells and memory T cells. In another aspect, inducing CD8 ⁺ effector T cells and CD4 ⁺ effector T cells includes inducing proliferation of IFNy and granzyme B producing CD8 ⁺ effector T cells, and/or the proliferation of IL-4 producing CD4 ⁺ effector T cells in the subject. In another aspect, inducing a humoral immune response includes inducing the production of IgG in the subject. In some aspects, IgG include IgGl and IgG2a. In one aspect, inducing the immune response includes inducing the secretion of broadly neutralizing antibodies.

[0012] In an additional embodiment, the invention provides a method of preventing or treating an influenza infection in a subject including administering to the subject a vaccine composition including: a) one or more non-naturally occurring recombinant influenza antigens; and b) a cationic lipid, thereby preventing or treating an influenza infection in a subject.

[0013] In one embodiment, the invention provides a method of enhancing immunogenicity of an influenza vaccine in a subject including administering to the subject a vaccine composition including: a) one or more non-naturally occurring recombinant influenza antigens; and b) a cationic lipid, thereby enhancing immunogenicity of an influenza vaccine.

[0014] In one aspect, the influenza vaccine is an inactivated flu vaccine, an attenuated flu vaccine or a recombinant flu vaccine. In another aspect, the influenza vaccine is a monovalent vaccine, a bivalent vaccine, a trivalent vaccine or a quadrivalent vaccine.

[0015] In another embodiment, the invention provides a method of inducing secretion of broadly neutralizing antibodies against influenza viruses in a subject including administering to the subject a vaccine composition including: a) one or more non-naturally occurring recombinant influenza antigens; and b) a cationic lipid, thereby inducing secretion of broadly neutralizing antibodies against influenza viruses in a subject. [0016] In a further embodiment, the invention provides a method of inducing a balanced Thl/Th2 immune response in a subject including administering to the subject a vaccine composition including: a) one or more non-naturally occurring recombinant influenza antigens; and b) a cationic lipid, thereby inducing secretion of broadly neutralizing antibodies against influenza viruses in a subject.

[0017] In one aspect, inducing a Thl immune response includes inducing proliferation of IFNy and granzyme B producing CD8+ effector T cells, and/or the proliferation of IL-4 producing CD4+ effector T cells in the subject. In some aspect, IFNy producing CD8+ effector T cells are associated with production of IgG2a, and IL-4 producing CD4+ effector T cells are associated with production of IgGl .

[0018] In an additional embodiment, the invention provides a method of inducing a polyfunctional CD4+/CD8+ T cell response against influenza viruses in a subject including administering to the subject an influenza vaccine composition including: a) one or more non- naturally occurring recombinant influenza antigens; and b) a cationic lipid, thereby inducing a polyfunctional CD4+/CD8+ T cell response against influenza viruses in the subject.

[0019] In one aspect, inducing a polyfunctional CD4+/CD8+ T cell response includes inducing the secretion of two or more cytokines. In sone aspects, the two or more cytokines arc selected from the group consisting of IFNy, granzyme B and IL-4.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIGURES 1A-1B illustrate the antibody mediated immune response induced by recombinant infuenza protein formulated with R-DOTAP. FIGURE 1A is a graph showing total IgG antibody titer obtained with R-DOTAP preparations. FIGURE IB is a graph showing total IgG antibody titer obtained with Y2-COBRA preparations and sucrose.

[0021] FIGURES 2A-2C illustrate average particle size for R-DOTAP liposomal nanoparticles and R-DOTAP admixed with COBRA Y2 or COBRA-NG2 protein antigens. FIGURE 2A is a graph illustrating particle size distribution. FIGURE 2B is a transmission electron microscopy (TEM) photograph illustrating R-DOTAP nanoparticles. FIGURE 2C is a TEM photograph illustrating R-DOTAP mixed with COBRA-Y2 and COBRA-NG2 mixtures resuspended in water.

[0022] FIGURES 3A-3D illustrate T cell responses to R-DOTAP formulations containing nucleoprotein and COBRA HA antigens FIGURE 3A is a graph showing the number of antigen specific IFN-y producing T cells in spleens in response to vaccines with influenza nucleoproteins. FIGURE 3B is a graph showing the number of antigen specific IFN-y producing T cells in spleens in response to monovalent and bivalent vaccines with influenza recombinant COBRA sequences. FIGURE 3C is a graph illustrating specific T cell response to H1N1. FIGURE 3D is a graph illustrating specific T cell response to H3N2.

[0023] FIGURES 4A-4E illustrate antibody mediated immune responses to COBRA proteins. FIGURE 4A a is a graph showing antigen specific antibody titers after vaccination with one or two doses. FIGURE 4B is a graph comparing anti-Y2 antigen specific antibody titers 35 and 62 days after vaccination using sucrose, R-DOTAP or Addavax™ as the adjuvant. FIGURE 4C is a graph comparing anti-NG2 antigen specific antibody titers 35 and 62 days after vaccination using sucrose, R-DOTAP or Addavax™ as the adjuvant. FIGURE 4D is a graph comparing anti-Y2 antigen specific IgGl and IgG2a antibody titers 35 and 62 days after vaccination using sucrose, R-DOTAP or Addavax™ as the adjuvant. FIGURE 4E is a graph comparing anti-NG2 antigen specific IgGl and IgG2a antibody titers 35 and 62 days after vaccination using sucrose, R-DOTAP or Addavax™ as the adjuvant.

[0024] FIGURES 5A-5B illustrate the ability of bivalent COBRA antigens formulated with R-DOTAP to induce Thl and Th2 antibody responses. FIGURE 5A is graph illustrating Thl specific antibody titers. FIGURE 5B is graph illustrating Thl specific antibody titers.

[0025] FIGURES 6A-6C illustrate HAI titer on day 35 after vaccination with influenza virus alone or in combination with R-DOTAP nanoparticles. FIGURE 6A is a graph illustrating HAI titers when the inactivated influenza vaccine, Fluzone® (2011-12 formulation) was a split virus hemagglutinin preparation derived from A/Calilbrnia/07/2009 X-179A (H1N1). FIGURE 6B is a graph illustrating HAI titers when the inactivated influenza vaccine, Fluzone® (2011-12 formulation) was a split virus hemagglutinin preparation derived from A/Victoria/210/2009 X- 187 (an A/Pcrth/16/2009- like virus) (H3N2). FIGURE 6C is a graph illustrating HAI titers when the inactivated influenza vaccine, Fluzone® (2011-12 formulation) was a split virus hemagglutinin preparation derived from and B/Brisbane/60/2008.

[0026] FIGURES 7A-7D illustrate the effects of the presence of R-DOTAP in the vaccine formulation after vaccination and viral challenge. FIGURE 7 A is a graph illustrating body weight changes over time after vaccination and infection. FIGURE 7B is a Kaplan Mayers graph illustrating survival post infection. FIGURE 7C is a graph illustrating virus titers 3 days post infection. FIGURE 7D is a graph illustrating virus titers 6 days post infection. [0027] FIGURE 8 is a schematic representation of the pre-immune ferret model.

[0028] FIGURES 9A-9C illustrate the results of H INI HAI response. FIGURE 9 A is a graph illustrating HAI titers in animals vaccinated with Y2+NG2 HA proteins. FIGURE 9B is a graph illustrating HAI titers in animals vaccinated with Mich/15 + Sing/16 HA proteins. FIGURE 9C is a schematic representation showing when during the experiment timeline blood was collected to measure the data in FIGURES 9 A and 9B.

[0029] FIGURES 10A-10C illustrate the results of H3N2 HAI response. FIGURE 10A is a graph illustrating HAI titers in animals vaccinated with Y2+NG2 HA proteins. FIGURE 10B is a graph illustrating HAI titers in animals vaccinated with Mich/ 15 + Sing/16 HA proteins. FIGURE 10C is a schematic representation showing when during the experiment timeline blood was collected to measure the data in FIGURES 10A and 10B.

[0030] FIGURE 11A-11C illustrate further characterization of the response in animals vaccinated with Y2+NG2 HA proteins. FIGURE 11A is a graph illustrating the body weight of the animals during the study. FIGURE 1 IB is a graph illustrating D3 nasal wash viral titers. FIGURE 11C is a schematic representation showing when during the experiment timeline nasal was performed to measure the data in FIGURE 11B.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention is based on the seminal discovery that that the use of a cationic lipid as an immunomodulator enhances immunity induced by influenza recombinant antigens in vaccine compositions.

[0032] Before the present compositions and methods are described, it is to be understood that this invention is not limited to particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only in the appended claims.

[0033] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” includes one or more methods, and/or steps of the type described herein which w ill become apparent to those persons skilled in the art upon reading this disclosure and so forth. [0034] As used herein, the term “about” in association with a numerical value is meant to include any additional numerical value reasonably close to the numerical value indicated. For example, and based on the context, the value can vary up or down by 5-10%. For example, for a value of about 100, means 90 to 110 (or any value between 90 and 110).

[0035] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0036] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, it will be understood that modifications and variations are encompassed within the spirit and scope of the instant disclosure. The preferred methods and materials are now described.

[0037] VACCINES COMPOSITIONS

[0038] In one embodiment, the present invention provides a vaccine composition including: one or more non-naturally occurring recombinant influenza antigens; and a cationic lipid.

[0039] As used herein the term composition is meant to include pharmaceutical compositions, which may also contain other therapeutic agents, and may be formulated, for example, by employing conventional pharmaceutically acceptable vehicles or diluents, as well as pharmaceutical additives of a type appropriate to the mode of desired administration (for example, excipients, preservatives, etc.) according to techniques known in the art of pharmaceutical formulation. In certain embodiments, the compositions disclosed herein are formulated with additional agents that promote entry into the desired cell or tissue. Such additional agents include micelles, liposomes, and dendrimers.

[0040] The term “pharmaceutically acceptable” refers to the fact that the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. For example, the carrier, diluent, or excipient or composition thereof may be administered to a subject along with a conjugate of the invention without causing any undesirable biological effects or interacting in an undesirable manner with any of the other components of the pharmaceutical composition in which it is contained.

[0041] Pharmaceutical compositions including the peptides or compositions described herein may be administered by any suitable means, for example, parenterally, such as by subcutaneous, intravenous, intramuscular, intrathecal, or intracistemal injection or infusion techniques (e.g., as sterile injectable aqueous or non-aqueous solutions or suspensions) in dosage formulations containing non-toxic, pharmaceutically acceptable vehicles or diluents. Depending on the condition being treated, these pharmaceutical compositions may be formulated and administered systemically or locally. Techniques for formulation and administration are generally known in the art. Suitable routes may, for example, parenteral delivery, including intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, or intraperitoneal. For injection, the pharmaceutical compositions of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as water, Hanks’ solution, Ringer’s solution, or physiologically buffered saline.

[0042] The compositions of the invention are “vaccine compositions”, as used herein the term

“vaccine” relates to a pharmaceutical preparation (pharmaceutical composition) or product that upon administration induces an immune response, in particular a cellular immune response, which recognizes and attacks a pathogen or a diseased cell such as a cancer cell. A vaccine may be used for the prevention or treatment of a disease.

[0043] The term “universal flu vaccine” concerns influenza vaccines in particular and is formulated to provide immune protection against at least two variants of an influenza virus. In one aspect, the vaccine compositions described herein are universal flu vaccines.

[0044] In one aspect, the vaccine compositions described herein provide immune protection against at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 variants of an influenza virus. The vaccine compositions described herein provide immune protection against at least 3variants of an influenza virus, the vaccine compositions described herein provide immune protection against at least 4 variants of an influenza virus. The vaccine compositions described herein provide immune protection against at least 5 variants of an influenza virus. The vaccine compositions described herein provide immune protection against at least 6 variants of an influenza virus. The vaccine compositions described herein provide immune protection against at least 7 variants of an influenza virus. The vaccine compositions described herein provide immune protection against at least 8 variants of an influenza virus. The vaccine compositions described herein provide immune protection against at least 9 variants of an influenza virus. The vaccine compositions described herein provide immune protection against at least 10 variants of an influenza virus. [0045] In one aspect, examples of influenza variants include, but are not limited to H1N1, H3N2, H5N1, and H7N9. In one aspect, the vaccine compositions described herein provide immune protection against at least H1N1 and/or H3N2 hemagglutinin influenza variants.

[0046] The vaccine compositions described herein include one or more recombinant influenza antigens.

[0047] In one aspect, the one or more recombinant influenza antigens are recombinant H1N1 and/or H3N2 hemagglutinin influenza proteins.

[0048] The terms “peptide”, “polypeptide” and “protein” are used interchangeably herein and refer to any chain of at least two amino acids, linked by a covalent chemical bound. As used herein polypeptide can refer to the complete amino acid sequence coding for an entire protein or to a portion thereof. A “protein coding sequence” or a sequence that “encodes” a particular polypeptide or peptide, is a nucleic acid sequence that is transcribed (in the case of DNA) and is translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5’ (amino) terminus and a translation stop codon at the 3’ (carboxyl) terminus. A coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence will usually be located 3’ to the coding sequence. [0049] An “antigen” according to the invention covers any substance that will elicit an immune response. In particular, an “antigen” relates to any substance, preferably a peptide or protein, that reacts specifically with antibodies or T-lymphocytes (T cells). According to the present invention, the term “antigen” comprises any molecule which comprises at least one epitope. Preferably, an antigen in the context of the present invention is a molecule which, optionally after processing, induces an immune reaction. According to the present invention, any suitable antigen may be used, which is a candidate for an immune reaction, wherein the immune reaction is preferably a cellular immune reaction. In the context of the embodiments of the present invention, the antigen is preferably presented by a cell, preferably by an antigen presenting cell which includes a diseased cell, in particular a cancer cell, in the context of MHC molecules, which results in an immune reaction against the antigen. An antigen is preferably a product which corresponds to or is derived from a naturally occurring antigen. Such naturally occurring antigens include tumor antigens.

[0050] In another aspect, the one or more recombinant influenza antigens include computationally optimized broadly reactive influenza antigen (COBRA) hemagglutinins (HA). The COBRA HAs described herein are further described in detail in International Patent

Application No. PCT/US2022/032799, U.S. Patent No. 9,212,207 and the scientific publications Allen and Ross (“Bivalent Hl and H3 COBRA Recombinant Hemagglutinin Vaccines Elicit Seroprotectective Antibodies against H1N1 and H3N2 Influenza Viruses from 2009 to 2019”; J. Virology (2022); 96(7)) and Henson et al. (“R-DOTAP Cationic Lipid Nanoparticles Outperform Squalene-Based Adjuvant Systems in Elicitation of CD4 T Cells after Recombinant Influenza Hemagglutinin”; Viruses (2023): 15:538), which are incorporated herein by reference in its entirety.

[0051] As used herein, the “non-naturally occurring” peptide or antigen means a peptide or antigen that is not found in nature and that is comprised of one or more peptides or antigens, naturally occurring or non-naturally occurring, combined into a single peptide or antigen.

[0052] In one aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence with at least 80%, 85% 90% or 95% sequence identity to any of SEQ ID NOs:3-22 and combinations thereof. In one aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence with at least 80%, 85% 90% or 95% sequence identity to SEQ ID NO: 3 or SEQ ID NO:4. In another aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence of any of SEQ ID NOs:3-22 and combinations thereof. In another aspect, the one or more non- naturally occurring recombinant influenza antigens include an amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4. In another aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence with at least 80%, 85% 90% or 95% sequence identity to of SEQ ID NOs:3 and SEQ ID NO:4. In one aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence of SEQ ID NO: 3 and SEQ ID NO:4. In one aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence of SEQ ID NO: 3 and SEQ ID NO:4.

[0053] The terms “sequence identity” or “percent identity” are used interchangeably herein. To determine the percent identity of two polypeptide molecules or two polynucleotide sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first polypeptide or polynucleotide for optimal alignment with a second polypeptide or polynucleotide sequence). The amino acids or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical positions/total number of positions (i.e., overlapping positions) x 100). In some embodiments the length of a reference sequence aligned for comparison purposes is at least 80% of the length of the comparison sequence, and in some embodiments is at least 90% or 100%. In an embodiment, the two sequences are the same length.

[0054] Ranges of desired degrees of sequence identity are approximately 80% to 100% and integer values in between. Percent identities between a disclosed sequence and a claimed sequence can be at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9%. In general, an exact match indicates 100% identity over the length of the reference sequence.

[0055] Polypeptides and polynucleotides that are about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 99.5% or more identical to polypeptides and polynucleotides described herein are embodied within the disclosure. For example, a polypeptide can have 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the sequences of the recombinant COBRA proteins described herein.

[0056] Variants of the disclosed sequences also include peptides, or full-length protein, that contain substitutions, deletions, or insertions into the protein backbone, that would still leave at least about 70% homology to the original protein over the corresponding portion. A yet greater degree of departure from homology is allowed if like-amino acids, i.e., conservative amino acid substitutions, do not count as a change in the sequence. Examples of conservative substitutions involve amino acids that have the same or similar properties. Illustrative amino acid conservative substitutions include the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isolcucinc to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine, or glutamate; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; valine to isoleucine to leucine.

[0057] In one aspect, the recombinant influenza antigens have a sequence with at least 80% sequence identity with the sequence of SEQ ID NO: 3. In one aspect, the recombinant influenza antigens have a sequence with at least 85% sequence identity with the sequence of SEQ ID NO: 3. In one aspect, the recombinant influenza antigens have a sequence with at least 95% sequence identity with the sequence of SEQ ID NO: 3. In one aspect, the recombinant influenza antigens have a sequence with at least 96% sequence identity with the sequence of SEQ ID NO: 3. In one aspect, the recombinant influenza antigens have a sequence with at least 97% sequence identity with the sequence of SEQ ID NO: 3. In one aspect, the recombinant influenza antigens have a sequence with at least 98% sequence identity with the sequence of SEQ ID NO: 3. In one aspect, the recombinant influenza antigens have a sequence with at least 99% sequence identity with the sequence of SEQ ID NO: 3.

[0058] In one aspect, the recombinant influenza antigens have a sequence with at least 80% sequence identity with the sequence of SEQ ID NO: 4. In one aspect, the recombinant influenza antigens have a sequence with at least 85% sequence identity with the sequence of SEQ ID NO:

4. In one aspect, the recombinant influenza antigens have a sequence with at least 95% sequence identity with the sequence of SEQ ID NO: 4. In one aspect, the recombinant influenza antigens have a sequence with at least 96% sequence identity with the sequence of SEQ ID NO: 4. In one aspect, the recombinant influenza antigens have a sequence with at least 97% sequence identity with the sequence of SEQ ID NO: 4. In one aspect, the recombinant influenza antigens have a sequence with at least 98% sequence identity with the sequence of SEQ ID NO: 4. In one aspect, the recombinant influenza antigens have a sequence with at least 99% sequence identity with the sequence of SEQ ID NO: 4.

[0059] The vaccine compositions described herein include a cationic lipid.

[0060] Adjuvants are essential components of subunit vaccines added to enhance immune responses to antigens through immunomodulation. Very few adjuvants have been approved for human use by regulatory agencies due to safety concerns. Current subunit vaccine adjuvants approved for human use are very effective in promoting humoral immune responses but are less effective at promoting T cell immunity. In this study, a novel pure cnantio-spccific cationic lipid l,2-dioleoyl-3-trimethylammonium-propane (R-DOTAP) was evaluated as an immunomodulator for subunit vaccines capable of inducing both humoral and cellular mediated immunity. Using recombinant protein antigens derived from computationally optimized broadly reactive influenza antigen (COBRA) proteins, it was demonstrated that R-DOTAP nanoparticles promoted strong cellular and antibody-mediated immune responses in both monovalent and bivalent vaccines. As further discussed below, R-DOTAP based vaccines induced antigen specific and polyfunctional CD8 ⁺ and CD4 ⁺ effector T cells and memory T cells, respectively. Antibody responses induced by R-DOTAP showed a balanced Thl/Th2 type immunity, neutralizing activity and protection of mice from challenge with live influenza viruses. R-DOTAP also facilitated significant dose sparing of the vaccine antigens. These studies demonstrate that R-DOTAP is an excellent immune stimulator for the production of next-generation subunit vaccines containing multiple recombinant proteins.

[0061] Adjuvants are often used to modify or augment the effects of a vaccine by stimulating the immune system to respond to the vaccine more vigorously, and thus providing increased immunity to a particular disease. Adjuvants accomplish this task by mimicking specific sets of evolutionarily conserved molecules, so called pathogen-associated molecular patterns, which include liposomes, lipopolysaccharide, molecular cages for antigens, components of bacterial cell walls, and endocylosed nucleic acids such as RNA, double-stranded RNA, single-stranded DNA, and unmethylated CpG dinucleotide-containing DNA. Because immune systems have evolved to recognize these specific antigenic moieties, the presence of an adjuvant in conjunction with the vaccine can greatly increase the innate immune response to the antigen by augmenting the activities of dendritic cells, lymphocytes, and macrophages by mimicking a natural infection.

[0062] The composition described herein can be formulated with a lipid nanoparticle as an adjuvant to enhance the presentation of the antigens to antigen presenting cells, and therefore to increase the immune response induce by the antigens.

[0063] In some aspects described herein, the adjuvant is a cationic lipid. As used herein, the term “cationic lipid” refers to any of a number of lipid species which carry a net positive charge at physiological pH or have a protonatable group and are positively charged at pH lower than the pKa.

[0064] Suitable cationic lipids according to the present disclosure include, but are not limited to: 3- p [4N1N, 8-diguanidino spermidine)-carbamoyl]cholesterol (BGSC); 3-[3 [N,N- diguanidinocthyl-aminocthanc)-carbamoyl]cholcstcrol (BGTC); N,N.lN2N3Tctra- methyltetrapalmitylspermine (cellfectin); N-t-butyl-N’- tetradecyl-3-tetradecyl-aminopropion- amidine (CLONfectin); dimethyldioctadecyl ammonium bromide (DDAB); 1,2- dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide (DMRIE); 2,3-dioleoyloxy- N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl- 1 -p- - ropanaminium trifluorocetate) (DOSPA); l,3-dioleoyloxy-2-(6-carboxyspermyl)-propyl amide (DOSPER); 4-(2,3-bis- palmitoyloxy-propyl)- 1 -methyl- 1 H-imidazole (DPIM) N,N,N’,N’- tetramethyl-N,N’-bis(2- hydroxyethyl)-2,3-dioleoyloxy-l,4-butane- diammonium iodide) (Tfx- 50); N-l-(2,3-dioleoyloxy) propyl-N,N,N-trimethyl ammonium chloride (DOTMA) or other N- (N,N-l-dialkoxy)-alkyl- N,N,N-trisubstituted ammonium surfactants; 1,2 dioleoyl-3-(4’- trimethylammonio) butanol- sn- glycerol (DOBT) or cholesteryl (4 ’trimethylammonia) butanoate (ChOTB) where the trimethylammonium group is connected via a butanol spacer arm to either the double chain (for DOTB) or cholesteryl group (for ChOTB); DORI (DL-l,2-dioleoyl-3- dimethylaminopropyl- .beta. -hydroxy ethylammonium) or DORIE (DL-l,2-0-dioleoyl-3- dimethylaminopropyl-.beta.- hydroxyethylammoniu- -m) (DORIE) or analogs thereof as disclosed in WO 93/03709; 1,2- dioleoyl-3 -succinyl- sn-glycerol choline ester (DOSC); cholesteryl hemisuccinate ester (ChOSC); lipopolyamines such aass dioctadecylamidoglycylspermine (DOGS) and dipalmitoyl phosphatidylethanolamylspermine (DPPES), cholesteryl- 3 P -carboxyl-amido- ethylenetrimethylammonium iodide, 1- dimethylamino-3-trimethylammonio-DL-2 -propylcholesteryl carboxylate iodide, cholesteryl- 3 -O-carboxyamidoethyleneamine, cho lesteryl-3 - .beta.-oxysuccinamido- ethylenetrimethylammonium iodide, l-dimethylamino-3- trimethylammonio-DL-2 -propyl- cho lesteryl-3- [3 -oxysu- ccinate iodide, 2-(2- trimethylammonio)-ethylmethylamino ethyl- cho lesteryl-3-. Beta, -oxysuccinate iodide, 3- p -N- (N’,N’ -dimethylaminoethane) carbamoyl cholesterol (DC-chol), and 3- P -N- (polycthylcnciminc)-carbamoylcholcstcrol; 0,0’- dimyristyl-N-lysyl aspartate (DMKE); 0,0’- dimyristyl-N-lysyl-glutamate (DMKD); 1 ,2- dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide (DMRIE); 1 ,2-dilauroyl- sn-glycero-3-ethylphosphocholine (DLEPC); 1 ,2- dimyristoyl-sn-glycero-3- ethylphosphocholine (DMEPC); l,2-dioleoyl-sn-glycero-3- ethylphosphocholine (DOEPC); 1,2- dipalmitoyl-sn-glycero-3-ethylphosphocholine (DPEPC); 1 ,2-distearoyl-sn-glycero-3- ethylphosphocholine (DSEPC); l,2-dioleoyl-3-trimethylammonium propane (DOTAP); dioleoyl dimethylaminopropane (DODAP); l,2-palmitoyl-3- trimethylammonium propane (DPTAP); l,2-distearoyl-3 -trimethylammonium propane (DSTAP), l,2-myristoyl-3- trimcthylammonium propane (DMTAP); and sodium dodecyl sulfate (SDS). Furthermore, structural variants and derivatives of the any of the described cationic lipids are also contemplated.

[0065] In some aspects, the cationic lipid is selected from the group consisting of DOTAP, DOTMA, DOEPC, and combinations thereof. In other aspects, the cationic lipid is DOTAP. In yet other aspects, the cationic lipid is DOTMA. In other aspects, the cationic lipid is DOEPC. In some aspects, the cationic lipid is purified. [0066] In some embodiments, the cationic lipid is an enantiomer of a cationic lipid. The term “enantiomer” refers to a stereoisomer of a cationic lipid which is a non-superimposable mirror image of its counterpart stereoisomer, for example R and S enantiomers. In various examples, the enantiomer is R-DOTAP or S-DOTAP. In one example, the enantiomer is R- DOTAP. In another example, the enantiomer is S-DOTAP. In some aspects, the enantiomer is purified.

[0067] In one aspect, the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R- DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, variations thereof or analogs thereof.

[0068] In another aspect, the one or more recombinant influenza antigens are encapsulated in liposomes including cationic lipids. In another aspect, the one or more recombinant influenza antigens mixed with preformed cationic lipid nanoparticles.

[0069] In some aspects, the one or more recombinant protein antigens and the preformed cationic lipid nanoparticles are mixed at a 1:1 ratio.

[0070] In one aspect, the one or more recombinant influenza antigens are present as micelles separate from the cationic lipid nanoparticles.

[0071] METHODS OF USE

[0072] In another embodiment, the invention provides a method of inducing an immune response against influenza viruses in a subject including administering to the subject a vaccine composition including: a) one or more non-naturally occurring recombinant influenza antigens; and b) a cationic lipid, thereby inducing an immune response against influenza viruses in a subject. [0073] The term “immune response” refers to an integrated bodily response to an antigen and preferably refers to a cellular immune response or a cellular as well as a humoral immune response. The immune response may be protective/preventive/prophylactic and/or therapeutic.

[0074] The immune system is a system of biological structures and processes within an organism that protects against disease. This system is a diffuse, complex network of interacting cells, cell products, and cell-forming tissues that protects the body from pathogens and other foreign substances, destroys infected and malignant cells, and removes cellular debris: the system includes the thymus, spleen, lymph nodes and lymph tissue, stem cells, white blood cells, antibodies, and lymphokines. B cells or B lymphocytes are a type of lymphocyte in the humoral immunity of the adaptive immune system and are important for immune surveillance. T cells or T lymphocytes are a type of lymphocyte that plays a central role in cell-mediated immunity. There are two major subtypes of T cells: the killer T cell and the helper T cell. In addition, there are suppressor T cells which have a role in modulating immune response. Killer T cells only recognize antigens coupled to Class I MHC molecules, while helper T cells only recognize antigens coupled to Class II MHC molecules. These two mechanisms of antigen presentation reflect the different roles of the two types of T cell. A third minor subtype are the y5 T cells that recognize intact antigens that are not bound to MHC receptors. In contrast, the B cell antigen-specific receptor is an antibody molecule on the B cell surface and recognizes whole pathogens without any need for antigen processing. Each lineage of B cell expresses a different antibody, so the complete set of B cell antigen receptors represent all the antibodies that the body can manufacture.

[0075] A “cellular immune response”, a “cellular response”, a “cellular response against an antigen” or a similar term is meant to include a cellular response directed to cells characterized by presentation of an antigen with class I or class II MHC. The cellular response relates to cells called T cells or T-lymphocytes which act as either “helpers” or “killers”. The helper T cells (also termed CD4+ T cells) play a central role by regulating the immune response and the killer cells (also termed cytotoxic T cells, cytolytic T cells, CD8+ T cells or CTLs) kill diseased cells such as cancer cells, preventing the production of more diseased cells. In preferred embodiments, the present invention involves the stimulation of an anti-tumor CTL response against tumor cells expressing one or more tumor expressed antigens and preferably presenting such tumor expressed antigens with class I MHC.

[0076] The terms “immunoreactive cell” “immune cells” or “immune effector cells” in the context of the present invention relate to a cell which exerts effector functions during an immune reaction. An “immunoreactive cell” preferably is capable of binding an antigen or a cell characterized by presentation of an antigen, or an antigen peptide derived from an antigen and mediating an immune response. For example, such cells secrete cytokines and/or chemokines, secrete antibodies, recognize cancerous cells, and optionally eliminate such cells. For example, immunoreactive cells comprise T cells (cytotoxic T cells, helper T cells, tumor infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells.

[0077] “Inducing an immune response” may mean that there was no immune response against a particular antigen before induction, but it may also mean that there was a certain level of immune response against a particular antigen before induction and after induction said immune response is enhanced. Thus, “inducing an immune response” also includes “enhancing an immune response”. Preferably, after inducing an immune response in a subject, said subject is protected from developing a disease such as the flu or the disease condition is ameliorated by inducing an immune response. For example, an immune response against an influenza antigen may be induced in a subject at risk of being infected by an influenza vims. Inducing an immune response in this case may mean that the disease condition of the subject is ameliorated, or that the subject does not develop the flu.

[0078] In one aspect, the immune response includes an induction of CD8 ⁺ effector T cells,

CD4 ⁺ effector T cells and memory T cells.

[0079] In one aspect, inducing CD8+ and CD4+ effector T cells includes inducing proliferation of IFNy and granzyme B producing CD8+ effector T cells, and/or the proliferation of IL-4 producing CD4+ effector T cells in the subject.

[0080] In another aspect, inducing a humoral immune response includes inducing the production of IgG in the subject. In some aspects, IgG include IgGl and IgG2a.

[0081] In one aspect, inducing the immune response includes inducing the secretion of broadly neutralizing antibodies.

[0082] In one aspect, the one or more non-naturally occurring recombinant influenza antigens include a computationally optimized broadly reactive influenza antigen (COBRA) hemagglutinins (HA). In another aspect, the one or more non-naturally occurring recombinant influenza antigens arc recombinant H1N1 and/or H3N2 hemagglutinin influenza proteins. In one aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence with at least 80%, 85% 90% or 95% sequence identity to any of SEQ ID NOs:3-22 and combinations thereof. In one aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence with at least 80%, 85% 90% or 95% sequence identity to SEQ ID NO: 3 or SEQ ID NO:4. In another aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence of any of SEQ ID NOs:3-22 and combinations thereof. In another aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4. In another aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence with at least 80%, 85% 90% or 95% sequence identity to of SEQ ID NOs:3 and SEQ ID NO:4. In one aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence of SEQ ID NO: 3 and SEQ ID NO:4. In one aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence of SEQ ID NO: 3 and SEQ ID NO:4. [0083] In another aspect, the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S- DDA, S-DOEPC, S-DOTMA, variations thereof or analogs thereof. In one aspect, the cationic lipid is R-DOTAP. In another aspect, the one or more recombinant influenza antigens are encapsulated in liposomes including cationic lipids. In another aspect, the one or more recombinant influenza antigens are mixed with preformed cationic lipid nanoparticles. In some aspects, the one or more recombinant influenza antigens and the preformed cationic lipid nanoparticles are mixed at a 1:1 ratio. In one aspect, the one or more recombinant influenza antigens are present as micelles separate from the cationic lipid nanoparticles.

[0084] In an additional embodiment, a method of preventing or treating an influenza infection in a subject including administering to the subject a vaccine composition including: a) one or more non-naturally occurring recombinant influenza antigens; and b) a cationic lipid, thereby preventing or treating an influenza infection in a subject.

[0085] In one aspect, the one or more non-naturally occurring recombinant influenza antigens include a computationally optimized broadly reactive influenza antigen (COBRA) hemagglutinins (HA). In another aspect, the one or more non-naturally occurring recombinant influenza antigens arc recombinant H1N1 and/or H3N2 hemagglutinin influenza proteins. In one aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence with at least 80%, 85% 90% or 95% sequence identity to any of SEQ ID NOs:3-22 and combinations thereof. In one aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence with at least 80%, 85% 90% or 95% sequence identity to SEQ ID NO: 3 or SEQ ID NO:4. In another aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence of any of SEQ ID NOs:3-22 and combinations thereof. In another aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4. In another aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence with at least 80%, 85% 90% or 95% sequence identity to of SEQ ID NOs:3 and SEQ ID NO:4. In one aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence of SEQ ID NO: 3 and SEQ ID NO:4. In one aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence of SEQ ID NO: 3 and SEQ ID NO:4. [0086] In another aspect, the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S- DDA, S-DOEPC, S-DOTMA, variations thereof or analogs thereof. In one aspect, the cationic lipid is R-DOTAP. In another aspect, the one or more recombinant influenza antigens are encapsulated in liposomes including cationic lipids. In another aspect, the one or more recombinant influenza antigens are mixed with preformed cationic lipid nanoparticles. In some aspects, the one or more recombinant influenza antigens and the preformed cationic lipid nanoparticles are mixed at a 1:1 ratio. In one aspect, the one or more recombinant influenza antigens are present as micelles separate from the cationic lipid nanoparticles.

[0087] In one embodiment, the invention provides a method of enhancing immunogenicity of an influenza vaccine in a subject including administering to the subject a vaccine composition including: a) one or more non-naturally occurring recombinant influenza antigens; and b) a cationic lipid, thereby enhancing immunogenicity of an influenza vaccine.

[0088] In some aspects administration can be in combination with one or more additional therapeutic agents. The phrases “combination therapy”, “combined with” and the like refer to the use of more than one medication or treatment simultaneously to increase the response. The cationic lipid of the present invention might for example be used in combination with existing influenza vaccine to increase the immune response generated by the influenza vaccine alone. The cationic lipid can be administered prior to, simultaneously with, or following administration of the influenza vaccine.

[0089] By “enhancing immunogenicity”, it is meant that the immunogenicity of the influenza vaccine is greater when it is administered in combination with the cationic lipid of the present invention, as compared to the immunogenicity induced by the influenza vaccine administered alone (e.g., in the absence of the administration of the cationic lipid as an immunomodulator).

[0090] In one aspect, the influenza vaccine is an inactivated flu vaccine, an attenuated flu vaccine or a recombinant flu vaccine.

[0091] Vaccines typically contain attenuated, inactivated or dead organisms or purified products derived from them. There are several types of vaccines in use, representing different strategies used to try to reduce the risk of illness while retaining the ability to induce a beneficial immune response. Influenza vaccines are usually “attenuated”, “inactivated” or “subunit” (e.g., recombinant). [0092] Live, attenuated microorganisms, such as active viruses that have been cultivated under conditions that disable their virulent properties, or that use closely related but less dangerous organisms to produce a broad immune response constitute attenuated vaccines. Although most attenuated vaccines are viral, some are bacterial in nature.

[0093] Inactivated, but previously virulent, micro-organisms that have been destroyed with chemicals, heat, or radiation - "ghosts", with intact but empty bacterial cell envelopes constitute inactivated vaccines. They are considered an intermediate phase between the inactivated and attenuated vaccines. Examples include IPV (polio vaccine), hepatitis A vaccine, rabies vaccine and most influenza vaccines.

[0094] Rather than introducing an inactivated or attenuated micro-organism to an immune system (which would constitute a "whole-agent" vaccine), a subunit vaccine uses a fragment of it to create an immune response. Only one protein of the virus (previously extracted from the blood serum of chronically infected patients but now produced by recombination of the viral genes into yeast), for example a surface protein is recomb inantly produced and used for the vaccine. The hemagglutinin and neuraminidase subunits of the influenza vims are examples or subunit proteins used in recombinant influenza vaccines.

[0095] In another aspect, the influenza vaccine is a monovalent vaccine, a bivalent vaccine, a trivalent vaccine or a quadrivalent vaccine.

[0096] In one aspect, the one or more non-naturally occurring recombinant influenza antigens include a computationally optimized broadly reactive influenza antigen (COBRA) hemagglutinins (HA). In another aspect, the one or more non-naturally occurring recombinant influenza antigens are recombinant H1N1 and/or H3N2 hemagglutinin influenza proteins. In one aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence with at least 80%, 85% 90% or 95% sequence identity to any of SEQ ID NOs:3-22 and combinations thereof. In one aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence with at least 80%, 85% 90% or 95% sequence identity to SEQ ID NO: 3 or SEQ ID NOT In another aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence of any of SEQ ID NOs:3-22 and combinations thereof. In another aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4. In another aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence with at least 80%, 85% 90% or 95% sequence identity to of SEQ ID NOs:3 and SEQ ID NON. In one aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence of SEQ ID NO: 3 and SEQ ID NON. In one aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence of SEQ ID NO: 3 and SEQ ID NON.

[0097] In another aspect, the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S- DDA, S-DOEPC, S-DOTMA, variations thereof or analogs thereof. In one aspect, the cationic lipid is R-DOTAP. In another aspect, the one or more recombinant influenza antigens are encapsulated in liposomes including cationic lipids. In another aspect, the one or more recombinant influenza antigens are mixed with preformed cationic lipid nanoparticles. In some aspects, the one or more recombinant influenza antigens and the preformed cationic lipid nanoparticles are mixed at a 1:1 ratio. In one aspect, the one or more recombinant influenza antigens are present as micelles separate from the cationic lipid nanoparticles.

[0098] Examples of influenza vaccines include, but are not limited to Afluria Quadrivalent,

Fluarix Quadrivalent, FluLaval Quadrivalent, Fluzone Quadrivalent, Flucelvax Quadrivalent, Fluzone High-Dose Quadrivalent, Fluad Quadrivalent, Flublok Quadrivalent, FluMist Quadrivalent and Fluzone®.

[0099] Tn another embodiment, the invention provides a method of inducing secretion of broadly neutralizing antibodies against influenza viruses in a subject including administering to the subject a vaccine composition including: a) one or more non-naturally occurring recombinant influenza antigens; and b) a cationic lipid, thereby inducing secretion of broadly neutralizing antibodies against influenza viruses in a subject.

[0100] Development of a universal influenza vaccine that can provide heterosubtypic immunity and protection against multiple clades has been a long-term goal to protect against Influenza infections. There are several strategies under investigation to achieve this goal, including the use of antigens from multiple subtypes to increase the coverage, targeting multiple proteins (e.g., inclusion of HA and non-HA proteins) as antigens, target conserved antigenic regions of influenza antigens, use of chimeric proteins containing stem and stalk HA from different subtypes, and use of consensuses based approaches such as COBRA sequences comprising multiple known mutations in the hemagglutinin or neuraminidase. In addition, vaccine technologies that induce both CDS T cell and antibody-mediated immunity are also actively being sought to achieve heterosubtypic protection against influenza. As described herein, using a prototype vaccine based on the R-DOTAP platform and COBRA H1N 1 and H3N2 derived HA or nucleoprotein antigen, that such a vaccine is capable of inducing antigen specific T cell responses, neutralizing antibodies against multiple clades of H1N1, and H3N2 strains, and protected mice against challenge with a lethal H1N1 strain. Hence, vaccine formulations containing R-DOTAP and COBRA HA, NA sequences, and nucleoprotein from influenza presents strong potential to further the goal of developing a safe and effective universal influenza vaccine.

[0101] The term “broadly neutralizing antibodies” or “bNAbs” as used herein is meant to refer to neutralizing antibodies which neutralize multiple influenza viral strains. bNAbs are unique in that they target conserved epitopes of the virus, meaning the virus may mutate, but the targeted epitopes will still exist. In contrast, non-bNAbs are specific for individual viral strains with unique epitopes.

[0102] In one aspect, the one or more non-naturally occurring recombinant influenza antigens include a computationally optimized broadly reactive influenza antigen (COBRA) hemagglutinins (HA). In another aspect, the one or more non-naturally occurring recombinant influenza antigens are recombinant H1N1 and/or H3N2 hemagglutinin influenza proteins. In one aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence with at least 80%, 85% 90% or 95% sequence identity to any of SEQ ID NOs:3-22 and combinations thereof. In one aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence with at least 80%, 85% 90% or 95% sequence identity to SEQ ID NO: 3 or SEQ ID NON. In another aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence of any of SEQ ID NOs:3-22 and combinations thereof. In another aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence of SEQ ID NON or SEQ ID NON. In another aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence with at least 80%, 85% 90% or 95% sequence identity to of SEQ ID NOs:3 and SEQ ID NON. In one aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence of SEQ ID NO: 3 and SEQ ID NON. In one aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence of SEQ ID NO: 3 and SEQ ID NON.

[0103] In another aspect, the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S- DDA, S-DOEPC, S-DOTMA, variations thereof or analogs thereof. In one aspect, the cationic lipid is R-DOTAP. In another aspect, the one or more recombinant influenza antigens are encapsulated in liposomes including cationic lipids. In another aspect, the one or more recombinant influenza antigens are mixed with preformed cationic lipid nanoparticles. In some aspects, the one or more recombinant influenza antigens and the prefomied cationic lipid nanoparticles are mixed at a 1:1 ratio. In one aspect, the one or more recombinant influenza antigens are present as micelles separate from the cationic lipid nanoparticles.

[0104] In a further embodiment, the invention provides a method of inducing a balanced Thl/Th2 immune response in a subject including administering to the subject a vaccine composition including: a) one or more non-naturally recombinant influenza antigens; and b) a cationic lipid, thereby inducing secretion of broadly neutralizing antibodies against influenza viruses in a subject.

[0105] Adjuvants can be broadly categorized into Thl, Th2, Thl7, and mixed Thl/Th2 or Thl/Thl7 types based on the cytokines and antibody subclasses induced by the vaccine. Thl type adjuvants for example show skewing towards IFN-y production and IgG2a/c antibody subtypes in mouse vaccinations. Th2 adjuvants stimulate more IL-4 production resulting in skewing towards IgGl antibody subtypes in mice.

[0106] In one aspect, inducing a Thl immune response includes inducing proliferation of IFNy and granzyme B producing CD8+ effector T cells, and/or the proliferation of TL-4 producing CD4+ effector T cells in the subj ect.

[0107] In some aspects, IFNy producing CD8+ effector T cells are associated with production of IgG2a, and IL-4 producing CD4+ effector T cells are associated with production of IgGl.

[0108] In one aspect, administering the vaccine composition to the subject includes subcutaneous administration or intramuscular administration.

[0109] The term “subject” as used herein refers to any individual or patient to which the subject methods arc performed. Generally, the subject is human, although as will be appreciated by those in the art, the subject may be an animal. Thus, other animals, including vertebrate such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, chickens, etc., and primates (including monkeys, chimpanzees, orangutans and gorillas) are included within the de flnition of subject.

[0110] The terms “administration of’ and or “administering” should be understood to mean providing a pharmaceutical composition in a therapeutically effective amount to the subject in need of treatment. Administration routes can be enteral, topical or parenteral. As such, administration routes include but are not limited to intracutaneous, subcutaneous, intravenous, intraperitoneal, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transdermal, transtracheal, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrastemal, oral, sublingual, buccal, rectal, vaginal, nasal, ocular administrations, as well infusion, inhalation, and nebulization. Preferably, the vaccine compositions described herein are administered through subcutaneous or intramuscular administration.

[0111] In another aspect, administering the vaccine composition includes administering two doses of the vaccines, and optionally administering a booster.

[0112] In one aspect, the one or more non-naturally recombinant influenza antigens include a computationally optimized broadly reactive influenza antigen (COBRA) hemagglutinins (HA). In another aspect, the one or more non-naturally recombinant influenza antigens are recombinant H1N1 and/or H3N2 hemagglutinin influenza proteins. In one aspect, the one or more non- naturally occurring recombinant influenza antigens include an amino acid sequence with at least 80%, 85% 90% or 95% sequence identity to any of SEQ ID NOs:3-22 and combinations thereof. In one aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence with at least 80%, 85% 90% or 95% sequence identity to SEQ ID NO: 3 or SEQ ID NON. In another aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence of any of SEQ ID NOs:3-22 and combinations thereof. In another aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence of SEQ ID NON or SEQ ID NON. In another aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence with at least 80%, 85% 90% or 95% sequence identity to of SEQ ID NOs:3 and SEQ ID NON. In one aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence of SEQ ID NO: 3 and SEQ ID NON. In one aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence of SEQ ID NO: 3 and SEQ ID NON.

[0113] In another aspect, the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S- DDA, S-DOEPC, S-DOTMA, variations thereof or analogs thereof. In one aspect, the cationic lipid is R-DOTAP. In another aspect, the one or more recombinant influenza antigens are encapsulated in liposomes including cationic lipids. In another aspect, the one or more recombinant influenza antigens are mixed with preformed cationic lipid nanoparticles. In some aspects, the one or more recombinant influenza antigens and the preformed cationic lipid nanoparticles are mixed at a 1:1 ratio. In one aspect, the one or more recombinant influenza antigens are present as micelles separate from the cationic lipid nanoparticles.

[0114] In an additional embodiment, the invention provides a method of inducing a polyfunctional CD4+/CD8+ T cell response against influenza viruses in a subject including administering to the subject an influenza vaccine composition including: a) one or more non- naturally occurring recombinant influenza antigens; and b) a cationic lipid, thereby inducing a polyfunctional CD4+/CD8+ T cell response against influenza viruses in the subject.

[0115] In one aspect, inducing a polyfunctional CD4+/CD8+ T cell response includes inducing the secretion of two or more cytokines. In sone aspects, the two or more cytokines are selected from the group consisting of IFNy, granzyme B and IL-4.

[0116] In one aspect, the one or more non-naturally occurring recombinant influenza antigens include a computationally optimized broadly reactive influenza antigen (COBRA) hemagglutinins (HA). In another aspect, the one or more non-naturally occurring recombinant influenza antigens are recombinant H1N1 and/or H3N2 hemagglutinin influenza proteins. In one aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence with at least 80%, 85% 90% or 95% sequence identity to any of SEQ ID NOs:3-22 and combinations thereof. In one aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence with at least 80%, 85% 90% or 95% sequence identity to SEQ ID NO: 3 or SEQ ID NON. In another aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence of any of SEQ ID NOs:3-22 and combinations thereof. In another aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence of SEQ ID NON or SEQ ID NON. In another aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence with at least 80%, 85% 90% or 95% sequence identity to of SEQ ID NOs:3 and SEQ ID NON. In one aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence of SEQ ID NO: 3 and SEQ ID NO:4. In one aspect, the one or more non-naturally occurring recombinant influenza antigens include an amino acid sequence of SEQ ID NO: 3 and SEQ ID NON.

[0117] In another aspect, the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S- DDA, S-DOEPC, S-DOTMA, variations thereof or analogs thereof. In one aspect, the cationic lipid is R-DOTAP. In another aspect, the one or more recombinant influenza antigens are encapsulated in liposomes including cationic lipids. In another aspect, the one or more recombinant influenza antigens are mixed with preformed cationic lipid nanoparticles. In some aspects, the one or more recombinant influenza antigens and the preformed cationic lipid nanoparticles are mixed at a 1:1 ratio. In one aspect, the one or more recombinant influenza antigens are present as micelles separate from the cationic lipid nanoparticles.

[0118] Presented below are examples discussing vaccines combination including influenza recombinant antigens and a cationic lipid contemplated for the discussed applications. The following examples are provided to further illustrate the embodiments of the present invention but are not intended to limit the scope of the invention. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

EXAMPLES

EXAMPLE 1

MATERIALS & METHODS

[0119] Animals and viruses: Six to twcnty-wcck-old C57BL/6J mice (B6 mice), BALB/cJ, KI 8-hACE2 mice (B6.Cg-Tg(K 18-ACE2)2Prlmn/J), and DBA/2J, mice were obtained from Jackson Laboratories. All animals were housed in specific-pathogen- free conditions at the Division of Laboratory Animal Resources (DEAR), University of Kentucky Medical Center, or in the animal research center at the University of Georgia. All animal protocols were reviewed and approved by the University of Kentucky (2019-3226) or The University of Georgia Institutional Animal Care and Use Committee following the National Institutes of Health Animal care guidelines (A2020 02-024-Y1-A5, A2018 06-018-Y3-A16). All studies were carried out in compliance with ARRIVE guidelines.

[0120] For influenza challenge studies (A/Brisbane/02/2018(Bris/18) was used. For HAI assays (A/California/07/2009 (Cal/09), A/Guangdong-Maonan/SWL1536/2019(GD19), A/Singapore/IFNIMH-16-00192016 (Singapore/16), A/'Hong Kong/4801/2014 (Hong Kong/14), A/Victoria/210/2009 X-187 (an A/Perth/16/2009- like virus) (H3N2), and B/Brisbane/60/2008 were used. Influenza virus were obtained from International influenza Resources (IRR). [0121] Reagents and antibodies:

[0122] cGMP grade R-DOTAP (1, 2-dioleoyl-3 -trimethyl ammonium-propane) was provided by Merck & Cie. Evonik produced cGMP grade R-DOTAP liposomal nanoparticles according to protocols described previously.

[0123] Influenza COBRA antigens: COBRA HA- Y2 (COBRA- Y2) (H1N1), and COBRA- HA-NG2 (COBRA-NG2) (H3N2), were synthesized at the University of Georgia Center for Vaccines and Immunology Research center. Influenza nucleoprotein from A/Puerto Rico/8/34/Mount Sinai) was obtained from Sino biologicals US Inc. Fluzone® vaccine formulation was obtained from the University of Kentucky Healthcare pharmacy.

[0124] Overlapping peptide pools from Influenza A/New York/383/2005 (H3N2) hemagglutinin protein and overlapping peptide pools from Influenza A/California/07/2009 (H1N1) pdm09 were obtained from BEI Resources.

[0125] Fluorochrome conjugated mouse monoclonal anti-mouse CD3 (Clone: 145-2cl 1), CD4 (Clone: GK1.5), CDS (Clone: YTS165.7.7), CD44 (clone: IM7), CD62L (clone: MED-14), IFNy (Clone: XMG1.2), TNFa (Clone: MP6-XT22), IL-2 (Clone: JES6.5H4), were purchased from BioLegend.

[0126] Preparation of R-DOTAP Nanoparticles and vaccine formulations:

[0127] cGMP grade R-DOTAP liposomal nanoparticles were produced by Evonik using thin film hydration method according to protocols described previously. Briefly, R-DOTAP thin films were formed by dissolving the lipid in 1:1 chloroform and methanol mixture in a round bottom flask. Organic solvents were then evaporated using steady stream of dry nitrogen gas followed by overnight vacuum desiccation. The dried R-DOTAP film is then hydrated by incubating films in water for 12 hr. The lipid suspensions were then sonicated for 10 minutes and extruded sequentially using 400, 200, and 100 nm polycarbonate membrane filters to obtain uniform sized liposomal nanoparticlcs (FIGURES 1A-1B). Groups of BALB/cJ (n=6-8) mice were immunized on day 0 and day 21 with two doses of monovalent COBRA -Y2 formulated with R-DOTAP nanoparticles or sucrose buffer (sucrose). Serum samples obtained from vaccinated mice on day 35 (14 days after the second dose) were measured for anti-COBRA-Y2 total IgG antibody titer. Data represents (a-b) mean ± SEM of half-max titers from each mouse. Comparisons between sucrose alone or R-DOTAP groups was performed using Student’s t-test (unpaired-two tailed) ** P <0.05. The nanoparticles were then diluted in 280mM Sucrose buffer and were stored at -80°C until use. For making vaccine formulations, concentrated antigens dissolved in PBS buffer was diluted to desired concentration in 280mM sucrose. Prior to vaccination, the vaccine components were brought to ambient temperature and antigen component is then mixed 1 : 1 ratio with the R- DOTAP nanoparticle using a pipette to form a uniform suspension. For subcutaneous vaccine delivery, lOOμl were used for each dose; for intramuscular delivery, 50 μl were used for each dose.

[0128] Physical Characterization of Vaccine formulation:

[0129] Particle size, polydispersity and zeta potential of R-DOTAP liposomes and vaccine formulations were measured at 23°C using Zeasizer nano equipped with a 4mW 632.8nm laser set at 90° angle. Dynamic light scattering was used to determine the fluctuations in dispersed light intensity. Distribution analysis and cumulants analysis to measure Z average and polydispersity was performed according to instructions and using apparatus software. A representative particle size distribution is shown in FIGURES 2A-2C. Average particle size, poly dispersity and zeta potential measurements are shown in Table 1.

[0130] Enzyme-Linked Immunosorbent Assay (ELISA):

[0131] Blood was collected into B.D. microtainer® serum separator tubes through tail vein bleed or cardiac puncture of euthanized mice. Isolated serum samples were stored at -80°C until analysis. For antibody titer measurement, 96 well plates were coated overnight at 4°C with 50μl /wcll recombinant RBD protein, COBRA-NG2, or COBRA-Y2 protein at 2 jag/ml concentration. After antigen coating, the wells were blocked for l -2hr at room temperature with 200pl/wcll of PBS-T buffer containing 3% non-fat dry milk and 0.1% tween-20. Following blocking, buffer was replaced with serum samples diluted in PBS-T buffer containing 1% non-fat dry milk. After 2hr incubation at R.T., the serum was removed, and wells were washed four times with PBS-T buffer. For detecting protein antibodies, wells were incubated for Ihr with lOOpl of PBS-T buffer containing 1% non-fat dry milk and HRP-conjugated anti-mouse IgG (1: 5000) (cat# 115-035-003 Jackson ImmunoResearch), anti-mouse IgGl(l:5000) (cat# 115-035-205; Jackson Immuno Research), anti-mousc IgG2c (1:5000) (Cat#l 15-035-206; Jackson ImmunoResearch) or anti-mouse IgG2a (1:5000) (Cat# 115-035-206; Jackson ImmunoResearch). Wells were then washed four times, and 100μl of SIGMAFAST OPD (o-phenylenediamine dihydrochloride) chromogenic substrate was added to each well. After 10 minutes of incubation, the reaction was stopped by adding 50μl of 3M HCL, and absorbance measured at 490nm (OD490) using a spectraMax M5 microplate reader. [0132] Enzyme-Linked Immunospot Assay (ELISpot):

[0133] 2.5 x 10 ⁵ processed spleen cells were stimulated for 18-24 hr at 37°C with T cell epitope peptides of interest or recombinant proteins, or no peptides (control) in a 96 well plate pre-coated with the mouse IFNy or IL-4 capture antibody (Mabtech). After stimulation, wells were washed with PBS and incubated with biotin-conjugated anti-IFNy or IL-4 antibody followed by streptavidin-HRP antibody. To visualize the antigen specific IFNy or IL-4 producing cells, wells were incubated for 6 minutes with TMB substrate, washed with water, and air dried. Spots were scanned and counted using CTL ImmunoSpot Analyzer and ImmunoSpot Ver.6 software. Spot counts were summarized as median values from triplicate samples. Each sample had unstimulated and PMA/Ionomycin control wells to detect background or as a positive control.

[0134] Intracellular Cytokine and Cell surface Staining:

[0135] For intracellular protein analysis, single cell suspensions of spleen cells were stimulated with indicated stimulatory antigenic peptide for 6hr at 37°C in cRPMI media supplemented with purified anti-mouse CD28 (2μg/ml), protein transport inhibitor Brefeldin A (5 μg/ml) and monensin (2.0pM). Following stimulation, cells were washed with FACS buffer and stained with fluorochrome conjugated anti-mouse CD3, CD4, CDS, CD44, and CD62L antibodies. The cells were then washed, fixed and pcrmcabilizcd using fixation/pcrmcabilization kit and stained with fluorochrome conjugated anti-mouse IFNy, TNFa, and TL-2. Cells were washed with FACS buffer after intracellular staining and analyzed immediately using flow cytometry.

[0136] Mouse vaccination:

[0137] Mice were anesthetized using isoflurane for all injections and implants. The injection site was shaved and cleaned with 70% ethanol prior to subcutaneous or intramuscular injection of a formulation. For subcutaneous (S.C.) vaccination, a lOOμl dose was delivered on a single flank of the hind limb and for intramuscular (I.M.) vaccination a 50 μl dose was delivered into the thigh muscle of the hind limb. For making R-DOTAP based vaccine formulations, R-DOTAP nanoparticles (4-6mg/ml) in 280 mM sucrose buffer were mixed 1 : 1 with indicated concentrations of recombinant proteins resuspended in 280mM sucrose buffer. For antigen-only vaccine formulations, recombinant protein was resuspended at the desired concentration in 280mM sucrose buffer. All vaccination regimens consisted of two doses delivered at 1-4-week intervals.

[0138] Hemagglutination inhibition assay:

[0139] The hemagglutination inhibition (HAI) assay was used to assess functional antibodies to the HA that are able to inhibit agglutination of guinea pig erythrocytes for H3N2 viruses, and turkey erythrocytes for H1N1 viruses. The protocols were adapted from the World Health Organization (WHO) laboratory influenza surveillance manual. Guinea pig red blood cells are frequently used to characterize contemporary A(H3N2) influenza strains that have developed a preferential binding to alpha (2,6) linked sialic acid receptors. To inactivate nonspecific inhibitors, sera samples were treated with receptor-destroying enzyme (RDE) prior to being tested. Briefly, three parts of RDE was added to one part of sera and incubated overnight at 37°C. RDE was inactivated by incubation at 56°C for 30 min.

[0140] RDE-treated sera were diluted in a series of two-fold serial dilutions in v-bottom microtiter plates. An equal volume of each A(H3N2) virus, adjusted to approximately 8 hemagglutination units (HAU)/50μl in the presence of 20nM Oseltamivir carboxylate, was added to each well. The plates were covered and incubated at room temperature for 30 mins, and then 0.75% guinea pig erythrocytes in PBS were added. Prior to use, the red blood cells (RBCs) were washed twice with PBS, stored at 4°C, and used within 24 h of preparation. The plates were mixed by gentle agitation, covered, and the RBCs were allowed to settle for 1 h at room temperature. The HAI titer was determined by the reciprocal dilution of the last well that contained nonagglutinated RBCs. Positive and negative serum controls were included for each plate.

[0141] In separate assays, RDE-trcatcd sera were diluted in a scries of two-fold serial dilutions in v-bottom microtiter plates. An equal volume of each influenza virus, adjusted to approximately 8 hemagglutination units (HAU)/50μl was added to each well. The plates were covered and incubated at room temperature for 20 mins with erythrocytes in phosphate buffered saline (PBS). Prior to use, the RBCs were washed twice with PBS, stored at 4°C, and used within 24 h of preparation. The plates were mixed by gentle agitation, covered, and the RBCs were allowed to settle for 30 mins at room temperature. The HAI titer was determined by the reciprocal dilution of the last well that contained non-agglutinated RBCs. Positive and negative serum controls were included for each plate.

[0142] All mice were negative (HAI < 1:10) for pre-existing antibodies to human influenza viruses prior to infection or vaccination, and for this study sero-protection was defined as HAI titer >1:40 and seroconversion as a 4-fold increase in titer compared to baseline, as per the WHO and European Committee for Medicinal Products to evaluate influenza vaccines. All mice were naive and seronegative at the time of vaccination, and thus sero-conversion and sero-protection rates are interchangeable for this study. [0143] Mouse Challenge experiments:

[0144] For influenza virus challenge studies, the DBA/2J mice (female, 7 to 9 weeks old) were immunized with indicated vaccine formulations intramuscularly on day 0 and day 28. On day 56, they were challenged with the H1N1 A/Brisbane/02/2018 (Bris/18) influenza vims at an 10X LD50 dose of 3.6* 10 ⁶ pfu/mouse intranasally with the volume of 50 ph. Mock vaccinated animals were inoculated intranasally with 50uL of PBS. All animals challenged with live vims were monitored twice daily, morning and evening, for weight loss and clinical signs (labored breathing, lethargy, hunched back, raffled fur, failure to respond to stimuli, and severe respiratory distress), for up to 14 days post infection. The body weight was tightly monitored until 14 days post- infection. Mice were humanely euthanized once they lost 20% of their original body weight or they reached clinical endpoints. Lungs in influenza challenge mice were collected from three pre-selected mice in each group on day 3 and day 6 post-infection for viral titer detection. Briefly, frozen lungs were processed and clarified supernatants containing vims were added to MDCK cells at 90% confluence and incubated for Ihr. following this step, cells were washed and supplemented with media containing 1.6% agarose. After 72 hr incubation at 37°C, plates were processed, dried and viral plaques were enumerated plaque forming units per gram of lung tissue.

[0145] Equipment, software and Statistical analysis:

[0146] Flow cytometry was performed using B.D. Symphony A3 flow cytometer equipped with BD FACSDiva™ software. All flow data was analyzed using FlowJo® version 10.0 software. Statistical analysis for all other studies were performed using GraphPad Prism 9.0 software and comparing means by a simple student’s T test or by ANOVA with Tukey multiple comparison correction. Mantel-Cox test was used for survival curves.

EXAMPLE 2

IMMUNOGENICITY OF RECOMBINANT INFLUENZA PROTEIN FORMULATED

WITH R-DOTAP NANOPARTICLES

[0147] The ability of R-DOTAP to enhance the immunogenicity of recombinant influenza proteins was assessed. For this, influenza nucleoprotein, or computationally optimized broadly reactive antigen (COBRA) hemagglutinins (HA) were used as vaccine antigens. In a first set of experiments, nucleoproteins were formulated with R-DOTAP nanoparticles, B6 mice were immunized with two doses of vaccine; the T cell immune response was then assessed using a validated H2-D ^b binding CD8 T cell epitope (NP366-74: ASNENMETM, SEQ ID NO:1) and a validated I-A ^b binding CD4 T cell epitope (NP-311-25: QVYSLIRPNENPAHK, SEQ ID NO:2). [0148] In a second set of experiments, monovalent and bivalent vaccine (R-DOTAP-Y2NG2) containing recombinant COBRA sequences representing H1N1 hemagglutinin (Y2) and H3N2 hemagglutinin (NG2) formulated with R-DOTAP nanoparticles were prepared, BALB/cJ mice were vaccinated intramuscularly with two doses of vaccine and T cell responses were measured 7 days after a booster dose using an IFN-y ELISpot assay.

[0149] to stimulate antigen-specific T cells in the ELISpot assay, whole COBRA proteins or overlapping peptides from A/Califomia 07/2009 (H1N1), or A/New York/384/05(H3N2) hemagglutinins, which share consensus with COBRA sequences and are relevant to the naturally circulating influenza viruses were used.

[0150] As shown in FIGURES 3A-3D, the R-DOTAP containing formulations induced strong T cell responses to both nucleoprotein and COBRA HA antigens. R-DOTAP based vaccines induced strong CD4 ⁺ and CD8 ⁺ T cell responses to nucleoprotein compared to antigen only vaccines (FIGURE 3A). Similarly, mice vaccinated with R-DOTAP -Y2NG2 bivalent vaccine showed robust T cell responses to COBRA antigens (FIGURES 3B-3D). T cell immune responses induced by R-DOTAP adjuvanted formulations were significantly higher compared Addvax® adjuvanted formulations, an oil-emulsion based adjuvants system. Importantly, T cells raised against COBRA antigens recognize and respond to multiple conserved T cell epitopes presented by naturally occurring hemagglutinins from H1N1 and H3N2 strains of viruses (FIGURES 3B- 3D).

EXAMPLE 3

EFFECT OF RECOMBINANT INFLUENZA PROTEIN FORMULATED WITH R-

DOTAP NANOPARTICLES ON ANTIGEN SPECIFIC ANTIBODY TITERS

[0151] To assess antibody mediated immune responses to COBRA proteins, serum samples from vaccinated mice were analyzed for antigen specific antibody titers. A significant enhancement of antibody titers with a single injection of the R-DOTAP based formulation was observed (FIGURE 4A). A second dose further increased antibody titers significantly as compared to antigen-only groups. Mixing two COBRA antigens in a single bivalent vaccine did not pose any formulation issues with R-DOTAP. The addition of NP to the bivalent vaccine was also evaluated and did not encounter any compatibility or stability limitations (data not shown). [0152] When compared to Addavax™ adjuvanted formulation, mice vaccinated with R- DOTAP containing formulations showed higher antibody titers measured at day 35 and day 62 (FIGURES 4B and 4C), indicating that R-DOTAP induced potent antibody induction comparable to Addavax™. Dose sparing potential was next evaluated by immunizing BALB/cJ mice with varying doses of COBRA-Y2 formulated with varying doses of R-DOTAP nanoparticles and Y2-specific antibody responses were measured. Mice immunized with 0.35-3.0 μg ofY2 antigen formulated with 300μg of R-DOTAP showed significantly increased Y2-specific total IgG titers measured 14 days after the boost vaccine (FIGURE 1). Animals vaccinated with 0.35 μg or 3. Ogg showed similar IgG titers. Similarly, mice vaccinated with 50-300jig of R- DOTAP added to the vaccine formulations induced similar elevation in total antibody titers without any significant differences between low dose and high dose of R-DOTAP (FIGURE 1). [0153] Thl type antibody mediated immune responses play an important role in protection against viral infections. To further assess R-DOTAP induced immune response, antibody subclass titers following vaccination were measured. It was observed that R-DOTAP induced class switching, as evidenced by presence of IgGl and IgG2a antibodies in day 35 serum samples, similar levels of IgGl and IgG2a were observed, indicating a balanced Thl/Th2 response (FIGURES 4D and 4E).

EXAMPLE 4

EFFECT OF RECOMBINANT INFLUENZA PROTEIN FORMULATED WITH R-

DOTAP NANOPARTICLES ON THE GENERATION OF NEUTRALIZING

ANTIBODIES

[0154] The functional ability of vaccine to elicit antibodies that block influenza virus interaction with sialic acids was next characterized. The hemagglutination inhibition (HAI) assay was used, to evaluate the generation of influenza virus neutralizing antibodies to different strains of H1N1 and H3N2 viruses.

[0155] BBiivvaalleenntt vvaacccciinneess (R-DOTAP-Y2NG2) formulated with R-DOTAP showed significantly enhanced titers compared to antigen only vaccines (FIGURES 5A-5B) to multiple drift variants of both H1N1 and H3N2 virus strains that share consensus with the COBRA Y-2, and COBRA-NG2 respectively. Robust HA1 titers above the 1 :40 threshold were observed for all H1N1 viruses, even at the lowest dose (0.12ug) of antigen tested. HAI titers against H3N2 drift variants were lower than HAI titers against H IN 1 viruses, but still showed significant HAI titers above the 1:40 threshold for the 3ug dose. As expected, very little neutralizing activity (HAI titer < 1 :40) of antibodies induced with antigen alone samples or from mock vaccinated mice was observed. Together these results show that vaccination with bivalent COBRA antigens formulated with R-DOTAP induced robust and balanced Thl and Th2 antibody responses and broadly cross- reactive antibodies that can neutralize several H1N1 and H3N2 strains drift variants.

EXAMPLE 5

R-DOTAP CAN ENHANCE IMMUNOGENICITY OF UNADJUV ANTED SEASONAL

INFLUENZA VACCINES

[0156] Whether R-DOTAP can be used to enhance immunogenicity to existing human influenza vaccines was evaluated. As a proof of concept, a trivalent inactivated influenza vaccine, Fluzone® (2011-12 formulation) consisting of a split virus hemagglutinin preparation derived from A/California/07/2009 X-179A (H1N1), A Victoria'210/2009 X-187 (an APerth/ 16/2009- like virus) (H3N2), and B/Brisbane/60/2008 was used. Vaccine formulations were prepared by mixing 1:1 ratio of R-DOTAP nanoparticles and varying doses of Fluzone®. C57BL/6J mice were vaccinated (0. Iml/dose) on day 0 and day 21 and blood was obtained for HAI titers on day 35.

[0157] It was observed that formulating Fluzonc® with R-DOTAP significantly enhanced the HAT titers to all viral strains (FIGURES 6A-6C) in vaccinated mice compared to Fluzone® only vaccinated groups. Importantly a significant dose sparing effect in R-DOTAP vaccine groups was observed.

EXAMPLE 6

RECOMBINANT PROTEINS FORMULATED WITH R-DOTAP PROTECTED MICE

FROM CHALLENGE INFLUENZA VIRUS

[0158] To determine the protective efficacy of bivalent influenza vaccine R-DOTAP-Y2NG2 in mice, DBA/2J mice were vaccinated with formulations containing varying doses of COBRA antigens with or without R-DOTAP in a two-dose regimen followed by challenge with A/Brisbane/2/2018 (H1N1) (3.6xl0 ⁶ pfu/dose) 28 days post vaccination. Weight loss was measured as predictor of protection from challenge.

[0159] 100% of both unvaccinated mice and mice vaccinated with antigen-only (3μg/HA) vaccine formulations rapidly lost body weight with 0 and 33% survival, respectively within 7 days (FIGURES 7 A and 7B). In contrast less than 5% weight loss and 100% survival were observed in all groups vaccinated with the bivalent vaccine formulated with R-DOTAP (FIGURES 7 A and 7B). Both low dose (0.12ug/HA) and high dose (3ug/HA) COBRA antigen formulated with R-

DOTAP provided complete protection indicating a significant dose sparing effect (FIGURE 7 A).

[0160] The viral clearance in the lungs following challenge was also evaluated. Vaccine formulations containing R-DOTAP completely cleared influenza virus in the lungs within 3 days (FIGURE 7C) and no virus was detected by day 6 (FIGURE 7D). In contrast, mice receiving antigen only and unvaccinated mice showed significant viral load both at day 3 and day 6. Together, these studies demonstrated that recombinant protein vaccines containing R-DOTAP as immune modulator induced immune responses capable of protecting mice from viral infections.

EXAMPLE 7

DISCUSSION

[0161] Cationic lipids are excellent delivery vehicles for transporting nucleic acids and protein/peptides into cells and have been widely used in human drug delivery. However, most cationic lipids are inert and do not activate immunological signals necessary for effective immune response to vaccine antigens. Here, it was demonstrated that R-DOTAP promotes robust antibody and T cell responses to a variety of viral proteins capable of providing neutralizing activity and protection from viral challenge.

[0162] Both TEM pictures (FIGURES 2A-2C) and physical characterization data (Table 1 & FIGURES 2A-2C) showed that R-DOTAP formed uniform smooth surface spherical structures ranging around 150 nm size in sucrose buffer. Mixing antigens with the nanoparticles did not significantly alter the size and polydispersity of the formulation (table 1). Changes in the surface and zeta potential of the nanoparticles following antigen addition were observed.

[0163] Table 1: Particle size, poly dispersity index (PDI) and zeta potential of R-DOTAP formulations.

[0164] In this study, the ability of R-DOTAP to enhance both humoral and cellular immune responses to large protein antigens using prototype vaccine formulations containing antigens derived from the respiratory virus Influenza was evaluated. By formulating R-DOTAP with the COBRA influenza antigens the potential for the R-DOTAP-COBRA vaccine to provide an effective universal flu vaccine by inducing a broadly protective immune response that may neutralize multiple strains of the flu virus was evaluated (FIGURES 5A-5B). It was demonstrated that co-formulating Influenza derived viral protein antigens with R-DOTAP significantly enhanced vaccine immunogenicity, generated robust antigen-specific cellular and antibody- mediated immune responses in mice, mediated significant antigen dose sparing, and protected vaccinated mice from influenza virus challenge.

[0165] The present results demonstrated that single component R-DOTAP nanoparticles can perform multiple tasks essential to generating broad and durable protective immune responses.

[0166] There is ample evidence that suggests CDS T cells play a crucial role in long-term protection against highly mutating viruses such as Influenza. While most approved adjuvants for recombinant proteins effectively induce humoral and Thl type immune responses, very few if any can induce robust and clinically effective cytotoxic CDS T cell immune responses in humans. The current study demonstrated the ability of R-DOTAP to generate CDS T cells to internal epitopes of large recombinant protein antigens. These T cells were poly functional with an effector phenotype. They were capable of producing multiple cytotoxic cytokines and persisted in vaccinated mice 28 days after a second vaccine, indicating the establishment of T cell memory responses.

[0167] It was observed that R-DOTAP containing vaccines induced both IFN-y and IL-4 producing T cells (data not shown) and generated both IgGl and IgG2a antibody subtypes. No strong skewing towards either the Thl or Th2 subtype was observed. Thus, from an antibody perspective, these results indicated that R-DOTAP induced a balanced Thl/Th2 type immunity optimal for effective vaccine induced antibody responses. However, among antigen specific Thl and CDS T -cells, R-DOTAP promoted the development of high levels of polyfunctional cytokine secreting cells shown to be optimal in promoting viral clearance.

[0168] In summary, these studies demonstrated that protein subunit vaccines against Influenza that are based on the R-DOTAP platform induced broadly protective cellular and humoral immune responses and provide a significant dose sparing effect on the antigen. The heterogeneity of recombinant proteins capable of being formulated and administered with R-DOTAP coupled with the ability to produce multivalent vaccines suggest that R-DOTAP is an excellent candidate for use in a variety of prophylactic vaccines against infectious disease. Furthermore, it has been demonstrated that R-DOTAP is highly effective in enhancing the potency of the currently licensed seasonal influenza vaccine, further expanding the potential utility of R-DOTAP. The safety and efficacy profiles of R-DOTAP have been successfully established in human clinical trials paving the way for future trials of R-DOTAP based universal influenza vaccines.

EXAMPLE 8

USE OF A R-DOTAP ADJUVANT FORMULATION FOR BIVALENT COBRA H1/H3

VACCINES IN A PRE-IMMUNE FERRET MODEL

[0169] In a pre-immune ferret model, as described in FIGURE 8, ferrets were primed with the virus, vaccinated, and boosted, before an influenza challenge was performed. The model mimic human response to vaccination by first infecting the ferrets with influenza viruses (H1N1 — A/Singapore/6/1986- and H3N2 - AvPanama/2007/1999).

[0170] As illustrated in FIGURES 9A-9C, the H1N 1 HAI response was evaluated. H1/H3 pre- immune ferrets were either vaccinated twice with Y2/NG2 rHA (15ug), with R-DOTAP alone, or with wild type rHA and R-DOTAP, and HAI titers were measured. As illustrated in FIGURES 10A-10C, the H3N2 HAI response was also evaluated. H1/H3 pre-immune ferrets were either vaccinated twice with Y2/NG2 rHA (15ug), with R-DOTAP alone, or with wild type rHA and R- DOTAP, and HAI titers were measured.

[0171] As illustrated in FIGURES 11A-11C, body weight was better maintained, and nasal wash viral titers were lower in the animals that were vaccinated with Y2/NG2 rHA.

[0172] This study demonstrated that COBRA rHA vaccines adjuvanted with Infectimune® (R- DOTAP) were capable of eliciting protective HAI antibody responses in pre-immune ferrets across panels of viruses from the last decade. It was also shown that they elicited HAI reactive antibodies against future drifted viral isolates from 2019-2020. The vaccination prevented weight loss and H1N1 viral replication in the lungs of vaccinated animals. In a population that has a more extensive prc-immunc background to influenza, like humans, it is expected that these vaccines generate a more broadly reactive antibody profile due to the recall of a more diverse population of memory B cells. [0173] List of sequences:

[0174] Although the invention has been described with reference to the above examples, it will be understood that modifications and variations arc encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.