Recombinant protein compositions formulated with enantiospecific cationic lipids and uses thereof

Abstract

Provided herein are vaccine compositions comprising a recombinant protein antigen derived from computationally optimized broadly reactive influenza antigen (COBRA) proteins and an immunomodulatory agent, and methods of using the same. The vaccine compositions include one or more COBRA proteins, and the immunomodulatory agent is a cationic lipid. The cationic lipid includes R-DOTAP. Methods of using the vaccine compositions include methods for inducing a humoral immune response against influenza virus, inducing polyfunctional CD8+ and CD4+ effector T cells against influenza virus, inducing memory T cells against influenza virus, enhancing immunity against influenza virus, and inducing a balanced Th1 / Th2 immune response against influenza virus in a subject.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 418,381, filed October 21, 2022, and U.S. Provisional Patent Application No. 63 / 539,066, filed September 18, 2023. The disclosures of the prior applications are considered part of the disclosure of this application and are incorporated herein by reference in their entirety in the disclosure of this application.

[0002] Incorporating a sequence listing The material in the attached Sequence Listing is hereby incorporated by reference into this application. The attached Sequence Listing xml file, named ST26.xml, was created on and is KB. [Background technology]

[0003] Background information Immunization is one of the most effective public health measures for combating viral infections. The increasing safety requirements for FDA approval, combined with the complexities associated with certain viral infections, have stimulated the search for a new generation of peptide / recombinant protein-based prophylactic vaccines. However, most recombinant proteins / peptides are non-immunogenic and induce only weak immune responses upon administration. Therefore, recombinant protein / peptide vaccines often require adjuvants to stimulate or enhance the immune response against the antigen.

[0004] A wide variety of adjuvants with varying immunomodulatory properties have been approved or are under investigation for recombinant protein-based vaccines. While many adjuvants, such as alum, squalene, or monophosphoryl lipid A, promote strong antibody responses, others, such as CpG and other TLR7, 8, or 9 agonists, promote stronger Th1 CD4 and CD8 T cell responses. However, even these distinctions are somewhat blurred, as a single adjuvant can promote different responses through distinct pathways. For example, squalene-based adjuvants induce CD8 T cell responses through a pathway distinct from their antibody-inducing properties. Thus, it is now understood that various adjuvants induce multiple distinct immunological signatures, making them more or less effective against different diseases, such as tuberculosis and influenza. Vaccines against respiratory viruses, such as influenza, primarily induce antibody responses, and in influenza, neutralizing antibody titers are primarily associated with immunity. However, natural infection also induces strong CD4 T cell and CD8 T cell responses, which are important for the development of long-lasting immunity. In situations where antigenic drift and shift prevent antibody recognition, CD8 T cell responses against internal proteins are also important. Therefore, it is now widely recognized that next-generation universal vaccines against respiratory viral pathogens should induce potent neutralizing antibodies and strong CD8 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 intracellular delivery of nucleic acids, including in clinical trials for human gene therapy. The novel mRNA vaccine technology used to combat the COVID-19 pandemic also uses lipid nanoparticles as a delivery vehicle to deliver mRNA into cells. In recent years, cationic lipids have become attractive targets for delivering proteins and peptides for use in immunotherapy and vaccines. Importantly, cationic lipid-mediated antigen uptake directs proteins and peptides into the MHC class I and class II processing pathways. Mechanistically, cationic lipid nanoparticles efficiently bind to the negatively charged cell membrane in a receptor-independent manner and are rapidly internalized into endosomes in amounts exceeding receptor-mediated uptake. Once inside the endosome, the cationic lipid fuses with the endosomal membrane, delivering a portion of its contents into the cytoplasm. Thus, certain cationic lipids are ideally suited as nonviral vectors for intracellular delivery of peptides, proteins, and inactivated whole viruses into the MHC class I and class II pathways. More recent studies of cationic lipids as delivery agents have also identified that certain cationic lipids have immunostimulatory properties and can activate pathways essential for an effective immune response after vaccination. However, these characteristics are not universally exhibited by all cationic lipids, as their immunostimulatory properties and mechanisms of action vary widely among cationic lipids.

[0006] The enantiospecific cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (R-DOTAP) has been shown to be particularly robust in inducing CD8 T cell responses to peptide-based vaccines. Further studies have revealed that R-DOTAP promotes cellular uptake and cross-presentation of CD8 epitopes from long peptides, promoting the formation of polyfunctional CD8 T cells. R-DOTAP alone has been shown to induce type I interferon in draining lymph nodes (LNs), and type I interferon was required for R-DOTAP-mediated induction of antigen-specific CD8 T cells. While R-DOTAP's ability to promote robust CD8 T cell responses to peptide-based vaccines has been well established, its ability to promote CD8 T cell responses to larger recombinant proteins is less clear. R-DOTAP has also been shown to induce robust antibody responses to the model antigen OVA, but no studies have examined its ability to promote antibody responses to vaccine-relevant recombinant proteins. Summary of the Invention

[0007] The present invention is based on the breakthrough discovery that the use of cationic lipids as immunomodulatory agents enhances the immunity induced by influenza recombinant proteins in vaccine compositions.

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

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

[0010] In another embodiment, the present invention provides a method of inducing an immune response to influenza virus in a subject, the method comprising administering to the subject a vaccine composition comprising: a) one or more non-naturally occurring recombinant influenza antigens; and b) a cationic lipid, thereby inducing an immune response to influenza virus in the subject.

[0011] In one embodiment, the immune response is CD8 + Effector T cells, CD4 + In another embodiment, the induction of CD8 + Effector T cells and CD4 + Induction of effector T cells is achieved by inducing CD8 T cells that produce IFNγ and granzyme B in a subject. + Proliferation of effector T cells and / or IL-4-producing CD4 + In another embodiment, inducing a humoral immune response comprises inducing the production of IgG in the subject. In some embodiments, IgG comprises IgG1 and IgG2a. In one embodiment, inducing an immune response comprises inducing the secretion of broadly neutralizing antibodies.

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

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

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

[0015] In another embodiment, the present invention provides a method for inducing the secretion of broadly neutralizing antibodies against influenza virus in a subject, the method comprising administering to the subject a vaccine composition comprising: a) one or more non-naturally occurring recombinant influenza antigens; and b) a cationic lipid, thereby inducing the secretion of broadly neutralizing antibodies against influenza virus in the subject.

[0016] In another embodiment, the present invention provides a method for inducing a balanced Th1 / Th2 immune response in a subject, the method comprising inducing secretion of broadly neutralizing antibodies against influenza virus in the subject by administering to the subject a vaccine composition comprising: a) one or more non-natural recombinant influenza antigens; and b) a cationic lipid.

[0017] In one embodiment, inducing a Th1 immune response comprises inducing proliferation of CD8+ effector T cells that produce IFNγ and granzyme B, and / or proliferation of CD4+ effector T cells that produce IL-4 in the subject. In some embodiments, IFNγ-producing CD8+ effector T cells are associated with IgG2a production, and IL-4-producing CD4+ effector T cells are associated with IgG1 production.

[0018] In a further embodiment, the present invention provides a method of inducing a polyfunctional CD4+ / CD8+ T cell response against influenza virus in a subject, the method comprising administering to the subject an influenza vaccine composition comprising: 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 virus in the subject.

[0019] In one embodiment, inducing a polyfunctional CD4+ / CD8+ T cell response comprises inducing the secretion of two or more cytokines, in some embodiments, the two or more cytokines are selected from the group consisting of IFNγ, granzyme B, and IL-4. [Brief explanation of the drawings]

[0020] [Figure 1A] Figures 1A-1B show the antibody-mediated immune responses induced by recombinant influenza proteins formulated with R-DOTAP. Figure 1A is a graph showing the total IgG antibody titers obtained with each R-DOTAP preparation. [Figure 1B] Figures 1A-1B show the antibody-mediated immune responses induced by recombinant influenza proteins formulated with R-DOTAP. Figure 1B is a graph showing the total IgG antibody titers obtained with each Y2-COBRA preparation and sucrose.

[0021] [Figure 2A] Figures 2A to 2C show the average particle size of liposomal nanoparticles of R-DOTAP and R-DOTAP mixed with COBRA Y2 or COBRA-NG2 protein antigens. Figure 2A is a graph showing the particle size distribution. [Figure 2B]Figures 2A to 2C show the average particle sizes of R-DOTAP liposomal nanoparticles and R-DOTAP mixed with COBRA Y2 or COBRA-NG2 protein antigens. Figure 2B is a transmission electron microscope (TEM) image showing R-DOTAP nanoparticles. [Figure 2C] Figures 2A-2C show the average particle size of R-DOTAP liposomal nanoparticles and R-DOTAP mixed with COBRA Y2 or COBRA-NG2 protein antigens. Figure 2C is a TEM image showing R-DOTAP mixed with a mixture of COBRA-Y2 and COBRA-NG2 and resuspended in water.

[0022] [Figure 3A] Figures 3A-3D show T cell responses to R-DOTAP formulations containing nucleoprotein and COBRA HA antigen. Figure 3A is a graph showing the number of antigen-specific IFN-γ-producing T cells in the spleen in response to a vaccine containing influenza nucleoprotein. [Figure 3B] Figures 3A-3D show T cell responses to R-DOTAP formulations containing nucleoprotein and COBRA HA antigen, and Figure 3B is a graph showing the number of antigen-specific IFN-γ-producing T cells in the spleen in response to monovalent and bivalent influenza vaccines containing recombinant COBRA sequences. [Figure 3C] Figures 3A-3D show T cell responses to R-DOTAP formulations containing nucleoprotein and COBRA HA antigen, and Figure 3C is a graph showing specific T cell responses to H1N1. [Figure 3D] Figures 3A-3D show T cell responses to R-DOTAP formulations containing nucleoprotein and COBRA HA antigen, and Figure 3D is a graph showing specific T cell responses to H3N2.

[0023] [Figure 4A]Figures 4A-4E show antibody-mediated immune responses to COBRA proteins: Figure 4A is a graph showing antigen-specific antibody titers after one or two vaccinations. [Figure 4B] Figures 4A-4E show antibody-mediated immune responses to COBRA proteins, and Figure 4B is a graph comparing anti-Y2 antigen-specific antibody titers 35 and 62 days after vaccination with sucrose, R-DOTAP, or Addavax™ as adjuvants. [Figure 4C] Figures 4A-4E show antibody-mediated immune responses to COBRA proteins, and Figure 4C is a graph comparing anti-NG2 antigen-specific antibody titers 35 and 62 days after vaccination with sucrose, R-DOTAP, or Addavax™ as adjuvants. [Figure 4D] Figures 4A-4E show antibody-mediated immune responses to COBRA proteins, and Figure 4D is a graph comparing anti-Y2 antigen-specific IgG1 and IgG2a antibody titers 35 and 62 days after vaccination with sucrose, R-DOTAP, or Addavax™ as adjuvants. [Figure 4E] Figures 4A-4E show antibody-mediated immune responses to COBRA proteins. Figure 4E is a graph comparing anti-NG2 antigen-specific IgG1 and IgG2a antibody titers 35 and 62 days after vaccination with sucrose, R-DOTAP, or Addavax™ as adjuvants.

[0024] [Figure 5A] Figures 5A-5B show the ability of bivalent COBRA antigen formulated with R-DOTAP to induce Th1 and Th2 antibody responses. Figure 5A is a graph showing Th1-specific antibody titers. [Figure 5B] Figures 5A-5B show the ability of bivalent COBRA antigen formulated with R-DOTAP to induce Th1 and Th2 antibody responses, and Figure 5B is a graph showing Th1-specific antibody titers.

[0025] [Figure 6A] Figures 6A-6C show HAI titers 35 days after vaccination with influenza virus alone or in combination with R-DOTAP nanoparticles. Figure 6A is a graph showing HAI titers when the inactivated influenza vaccine Fluzone® (2011-12 formulation) was a split virus hemagglutinin formulation derived from A / California / 07 / 2009 X-179A (H1N1). [Figure 6B] Figures 6A-6C show HAI titers 35 days after vaccination with influenza virus alone or in combination with R-DOTAP nanoparticles. Figure 6B is a graph showing HAI titers when the inactivated influenza vaccine Fluzone® (2011-12 formulation) was a split virus hemagglutinin formulation derived from A / Victoria / 210 / 2009 X-187 (A / Perth / 16 / 2009-like virus) (H3N2). [Figure 6C] Figures 6A-6C show HAI titers 35 days after vaccination with influenza virus alone or in combination with R-DOTAP nanoparticles. Figure 6C is a graph showing HAI titers when the inactivated influenza vaccine Fluzone® (2011-12 formulation) was a split virus hemagglutinin formulation derived from B / Brisbane / 60 / 2008.

[0026] [Figure 7A] Figures 7A-7D show the effect of the presence of R-DOTAP in the vaccine formulation after vaccination and virus challenge: Figure 7A is a graph showing weight change over time after vaccination and infection. [Figure 7B] Figures 7A-7D show the effect of the presence of R-DOTAP in the vaccine formulation after vaccination and virus challenge. Figure 7B is a Kaplan-Meier graph showing survival after infection. [Figure 7C]Figures 7A-7D show the effect of the presence of R-DOTAP in the vaccine formulation after vaccination and virus challenge, and Figure 7C is a graph showing virus titers 3 days after infection. [Figure 7D] Figures 7A-7D show the effect of the presence of R-DOTAP in the vaccine formulation after vaccination and virus challenge. Figure 7D is a graph showing virus titers 6 days after infection.

[0027] [Figure 8] FIG. 8 is a schematic diagram of the pre-immune ferret model.

[0028] [Figure 9A] Figures 9A-9C show the results of H1N1 HAI responses: Figure 9A is a graph showing HAI titers in animals vaccinated with Y2+NG2 HA proteins. [Figure 9B] Figures 9A-9C show the results of H1N1 HAI responses. Figure 9B is a graph showing HAI titers in animals vaccinated with Michigan / 15+Singapore / 16 HA proteins. [Figure 9C] Figures 9A-9C show the results of the H1N1 HAI response, and Figure 9C is a schematic diagram showing when blood was drawn in the experimental timeline to measure the data in Figures 9A and 9B.

[0029] [Figure 10A] Figures 10A-10C show the results of H3N2 HAI responses: Figure 10A is a graph showing HAI titers in animals vaccinated with Y2+NG2 HA proteins. [Figure 10B] Figures 10A-10C show the results of H3N2 HAI responses. Figure 10B is a graph showing HAI titers in animals vaccinated with Michigan / 15+Singapore / 16 HA proteins. [Figure 10C]Figures 10A-10C show the results of the H3N2 HAI response, and Figure 10C is a schematic diagram showing when blood was taken in the experimental timeline to measure the data in Figures 10A and 10B.

[0030] [Figure 11A] Figures 11A-11C show further characterization of the response in animals vaccinated with the Y2+NG2 HA protein: Figure 11A is a graph showing the body weight of the animals during the study. [Figure 11B] Figures 11A-C show further characterization of responses in animals vaccinated with Y2+NG2 HA proteins. Figure 11B is a graph showing virus titers in D3 nasal washes. [Figure 11C] Figures 11A-11C show further characterization of responses in animals vaccinated with Y2+NG2 HA protein, and Figure 11C is a schematic diagram showing when in the experimental timeline nasal washes were taken to measure the data in Figure 11B. DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention is based on the breakthrough discovery that the use of cationic lipids as immunomodulatory agents enhances the 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 the particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. Also, since the scope of the present invention is defined only by the appended claims, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0033] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. Thus, for example, reference to a "method" includes one or more methods, and / or steps of the type described herein that will become apparent to those skilled in the art upon reading this disclosure.

[0034] As used herein, the term "about" in connection with a numerical value is intended to include any additional numerical value reasonably close to the stated numerical value. For example, depending on the context, the value may vary above or below 5-10%. For example, a value of about 100 means 90-110 (or any value between 90-110).

[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 otherwise defined, 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. Any methods and materials similar or equivalent to those described herein can also be used in practicing or testing the present invention, but it is understood that modifications and variations are encompassed within the spirit and scope of this disclosure. Preferred methods and materials are described below.

[0037] Vaccine Composition

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

[0039] As used herein, the term composition is intended to encompass pharmaceutical compositions that may also contain other therapeutic agents and may be formulated, for example, according to techniques known in the pharmaceutical formulation art, by using conventional pharmaceutically acceptable solvents or diluents and types of pharmaceutical additives (e.g., excipients, preservatives, etc.) appropriate for the desired mode of administration. In certain embodiments, the compositions disclosed herein are formulated with additional agents that facilitate entry into desired cells or tissues. Such additional agents include micelles, liposomes, and dendrimers.

[0040] The term "pharmaceutically acceptable" means 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, could be administered to a subject, together with a conjugate of the present invention, without causing any undesired biological effects or interacting in an undesired manner with any of the other ingredients of the pharmaceutical composition contained therein.

[0041] Pharmaceutical compositions containing the peptides or compositions described herein may be administered by any suitable means, for example, parenterally, e.g., by subcutaneous, intravenous, intramuscular, intrathecal, or intracisternal injection or infusion (e.g., as a sterile injectable aqueous or non-aqueous solution or suspension) in a dosage formulation containing a non-toxic pharmaceutically acceptable solvent or diluent. 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. A suitable route may be, for example, parenteral delivery, including intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, or intraperitoneal delivery. For injection, the pharmaceutical compositions of the present invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as water, Hank's solution, Ringer's solution, or physiologically buffered saline.

[0042] The composition of the present invention is a "vaccine composition," and as used herein, the term "vaccine" relates to a pharmaceutical preparation (composition) or drug product that, after administration, induces an immune response, particularly a cellular immune response, that recognizes and attacks pathogens or abnormal cells, such as cancer cells. Vaccines can be used to prevent or treat disease.

[0043] The term "universal influenza vaccine" particularly relates to influenza vaccines that are formulated to provide immune protection against at least two influenza virus variants. In one embodiment, the vaccine composition described herein is a universal influenza vaccine.

[0044] In one aspect, the vaccine compositions described herein provide immune protection against at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten influenza virus variants. The vaccine compositions described herein provide immune protection against at least three influenza virus variants. The vaccine compositions described herein provide immune protection against at least four influenza virus variants. The vaccine compositions described herein provide immune protection against at least five influenza virus variants. The vaccine compositions described herein provide immune protection against at least six influenza virus variants. The vaccine compositions described herein provide immune protection against at least seven influenza virus variants. The vaccine compositions described herein provide immune protection against at least eight influenza virus variants. The vaccine compositions described herein provide immune protection against at least nine influenza virus variants. The vaccine compositions described herein provide immune protection against at least ten influenza virus variants.

[0045] In one embodiment, examples of influenza variants include, but are not limited to, H1N1, H3N2, H5N1, and H7N9. In one embodiment, the vaccine compositions described herein provide immune protection against at least H1N1 and / or H3N2 hemagglutinin influenza variants.

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

[0047] In one embodiment, 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 bond. As used herein, a polypeptide may refer to a complete amino acid sequence encoding an entire protein, or a portion thereof. A "protein coding sequence," or a sequence "encoding" a particular polypeptide or peptide, is a nucleic acid sequence that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide, either 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 typically be located 3' to the coding sequence.

[0049] An "antigen" according to the present invention includes any substance that induces an immune response. In particular, "antigen" relates to any substance, preferably a peptide or protein, that specifically reacts with antibodies or T lymphocytes (T cells). In the present invention, the term "antigen" includes any molecule that contains at least one epitope. Preferably, an antigen in the context of the present invention is a molecule that, optionally after processing, induces an immune response. Any suitable antigen that is a candidate for an immune response, preferably a cellular immune response, may be used in the present invention. In the context of embodiments of the present invention, the antigen is preferably presented by cells, preferably by antigen-presenting cells, including abnormal cells, in the context of MHC molecules, in order to elicit an immune response against the antigen. Preferably, the antigen corresponds to a naturally occurring antigen or is a product derived from a naturally occurring antigen. Such naturally occurring antigens include tumor antigens.

[0050] In another embodiment, the one or more recombinant influenza antigens comprise a computationally optimized broadly reactive influenza antigen (COBRA) hemagglutinin (HA). The COBRA HA described herein is further described in International Application No. PCT / US2022 / 032799, U.S. Patent No. 9,212,207, and the following scientific publications: Allen and Ross (“Bivalent H1 and H3 COBRA Recombinant Hemagglutinin Vaccines Elicit Seroprotective 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 their entireties.

[0051] As used herein, a "non-naturally occurring" peptide or antigen means a peptide or antigen that does not occur in nature and is composed of one or more naturally occurring or non-naturally occurring peptides or antigens that are combined into a single peptide or antigen.

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

[0053] The terms "sequence identity" and "percent identity" are used interchangeably herein. To determine percent identity between two polypeptide molecules or two polynucleotide sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into a first polypeptide or polynucleotide sequence to optimally align it 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, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical positions / total number of positions (i.e., overlapping positions) × 100). In some embodiments, the length of a reference sequence aligned for comparison purposes is at least 80% of the length of the sequence being compared, and in some embodiments, at least 90% or 100%. In certain embodiments, the two sequences are the same length.

[0054] Desirable ranges of sequence identity are approximately 80% to 100% and integer values ​​therebetween. The percent identity between the disclosed and claimed sequences 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%. Generally, an exact match indicates 100% identity over the entire length of the reference sequence.

[0055] Also encompassed by the present disclosure are polypeptides and polynucleotides that have about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5% or more identity to the polypeptides and polynucleotides described herein. For example, a polypeptide can have 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the sequence of a recombinant COBRA protein described herein.

[0056] Variants of the disclosed sequences also include peptides or full-length proteins that contain substitutions, deletions, or insertions in the protein backbone but still retain at least about 70% homology to the original protein over the corresponding portions. Greater degrees of deviation from homology are permitted, provided that similar amino acids, i.e., conservative amino acid substitutions, are not counted as changes in the sequence. Examples of conservative substitutions involve amino acids with identical or similar properties. Exemplary conservative amino acid substitutions include the following changes: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glutamine to asparagine; glutamic acid to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine, or glutamic acid; 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 embodiment, the recombinant influenza antigen has a sequence having at least 80% sequence identity to the sequence of SEQ ID NO:3. In one embodiment, the recombinant influenza antigen has a sequence having at least 85% sequence identity to the sequence of SEQ ID NO:3. In one embodiment, the recombinant influenza antigen has a sequence having at least 95% sequence identity to the sequence of SEQ ID NO:3. In one embodiment, the recombinant influenza antigen has a sequence having at least 96% sequence identity to the sequence of SEQ ID NO:3. In one embodiment, the recombinant influenza antigen has a sequence having at least 97% sequence identity to the sequence of SEQ ID NO:3. In one embodiment, the recombinant influenza antigen has a sequence having at least 98% sequence identity to the sequence of SEQ ID NO:3. In one embodiment, the recombinant influenza antigen has a sequence having at least 99% sequence identity to the sequence of SEQ ID NO:3.

[0058] In one embodiment, the recombinant influenza antigen has a sequence having at least 80% sequence identity to the sequence of SEQ ID NO:4. In one embodiment, the recombinant influenza antigen has a sequence having at least 85% sequence identity to the sequence of SEQ ID NO:4. In one embodiment, the recombinant influenza antigen has a sequence having at least 95% sequence identity to the sequence of SEQ ID NO:4. In one embodiment, the recombinant influenza antigen has a sequence having at least 96% sequence identity to the sequence of SEQ ID NO:4. In one embodiment, the recombinant influenza antigen has a sequence having at least 97% sequence identity to the sequence of SEQ ID NO:4. In one embodiment, the recombinant influenza antigen has a sequence having at least 98% sequence identity to the sequence of SEQ ID NO:4. In one embodiment, the recombinant influenza antigen has a sequence having at least 99% sequence identity to the sequence of SEQ ID NO:4.

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

[0060] Adjuvants are essential components of subunit vaccines, added to enhance immune responses to antigens through immunomodulation. Due to safety concerns, few adjuvants have been approved for human use by regulatory authorities. Current subunit vaccine adjuvants approved for human use are highly effective at promoting humoral immune responses but less effective at promoting T cell immunity. In this study, we evaluated a novel pure enantiospecific cationic lipid, 1,2-dioleoyl-3-trimethylammonium-propane (R-DOTAP), as an immunomodulatory agent for subunit vaccines capable of inducing both humoral and cellular immunity. Using a recombinant protein antigen derived from the computationally optimized broadly reactive influenza antigen (COBRA) protein, we demonstrated that R-DOTAP nanoparticles promoted potent cellular and antibody-mediated immune responses in both monovalent and bivalent vaccines. As discussed further below, R-DOTAP-based vaccines were developed to induce antigen-specific and multifunctional CD8 and CD8+ T cell responses, respectively. + Effector T cells and CD4 + R-DOTAP induced effector and memory T cells. The antibody responses induced by R-DOTAP demonstrated balanced Th1 / Th2 immunity, neutralizing activity, and protection of mice from challenge with live influenza virus. R-DOTAP also significantly promoted dose sparing of vaccine antigens. These studies demonstrate that R-DOTAP is an excellent immunostimulant for producing next-generation subunit vaccines containing multiple recombinant proteins.

[0061] Adjuvants are often used to modulate or enhance the efficacy of vaccines by stimulating the immune system to respond more vigorously to the vaccine, thereby providing enhanced immunity against specific diseases. Adjuvants accomplish this task by mimicking a specific set of evolutionarily conserved molecules, so-called pathogen-associated molecular patterns (PAMs). This set includes liposomes, lipopolysaccharides, molecular cages for antigens, components of bacterial cell walls, and endocytosed nucleic acids such as RNA, double-stranded RNA, single-stranded DNA, and unmethylated CpG dinucleotide-containing DNA. Because the immune system has evolved to recognize these specific antigenic moieties, the presence of adjuvants with vaccines can significantly enhance the innate immune response to antigens by mimicking natural infection and enhancing the activity of dendritic cells, lymphocytes, and macrophages.

[0062] The compositions described herein can be formulated with lipid nanoparticles as adjuvants to enhance the presentation of the antigens to antigen-presenting cells and increase the immune response induced by the antigen.

[0063] In some embodiments described herein, the adjuvant is a cationic lipid. As used herein, the term "cationic lipid" refers to any of a number of lipid species that have a net positive charge at physiological pH or that have a protonatable group and have a positive charge at a pH lower than the pKa.

[0064] Suitable cationic lipids according to the present disclosure include, but are not limited to: 3-β [4N1N,8-diguanidinospermidine)-carbamoyl] cholesterol (BGSC); 3-β [N,N-Diguanidinoethyl-aminoethane)-carbamoyl]cholesterol (BGTC); N,N,1N2N3 tetra-methyltetrapalmitylspermine (Cellfectin); Nt-butyl-N'-tetradecyl-3-tetradecyl-aminopropionamidine (CLONfectin); dimethyldioctadecylammonium bromide (DDAB); 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE); 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate trifluorocetate) (DOSPA); 1,3-dioleoyloxy-2-(6-carboxyspermyl)-propylamide (DOSPER); 4-(2,3-bis-palmitoyloxy-propyl)-1-methyl-1H-imidazole (DPIM); N,N,N',N'-tetramethyl-N,N'-bis(2-hydroxyethyl)-2,3-dioleoyloxy-1,4-butane-diammonium iodide) (Tfx-50); N-1-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTMA), or other N-(N,N-1-dialkoxy)-alkyl-N,N,N-trisubstituted ammonium surfactants; 1,2 dioleoyl-3-(4'-trimethylammonio)butanol- sn -glycerol (DOBT) or cholesteryl (4'-trimethylammonium) butanoate (ChOTB) (the trimethylammonium group is connected to the duplex (in the case of DOTB) or cholesteryl group (in the case of ChOTB) via a butanol spacer arm);DORI (DL-1,2-dioleoyl-3-dimethylaminopropyl-β-hydroxyethylammonium) or DORIE (DL-1,2-O-dioleoyl-3-dimethylaminopropyl-β-hydroxyethylammonium) (DORIE) or analogs thereof as disclosed in WO 93 / 03709; 1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC); cholesteryl hemisuccinate (ChOSC); lipopolyamines, such as dioctadecylamidoglycylspermine (DOGS) and dipalmitoylphosphatidylethanolamylspermine (DPPES), cholesteryl-3 β-Carboxyl-amido-ethylenetrimethylammonium iodide, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesterylcarboxylate iodide, cholesteryl-3-O-carboxyamidoethyleneamine, cholesteryl-3-β-oxysuccinamido-ethylenetrimethylammonium iodide, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl-3-β-oxysuccinate iodide, 2-(2-trimethylammonio)-ethylmethylaminoethyl-cholesteryl-3-β-oxysuccinate iodide, 3-β-N-(N',N'-dimethylaminoethane)carbamoylcholesterol (DC-chol), and 3-β-N-(polyethyleneimine)-carbamoylcholesterol. O,O'-Dimyristyl-N-lysyl aspartate (DMKE); O,O'-Dimyristyl-N-lysyl glutamate (DMKD); 1,2-Dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE); 1,2-Dilauroyl-sn-glycero-3-ethylphosphocholine (DLEPC); 1,2-Dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC); 1,2-Dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC); 1,2-Dipalmitoyl-sn-glycero-3-ethylphosphocholine (DPEPC); 1,2-Distearoyl-sn-glycero-3-ethylphosphocholine (DSEPC); 1,2-Dioleoyl-3-trimethylammonium propane (DOTAP);Dioleoyldimethylaminopropane (DODAP); 1,2-palmitoyl-3-trimethylammoniumpropane (DPTAP); 1,2-distearoyl-3-trimethylammoniumpropane (DSTAP), 1,2-myristoyl-3-trimethylammoniumpropane (DMTAP); and sodium dodecyl sulfate (SDS). Additionally, structural variants and derivatives of any of the above cationic lipids are contemplated.

[0065] In some embodiments, the cationic lipid is selected from the group consisting of DOTAP, DOTMA, DOEPC, and combinations thereof. In other embodiments, the cationic lipid is DOTAP. In yet other embodiments, the cationic lipid is DOTMA. In other embodiments, the cationic lipid is DOEPC. In some embodiments, the cationic lipid is purified.

[0066] In some embodiments, the cationic lipid is an enantiomer of the cationic lipid. The term "enantiomer" refers to a stereoisomer of the cationic lipid that is a non-superimposable mirror image of its counterpart, such as the 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 embodiments, the enantiomer is purified.

[0067] In one embodiment, the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, a variant thereof, or an analog thereof.

[0068] In another embodiment, the one or more recombinant influenza antigens are encapsulated within liposomes comprising cationic lipids, hi another embodiment, the one or more recombinant influenza antigens are mixed with preformed cationic lipid nanoparticles.

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

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

[0071] Usage

[0072] In another embodiment, the present invention provides a method of inducing an immune response to influenza virus in a subject, the method comprising administering to the subject a vaccine composition comprising: a) one or more non-naturally occurring recombinant influenza antigens; and b) a cationic lipid, thereby inducing an immune response to influenza virus in the subject.

[0073] The term "immune response" refers to the body's integrated reaction to an antigen, preferably a cellular immune response, or a cellular and humoral immune response. The immune response can 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 defend the body against foreign agents, such as pathogens, destroy infected and malignant cells, and remove necrotic debris. It includes the thymus, spleen, lymph nodes and lymphoid tissues, stem cells, white blood cells, antibodies, and lymphokines. B cells, or B lymphocytes, are a type of lymphocyte in the humoral immune system of the adaptive immune system and are important in 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: killer T cells and helper T cells. In addition, there are suppressor T cells, which are involved in regulating the immune response. Killer T cells recognize only antigens bound to MHC class I molecules, while helper T cells recognize only antigens bound to MHC class II molecules. These two antigen-presenting mechanisms reflect the different roles of these two types of T cells. A third, minor subtype, gamma delta T cells, recognize intact antigens not bound to MHC receptors. In contrast, B cell antigen-specific receptors are antibody molecules on the surface of B cells that recognize whole pathogens and do not require antigen processing. Because each lineage of B cells expresses different antibodies, the complete set of B cell antigen receptors represents all the antibodies the body can produce.

[0075] The terms "cellular immune response," "cellular response," "cellular response to antigen," or similar terms are intended to encompass cellular responses directed against cells characterized by antigen presentation by class I or class II MHC. Cellular responses involve cells called T cells or T lymphocytes, which act as "helpers" or "killers." Helper T cells (also known as CD4+ T cells) play a central role by regulating the immune response, while killer cells (also known as cytotoxic T cells, cytolytic T cells, CD8+ T cells, or CTLs) kill abnormal cells, such as cancer cells, and prevent the production of more abnormal cells. In a preferred embodiment, the invention involves stimulating an antitumor CTL response against tumor cells expressing one or more tumor-expressed antigens, preferably presenting such tumor-expressed antigens by class I MHC.

[0076] In the context of the present invention, the terms "immune response cell," "immune cell," or "immune effector cell" refer to cells that perform effector functions during an immune response. "Immune response cells" are preferably cells characterized by binding to an antigen or antigen-presenting antigen, or antigenic peptides derived from an antigen, and are capable of mediating an immune response. For example, such cells secrete cytokines and / or chemokines, secrete antibodies, recognize cancerous cells, and, if necessary, eliminate such cancerous cells. For example, immune response cells include 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 to a specific antigen before induction, or that a certain level of immune response to a specific antigen existed before induction and that the immune response was strengthened after induction. That is, "inducing an immune response" also encompasses "strengthening an immune response." After inducing an immune response in a subject, it is preferable that the subject is protected from developing a disease such as influenza, or that the induction of an immune response improves the condition of the subject. For example, an immune response to an influenza antigen can be induced in a subject at risk of infection with an influenza virus. In this case, the induction of an immune response may be intended to improve the condition of the subject or prevent the subject from developing influenza.

[0078] In one embodiment, the immune response is CD8 + Effector T cells, CD4 + This includes the induction of effector T cells and memory T cells.

[0079] In one embodiment, inducing CD8+ effector T cells and CD4+ effector T cells comprises inducing proliferation of CD8+ effector T cells that produce IFNγ and granzyme B, and / or proliferation of CD4+ effector T cells that produce IL-4 in the subject.

[0080] In another embodiment, inducing a humoral immune response comprises inducing the production of IgG in the subject. In some embodiments, IgG comprises IgG1 and IgG2a.

[0081] In one embodiment, inducing an immune response comprises inducing the secretion of broadly neutralizing antibodies.

[0082] In one embodiment, the one or more non-naturally occurring recombinant influenza antigens comprise a computationally optimized broadly reactive influenza antigen (COBRA) hemagglutinin (HA). In another embodiment, the one or more non-naturally occurring recombinant influenza antigens are recombinant H1N1 and / or H3N2 hemagglutinin influenza proteins. In one embodiment, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to any of SEQ ID NOs: 3-22, and combinations thereof. In one embodiment, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4. In another embodiment, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence of any of SEQ ID NOs: 3-22, and combinations thereof. In another embodiment, the one or more non-naturally occurring recombinant influenza antigens comprise the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. In another embodiment, the one or more non-naturally occurring recombinant influenza antigens comprise amino acid sequences having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 3 and SEQ ID NO: 4. In one embodiment, the one or more non-naturally occurring recombinant influenza antigens comprise the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 4. In one embodiment, the one or more non-naturally occurring recombinant influenza antigens comprise the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 4.

[0083] In another embodiment, 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 or analogs thereof. In one embodiment, the cationic lipid is R-DOTAP. In another embodiment, one or more recombinant influenza antigens are encapsulated within liposomes comprising the cationic lipid. In another embodiment, one or more recombinant influenza antigens are mixed with preformed cationic lipid nanoparticles. In some embodiments, one or more recombinant influenza antigens and preformed cationic lipid nanoparticles are mixed in a 1:1 ratio. In one embodiment, one or more recombinant influenza antigens are present as micelles separate from the cationic lipid nanoparticles.

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

[0085] In one embodiment, the one or more non-naturally occurring recombinant influenza antigens comprise a computationally optimized broadly reactive influenza antigen (COBRA) hemagglutinin (HA). In another embodiment, the one or more non-naturally occurring recombinant influenza antigens are recombinant H1N1 and / or H3N2 hemagglutinin influenza proteins. In one embodiment, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to any of SEQ ID NOs: 3-22, and combinations thereof. In one embodiment, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4. In another embodiment, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence of any of SEQ ID NOs: 3-22, and combinations thereof. In another embodiment, the one or more non-naturally occurring recombinant influenza antigens comprise the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. In another embodiment, the one or more non-naturally occurring recombinant influenza antigens comprise amino acid sequences having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 3 and SEQ ID NO: 4. In one embodiment, the one or more non-naturally occurring recombinant influenza antigens comprise the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 4. In one embodiment, the one or more non-naturally occurring recombinant influenza antigens comprise the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 4.

[0086] In another embodiment, 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 or analogs thereof. In one embodiment, the cationic lipid is R-DOTAP. In another embodiment, one or more recombinant influenza antigens are encapsulated within liposomes comprising the cationic lipid. In another embodiment, one or more recombinant influenza antigens are mixed with preformed cationic lipid nanoparticles. In some embodiments, one or more recombinant influenza antigens and preformed cationic lipid nanoparticles are mixed in a 1:1 ratio. In one embodiment, one or more recombinant influenza antigens are present as micelles separate from the cationic lipid nanoparticles.

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

[0088] In some embodiments, administration can be combined with one or more additional therapeutic agents. The terms "combination therapy," "combined with," and the like refer to the simultaneous use of two or more drugs or therapies to enhance a response. For example, the cationic lipids of the present invention can be used in combination with an existing influenza vaccine to enhance the immune response generated by the existing influenza vaccine alone. The cationic lipids can be administered before, simultaneously with, or after the administration of the influenza vaccine.

[0089] By "enhancing immunogenicity" is meant that the immunogenicity of an influenza vaccine is greater when administered in combination with a cationic lipid of the present invention compared to the immunogenicity induced by the influenza vaccine administered alone (e.g., without administration of a cationic lipid as an immunomodulator).

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

[0091] Vaccines typically contain attenuated, inactivated, or killed organisms or purified products derived therefrom. There are several types of vaccines currently in use, representing different strategies used to reduce the risk of disease while maintaining the ability to induce a beneficial immune response. Influenza vaccines are usually "attenuated," "inactivated," or "subunit" (e.g., recombinant) influenza vaccines.

[0092] Live, weakened microorganisms, such as active viruses cultured under conditions that neutralize their pathogenicity, or closely related but less dangerous organisms that elicit a broad immune response, constitute attenuated vaccines. Most attenuated vaccines are viral, but some are bacterial in nature.

[0093] Inactivated vaccines consist of inactivated but formerly pathogenic microorganisms that have been destroyed by chemicals, heat, or radiation—structural but empty bacterial cell envelopes, or "ghosts." They are considered an intermediate step between inactivated and attenuated vaccines. Examples include IPV (polio vaccine), hepatitis A vaccine, rabies vaccine, and most influenza vaccines.

[0094] Subunit vaccines do not introduce inactivated or attenuated microorganisms into the immune system (which constitutes a "whole drug" vaccine), but rather use fragments of them to provoke an immune response. Only one protein of the virus (previously extracted from the serum of chronically infected patients, but now produced by recombinantly transfecting viral genes into yeast), such as the surface protein, is recombinantly produced and used in the vaccine. The hemagglutinin and neuraminidase subunits of the influenza virus are examples of subunit proteins used in recombinant influenza vaccines.

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

[0096] In one embodiment, the one or more non-naturally occurring recombinant influenza antigens comprise a computationally optimized broadly reactive influenza antigen (COBRA) hemagglutinin (HA). In another embodiment, the one or more non-naturally occurring recombinant influenza antigens are recombinant H1N1 and / or H3N2 hemagglutinin influenza proteins. In one embodiment, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to any of SEQ ID NOs: 3-22, and combinations thereof. In one embodiment, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4. In another embodiment, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence of any of SEQ ID NOs: 3-22, and combinations thereof. In another embodiment, the one or more non-naturally occurring recombinant influenza antigens comprise the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. In another embodiment, the one or more non-naturally occurring recombinant influenza antigens comprise amino acid sequences having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 3 and SEQ ID NO: 4. In one embodiment, the one or more non-naturally occurring recombinant influenza antigens comprise the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 4. In one embodiment, the one or more non-naturally occurring recombinant influenza antigens comprise the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 4.

[0097] In another embodiment, 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 or analogs thereof. In one embodiment, the cationic lipid is R-DOTAP. In another embodiment, one or more recombinant influenza antigens are encapsulated within liposomes comprising the cationic lipid. In another embodiment, one or more recombinant influenza antigens are mixed with preformed cationic lipid nanoparticles. In some embodiments, one or more recombinant influenza antigens and preformed cationic lipid nanoparticles are mixed in a 1:1 ratio. In one embodiment, 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] In another embodiment, the present invention provides a method for inducing the secretion of broadly neutralizing antibodies against influenza virus in a subject, the method comprising administering to the subject a vaccine composition comprising: a) one or more non-naturally occurring recombinant influenza antigens; and b) a cationic lipid, thereby inducing the secretion of broadly neutralizing antibodies against influenza virus in the subject.

[0100] The development of a universal influenza vaccine capable of providing heterosubtypic immunity and protection against multiple clades has been a long-term goal for preventing influenza infection. To achieve this goal, several strategies are under investigation, including using antigens from multiple subtypes to increase coverage, targeting multiple proteins as antigens (e.g., including HA and non-HA proteins), targeting conserved antigenic regions of influenza antigens, using chimeric proteins containing stem and stalk HAs from different subtypes, and using consensus-based approaches such as COBRA sequences that contain multiple known mutations in hemagglutinin and neuraminidase. Furthermore, vaccine technologies that induce both CD8 T cell and antibody-mediated immunity are also being actively explored to achieve heterosubtypic protection against influenza. As described herein, using a prototype vaccine based on the R-DOTAP platform and COBRA H1N1- and H3N2-derived HA or nucleoprotein antigens, such vaccines were able to induce antigen-specific T cell responses, neutralizing antibodies against multiple clades of H1N1 and H3N2 strains, and protected mice from challenge with a lethal H1N1 strain. Thus, vaccine formulations containing R-DOTAP and influenza-derived COBRA HA, NA sequences, and nucleoproteins show strong potential for advancing the goal of developing a safe and effective universal influenza vaccine.

[0101] The term "broadly neutralizing antibodies" or "bNAbs," as used herein, is intended to refer to neutralizing antibodies that neutralize multiple influenza virus strains. bNAbs are unique in that they target conserved epitopes of the virus, meaning that even as the virus mutates, the targeted epitopes remain present. In contrast, non-bNAbs are specific to individual virus strains bearing unique epitopes.

[0102] In one embodiment, the one or more non-naturally occurring recombinant influenza antigens comprise a computationally optimized broadly reactive influenza antigen (COBRA) hemagglutinin (HA). In another embodiment, the one or more non-naturally occurring recombinant influenza antigens are recombinant H1N1 and / or H3N2 hemagglutinin influenza proteins. In one embodiment, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to any of SEQ ID NOs: 3-22, and combinations thereof. In one embodiment, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4. In another embodiment, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence of any of SEQ ID NOs: 3-22, and combinations thereof. In another embodiment, the one or more non-naturally occurring recombinant influenza antigens comprise the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. In another embodiment, the one or more non-naturally occurring recombinant influenza antigens comprise amino acid sequences having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 3 and SEQ ID NO: 4. In one embodiment, the one or more non-naturally occurring recombinant influenza antigens comprise the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 4. In one embodiment, the one or more non-naturally occurring recombinant influenza antigens comprise the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 4.

[0103] In another embodiment, 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 or analogs thereof. In one embodiment, the cationic lipid is R-DOTAP. In another embodiment, one or more recombinant influenza antigens are encapsulated within liposomes comprising the cationic lipid. In another embodiment, one or more recombinant influenza antigens are mixed with preformed cationic lipid nanoparticles. In some embodiments, one or more recombinant influenza antigens and preformed cationic lipid nanoparticles are mixed in a 1:1 ratio. In one embodiment, one or more recombinant influenza antigens are present as micelles separate from the cationic lipid nanoparticles.

[0104] In another embodiment, the present invention provides a method for inducing a balanced Th1 / Th2 immune response in a subject, the method comprising inducing secretion of broadly neutralizing antibodies against influenza virus in the subject by administering to the subject a vaccine composition comprising: a) one or more non-natural recombinant influenza antigens; and b) a cationic lipid.

[0105] Adjuvants can be broadly classified into Th1, Th2, Th17, and mixed Th1 / Th2 or Th1 / Th17 types based on the cytokines and antibody subclasses induced by the vaccine. For example, Th1-type adjuvants induce IFN-γ production and a bias toward the IgG2a / c antibody subtype in mice vaccinated with them. Th2 adjuvants induce greater IL-4 production and a bias toward the IgG1 antibody subtype in mice.

[0106] In one aspect, inducing a Th1 immune response comprises inducing proliferation of IFNγ and granzyme B-producing CD8+ effector T cells and / or proliferation of IL-4-producing CD4+ effector T cells in the subject.

[0107] In some embodiments, CD8+ effector T cells that produce IFNγ are associated with the production of IgG2a, and CD4+ effector T cells that produce IL-4 are associated with the production of IgG1.

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

[0109] The term "subject," as used herein, refers to any individual or patient on whom the method is performed. Typically, the subject is a human, although, as will be understood by those skilled in the art, the subject may also be an animal. That is, other animals, including farm animals, including rodents (including mice, rats, hamsters, and guinea pigs), cats, dogs, rabbits, cows, horses, goats, sheep, pigs, chickens, etc., and vertebrates such as primates (including monkeys, chimpanzees, orangutans, and gorillas), are also included within the definition of a subject.

[0110] The terms "administration of" and / or "administering" should be understood to mean providing a therapeutically effective amount of a pharmaceutical composition to a subject in need of treatment. The route of administration can be enteral, topical, or parenteral. Thus, routes of administration include, but are not limited to, intradermal, subcutaneous, intravenous, intraperitoneal, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transdermal, transtracheal, subcuticular, intraarticular, subcapsular, intrathecal, intraspinal, and intrasternal administration, oral, sublingual, buccal, rectal, vaginal, intranasal, and ocular administration, as well as infusion, inhalation, and nebulization. Preferably, the vaccine compositions described herein are administered subcutaneously or intramuscularly.

[0111] In another embodiment, administering the vaccine composition comprises administering two doses of the vaccine and, optionally, one booster dose.

[0112] In one embodiment, the one or more non-naturally recombinant influenza antigens comprise a computationally optimized broadly reactive influenza antigen (COBRA) hemagglutinin (HA). In another embodiment, the one or more non-naturally recombinant influenza antigens are recombinant H1N1 and / or H3N2 hemagglutinin influenza proteins. In one embodiment, the one or more non-naturally recombinant influenza antigens comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to any of SEQ ID NOs: 3-22, and combinations thereof. In one embodiment, the one or more non-naturally recombinant influenza antigens comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4. In another embodiment, the one or more non-naturally recombinant influenza antigens comprise the amino acid sequence of any of SEQ ID NOs: 3-22, and combinations thereof. In another embodiment, the one or more non-naturally recombinant influenza antigens comprise the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. In another embodiment, the one or more non-naturally occurring recombinant influenza antigens comprise amino acid sequences having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 3 and SEQ ID NO: 4. In one embodiment, the one or more non-naturally occurring recombinant influenza antigens comprise the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 4. In one embodiment, the one or more non-naturally occurring recombinant influenza antigens comprise the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 4.

[0113] In another embodiment, 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 or analogs thereof. In one embodiment, the cationic lipid is R-DOTAP. In another embodiment, one or more recombinant influenza antigens are encapsulated within liposomes comprising the cationic lipid. In another embodiment, one or more recombinant influenza antigens are mixed with preformed cationic lipid nanoparticles. In some embodiments, one or more recombinant influenza antigens and preformed cationic lipid nanoparticles are mixed in a 1:1 ratio. In one embodiment, one or more recombinant influenza antigens are present as micelles separate from the cationic lipid nanoparticles.

[0114] In a further embodiment, the present invention provides a method of inducing a polyfunctional CD4+ / CD8+ T cell response against influenza virus in a subject, the method comprising administering to the subject an influenza vaccine composition comprising: 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 virus in the subject.

[0115] In one embodiment, inducing a polyfunctional CD4+ / CD8+ T cell response comprises inducing the secretion of two or more cytokines, in some embodiments, the two or more cytokines are selected from the group consisting of IFNγ, granzyme B, and IL-4.

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

[0117] In another embodiment, 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 or analogs thereof. In one embodiment, the cationic lipid is R-DOTAP. In another embodiment, one or more recombinant influenza antigens are encapsulated within liposomes comprising the cationic lipid. In another embodiment, one or more recombinant influenza antigens are mixed with preformed cationic lipid nanoparticles. In some embodiments, one or more recombinant influenza antigens and preformed cationic lipid nanoparticles are mixed in a 1:1 ratio. In one embodiment, one or more recombinant influenza antigens are present as micelles separate from the cationic lipid nanoparticles.

[0118] Set forth below are examples that discuss vaccine combinations comprising influenza recombinant antigens and cationic lipids, as contemplated for the uses discussed above. The following examples are provided to further illustrate embodiments of the present invention and are not intended to limit the scope of the invention. The following examples are typical of those that might be used, although other procedures, methodologies, or techniques known to those of skill in the art may alternatively be used. [Example]

[0119] Example 1 Materials and Methods Animals and Viruses: 6- to 20-week-old C57BL / 6J mice (B6 mice), BALB / cJ mice, K18-hACE2 mice (B6.Cg-Tg(K18-ACE2)2Prlmn / J), and DBA / 2J mice were obtained from Jackson Laboratory. All animals were housed under specific pathogen-free conditions at the Division of Laboratory Animal Resources (DLAR) at the University of Kentucky Medical Center or the University of Georgia Animal Research Center. All animal protocols followed the National Institutes of Health Animal Care Guidelines (A2020 02-024-Y1-A5, A2018 06-018-Y3-A16) and were reviewed and approved by the University of Kentucky (2019-3226) or the University of Georgia Institutional Animal Care and Use Committee. All studies were conducted in accordance with the ARRIVE guidelines.

[0120] For influenza challenge testing, we used A / Brisbane / 02 / 2018 (Brisbane / 18). For HAI assays, we used A / California / 07 / 2009 (California / 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 / 2009X-187 (A / Perth / 16 / 2009-like virus) (H3N2), and B / Brisbane / 60 / 2008. Influenza viruses were obtained from the International Influenza Resource Center (IRR).

[0121] Reagents and antibodies:

[0122] cGMP-grade R-DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) was provided by Merck & Co. cGMP-grade R-DOTAP liposomal nanoparticles were manufactured by Evonik according to the protocol 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 Vaccine and Immunology Center. Influenza nucleoprotein from A / Puerto Rico / 8 / 34 / Mount Sinai was obtained from Sino Biologicals, Inc. (USA). Fluzone® vaccine formulation was obtained from the University of Kentucky HealthCare pharmacy.

[0124] The overlapping peptide pool of influenza A / New York / 383 / 2005 (H3N2) hemagglutinin protein and the overlapping peptide pool of influenza A / California / 07 / 2009 (H1N1) pdm09 were obtained from BEI Resources.

[0125] Fluorescent dye-conjugated mouse monoclonal anti-mouse CD3 (clone: ​​145-2c11), CD4 (clone: ​​GK1.5), CD8 (clone: ​​YTS165.7.7), CD44 (clone: ​​IM7), CD62L (clone: ​​MED-14), IFNγ (clone: ​​XMG1.2), TNFα (clone: ​​MP6-XT22), and 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 the thin film hydration method according to a previously described protocol. Briefly, R-DOTAP thin films were formed by dissolving lipids in a 1:1 mixture of chloroform and methanol in a round-bottom flask. The organic solvent was then evaporated with a steady stream of dry nitrogen gas and dried overnight under vacuum. The dried R-DOTAP film was then hydrated by incubating in water for 12 hours. The lipid suspension was then sonicated for 10 minutes and sequentially extruded through 400 nm, 200 nm, and 100 nm polycarbonate membrane filters to obtain uniformly sized liposomal nanoparticles (Figure 1A-B). Groups of BALB / cJ mice (n = 6-8) were immunized twice on days 0 and 21 with monovalent COBRA-Y2 formulated with R-DOTAP nanoparticles or sucrose buffer (sucrose). Anti-COBRA-Y2 total IgG antibody titers were measured in serum samples obtained from vaccinated mice on day 35 (14 days after the second dose). Data (ab) represent the mean ± SEM of median maximal antibody titers from each mouse. Comparisons between the sucrose-only group and the R-DOTAP group were performed using a Student's t-test (two-tailed, unpaired test). **P ≤ 0.05. The nanoparticles were then diluted with 280 mM sucrose buffer and stored at -80°C until use. To prepare the vaccine formulation, concentrated antigen dissolved in PBS buffer was diluted to the desired concentration with 280 mM sucrose. Prior to vaccination, the vaccine components were allowed to warm to room temperature. The antigen components were mixed with the R-DOTAP nanoparticles in a 1:1 ratio using a pipette to form a uniform suspension. For subcutaneous vaccine delivery, 100 μl was used for each dose, and for intramuscular delivery, 50 μl was used for each dose.

[0128] Physical characterization of vaccine formulations:

[0129] The particle size, polydispersity, and zeta potential of R-DOTAP liposomes and vaccine formulations were measured at 23 °C using a Zeasizer nano equipped with a 4 mW 632.8 nm laser set at a 90° angle. Dynamic light scattering was used to measure the fluctuations in scattered light intensity. Distribution and cumulant analysis to measure Z-average and polydispersity were performed using the instrument software according to the manufacturer's instructions. Representative particle size distributions are shown in Figures 2A-2C. The measured values ​​of mean particle size, polydispersity, and zeta potential are listed in Table 1.

[0130] Enzyme-linked immunosorbent assay (ELISA):

[0131] Blood was collected from euthanized mice via tail vein bleeding or cardiac puncture into BD Microtainer® serum separator tubes. Separated serum samples were stored at -80°C until analysis. For antibody titer measurements, 96-well plates were coated with 50 μl / well of recombinant RBD protein, COBRA-NG2, or COBRA-Y2 protein at a concentration of 2 μg / ml overnight at 4°C. After antigen coating, wells were blocked with 200 μl / well of PBS-T buffer containing 3% nonfat dry milk and 0.1% Tween-20 for 1-2 hours at room temperature. After blocking, the buffer was replaced with serum samples diluted in PBS-T buffer containing 1% nonfat dry milk. After 2 hours of incubation at room temperature, serum was removed, and wells were washed four times with PBS-T buffer. To detect protein antibodies, wells were incubated for 1 hour with 100 μl of PBS-T buffer containing 1% nonfat dry milk and HRP-conjugated anti-mouse IgG (1:5000) (catalog no. 115-035-003, Jackson ImmunoResearch), anti-mouse IgG1 (1:5000) (catalog no. 115-035-205, Jackson ImmunoResearch), anti-mouse IgG2c (1:5000) (catalog no. 115-035-206, Jackson ImmunoResearch), or anti-mouse IgG2a (1:5000) (catalog no. 115-035-206, Jackson ImmunoResearch). The wells were then washed four times, and 100 μl of SIGMAFAST OPD (o-phenylenediamine dihydrochloride) colorimetric substrate was added to each well. After 10 min of incubation, the reaction was stopped by adding 50 μl of 3 M HCl, and the optical density at 490 nm (OD490) was measured using a spectraMax M5 microplate reader.

[0132] Enzyme-linked immunosorbent spot assay (ELISpot):

[0133] 2.5×10 5Treated splenocytes were stimulated with the desired T cell epitope peptide or recombinant protein, or no peptide (control), for 18–24 hours at 37°C in 96-well plates precoated with mouse IFNγ or IL-4 capture antibodies (Mabtec). After stimulation, wells were washed with PBS and incubated with biotin-conjugated anti-IFNγ or IL-4 antibodies, followed by streptavidin-HRP antibodies. To visualize antigen-specific IFNγ- or IL-4-producing cells, wells were incubated with TMB substrate for 6 minutes, washed with water, and air-dried. Spots were scanned and counted using a CTL ImmunoSpot Analyzer and ImmunoSpot version 6 software. Spot counts were summarized as the median value from triplicate samples. Each sample included unstimulated wells and PMA / ionomycin control wells to detect background or as positive controls.

[0134] Intracellular cytokine and cell surface staining:

[0135] For intracellular protein analysis, single-cell suspensions of splenocytes were stimulated with the indicated stimulating antigen peptides for 6 hours at 37°C in cRPMI medium supplemented with purified anti-mouse CD28 (2 μg / ml), the protein transport inhibitor brefeldin A (5 μg / ml), and monensin (2.0 μM). After stimulation, cells were washed with FACS buffer and stained with fluorochrome-conjugated anti-mouse CD3, CD4, CD8, CD44, and CD62L antibodies. Cells were then washed, fixed, and permeabilized using a fixation / permeabilization kit and stained with fluorochrome-conjugated anti-mouse IFNγ, TNFα, and IL-2. After intracellular staining, cells were washed with FACS buffer and immediately analyzed by flow cytometry.

[0136] Vaccination of mice:

[0137] For all injections and implants, mice were anesthetized with isoflurane. The injection site was shaved and cleaned with 70% ethanol before the formulation was injected subcutaneously or intramuscularly. For subcutaneous (SC) vaccination, a 100 μl dose was delivered into one hind limb flank, and for intramuscular (IM) vaccination, a 50 μl dose was delivered into the thigh muscle of the hind limb. To generate R-DOTAP-based vaccine formulations, R-DOTAP nanoparticles (4–6 mg / ml) in 280 mM sucrose buffer were mixed 1:1 with the indicated concentration of recombinant protein resuspended in 280 mM sucrose buffer. For antigen-only vaccine formulations, the recombinant protein was resuspended at the desired concentration in 280 mM sucrose buffer. All vaccination regimens consisted of two doses delivered 1–4 weeks apart.

[0138] Hemagglutination inhibition assay:

[0139] A hemagglutination inhibition (HAI) assay was used to evaluate functional antibodies against HA that could inhibit agglutination of guinea pig red blood cells for H3N2 viruses and turkey red blood cells for H1N1 viruses. The protocol was adapted from the World Health Organization (WHO) Laboratory Influenza Surveillance Manual. Guinea pig red blood cells are frequently used to characterize modern A(H3N2) influenza strains that have developed preferential binding to α(2,6)-linked sialic acid receptors. To inactivate nonspecific inhibitors, serum samples were treated with receptor-destroying enzyme (RDE) before testing. Briefly, RDE was added to serum at a ratio of 3:1 and incubated overnight at 37°C. RDE was inactivated by incubation at 56°C for 30 minutes.

[0140] RDE-treated serum was 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) / 50 μl in the presence of 20 nM oseltamivir carboxylate, was added to each well. The plates were covered and incubated at room temperature for 30 minutes, after which 0.75% guinea pig red blood cells in PBS were added. Prior to use, red blood cells (RBCs) were washed twice with PBS, stored at 4°C, and used within 24 hours of preparation. The plates were mixed by gentle agitation, covered, and the RBCs were allowed to settle at room temperature for 1 hour. HAI titers were determined by inverse dilution of the last well containing unagglutinated RBCs. Each plate included positive and negative serum controls.

[0141] In a separate assay, RDE-treated serum was diluted in a series of two-fold serial dilutions in V-bottom microtiter plates. An equal volume of each influenza virus, adjusted to approximately 8 hemagglutination units (HAU) / 50 μl, was added to each well. The plates were covered and incubated with red blood cells in phosphate-buffered saline (PBS) for 20 minutes at room temperature. Prior to use, the red blood cells were washed twice with PBS, stored at 4°C, and used within 24 hours of preparation. The plates were mixed by gentle agitation, covered, and the red blood cells were allowed to settle for 30 minutes at room temperature. The HAI titer was determined by inverse dilution of the last well containing unagglutinated red blood cells. Each plate included positive and negative serum controls.

[0142] All mice were negative for pre-existing antibodies to human influenza virus (HAI ≤ 1:10) before infection or vaccination. In this study, seroprotection was defined as an HAI titer of > 1:40, and seroconversion was defined as a four-fold increase in antibody titer compared to baseline, in accordance with the WHO and European Commission guidelines for evaluating influenza vaccines. Because all mice were naive and seronegative at the time of vaccination, seroconversion and seroprevalence are interchangeable in this study.

[0143] Mouse exposure experiments:

[0144] For influenza virus challenge studies, DBA / 2J mice (female, 7-9 weeks old) were immunized intramuscularly with the indicated vaccine formulations on days 0 and 28. On day 56, mice were challenged with H1N1 A / Brisbane / 02 / 2018 (Bris / 18) influenza virus at a 10-fold LD50 dose (3.6 × 10 6 Mock-vaccinated animals were intranasally challenged with 50 μL of 1000 pfu / mouse in a volume of 50 μL. Mock-vaccinated animals were intranasally inoculated with 50 μL of PBS. All animals exposed to live virus were monitored twice daily, morning and evening, for weight loss and clinical signs (labored breathing, lethargy, hunched back, ruffled fur, unresponsiveness to stimuli, and severe respiratory distress) until 14 days post-infection. Body weight was closely monitored until 14 days post-infection. Mice were humanely euthanized when they lost 20% of their original body weight or reached clinical endpoints. For virus titer detection, lungs from influenza-challenged mice were harvested from three pre-selected mice per group on days 3 and 6 post-infection. Briefly, frozen lungs were processed, and the clarified virus-containing supernatant was added to 90% confluent MDCK cells and incubated for 1 hour. After this step, the cells were washed and medium containing 1.6% agarose was added. After 72 hours of incubation at 37°C, plates were processed, dried, and viral plaques were counted as plaque-forming units per gram of lung tissue.

[0145] Equipment, software, and statistical analysis:

[0146] Flow cytometry was performed using a BD Symphony A3 flow cytometer equipped with BD FACSDiva™ software. All flow data were analyzed using FlowJo® version 10.0 software. Statistical analysis for all other studies was performed using GraphPad Prism 9.0 software, with means compared by simple Student's t-test or ANOVA with Tukey's multiple comparisons correction. The Mantel-Cox test was used for survival curves.

[0147] Example 2 Immunogenicity of recombinant influenza proteins formulated with R-DOTAP nanoparticles The ability of R-DOTAP to enhance the immunogenicity of recombinant influenza proteins was evaluated. To this end, influenza nucleoprotein or computationally optimized broadly reactive antigen (COBRA) hemagglutinin (HA) was used as the vaccine antigen. In the first set of experiments, the nucleoprotein was formulated with R-DOTAP nanoparticles, and B6 mice were immunized with two doses of the vaccine. T cell immune responses were then assessed using a validated H2-Db-binding CD8 T cell epitope (NP366-74:ASNENMETM, SEQ ID NO: 1) and a validated I-Ab-binding CD4 T cell epitope (NP-311-25:QVYSLIRPNENPAHK, SEQ ID NO: 2).

[0148] In a second set of experiments, monovalent and bivalent vaccines (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 with two intramuscular doses of the vaccine, and T cell responses were measured 7 days after the booster dose using an IFN-γ ELISpot assay.

[0149] To stimulate antigen-specific T cells in ELISpot assays, we used either the entire COBRA protein or overlapping peptides from A / California 07 / 2009 (H1N1) hemagglutinin or A / New York / 384 / 05 (H3N2) hemagglutinin, which share consensus with the COBRA sequence and are related to influenza viruses circulating in nature.

[0150] As shown in Figures 3A-3D, the R-DOTAP-containing formulation induced strong T cell responses against both the nucleoprotein antigen and the COBRA HA antigen. The R-DOTAP-based vaccine induced strong CD4 T cell responses against the nucleoprotein antigen compared to the antigen-only vaccine. + T cell responses and CD8 +The R-DOTAP-Y2NG2 bivalent vaccine induced a robust T cell response against the COBRA antigen (Figures 3B-3D). The T cell immune response induced by the R-DOTAP-adjuvanted formulation was significantly higher than that induced by the formulation adjuvanted with Addvax®, an oil-emulsion-based adjuvant system. Importantly, the T cells raised against the COBRA antigen recognized and responded to multiple conserved T cell epitopes presented by naturally occurring hemagglutinins from H1N1 and H3N2 virus strains (Figures 3B-3D).

[0151] Example 3 Effect of recombinant influenza proteins formulated with R-DOTAP nanoparticles on antigen-specific antibody titers To evaluate the antibody-mediated immune response to the COBRA protein, serum samples from vaccinated mice were analyzed for antigen-specific antibody titers. A significant increase in antibody titers was observed after a single injection of the R-DOTAP-based formulation (Figure 4A). A further significant increase in antibody titers was observed after a second injection compared with the antigen-only group. Combining two COBRA antigens into a single bivalent vaccine did not pose any formulation issues with R-DOTAP. The addition of NP to the bivalent vaccine was also evaluated, but no compatibility or stability limitations were encountered (data not shown).

[0152] Compared with the formulation containing Addavax™ as an adjuvant, mice vaccinated with the R-DOTAP-containing formulation had higher antibody titers measured on days 35 and 62 (Figures 4B and 4C), indicating that R-DOTAP induces potent antibody induction comparable to Addavax™. We next assessed the potential for dose-sparing by immunizing BALB / cJ mice with various doses of COBRA-Y2 formulated with various doses of R-DOTAP nanoparticles and measuring Y2-specific antibody responses. Mice immunized with 0.35–3.0 μg of Y2 antigen formulated with 300 μg of R-DOTAP showed significantly increased Y2-specific total IgG titers measured 14 days after the booster vaccination (Figure 1). Animals vaccinated with 0.35 μg or 3.0 μg also showed similar IgG titers. Similarly, mice vaccinated with 50–300 μg of R-DOTAP added to the vaccine formulation also induced a similar increase in total antibody titers, with no significant difference between low- and high-dose R-DOTAP (Figure 1).

[0153] Th1-type antibody-mediated immune responses play an important role in defense against viral infection. To further evaluate the immune response induced by R-DOTAP, we measured antibody subclass titers after vaccination. R-DOTAP induced class switching, as evidenced by the presence of IgG1 and IgG2a antibodies in serum samples on day 35. Similar levels of IgG1 and IgG2a were observed, indicating a balanced Th1 / Th2 response (Figures 4D and 4E).

[0154] Example 4 Effect of recombinant influenza proteins formulated with R-DOTAP nanoparticles on the development of neutralizing antibodies Next, we evaluated the functional ability of the vaccine to induce antibodies that block the interaction of influenza virus with sialic acid.Hemagglutination inhibition (HAI) assays were used to evaluate the development of influenza virus-neutralizing antibodies against different H1N1 and H3N2 virus strains.

[0155] The bivalent vaccine formulated with R-DOTAP (R-DOTAP-Y2NG2) showed significantly enhanced titers against multiple drift variants of H1N1 and H3N2 viruses that share consensus with COBRA Y-2 and COBRA-NG2, respectively, compared with the antigen-only vaccine (Figures 5A-5B). Robust HAI titers exceeding the 1:40 threshold were observed for all H1N1 viruses, even at the lowest dose (0.12 μg) tested. HAI titers against H3N2 drift variants were lower than those against H1N1 viruses, but still demonstrated significant HAI titers exceeding the 1:40 threshold at the 3 μg dose. As expected, little neutralizing activity (HAI titers <1:40) was observed with antibodies induced by antigen-only preparations or from mock-vaccinated mice. These results indicate that vaccination with bivalent COBRA antigen formulated with R-DOTAP induced robust and balanced Th1 and Th2 antibody responses and broadly cross-reactive antibodies capable of neutralizing several H1N1 and H3N2 strain drift variants.

[0156] Example 5 R-DOTAP can enhance the immunogenicity of unadjuvanted seasonal influenza vaccines We evaluated whether R-DOTAP could be used to enhance the immunogenicity of existing human influenza vaccines. As a proof-of-concept study, we used the 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 (A / Perth / 16 / 2009-like virus) (H3N2), and B / Brisbane / 60 / 2008. Vaccine formulations were prepared by mixing R-DOTAP nanoparticles with various doses of Fluzone® in a 1:1 ratio. C57BL / 6J mice were vaccinated (0.1 ml / dose) on days 0 and 21, and blood was collected on day 35 for HAI titers.

[0157] We observed that the incorporation of R-DOTAP into Fluzone® significantly enhanced HAI titers against all virus strains in vaccinated mice compared with the Fluzone®-only group (Figures 6A-6C). Importantly, a significant dose-sparing effect was observed in the R-DOTAP vaccine group.

[0158] Example 6 Recombinant proteins formulated with R-DOTAP protected mice from influenza virus challenge To investigate the protective efficacy of the bivalent influenza vaccine R-DOTAP-Y2NG2 in mice, DBA / 2J mice were vaccinated with a formulation containing various doses of COBRA antigen, with or without R-DOTAP, in a two-dose regimen and challenged with A / Brisbane / 2 / 2018(H1N1) (3.6 × 10 6 pfu / dose). Weight loss was measured as a predictor of protection from challenge.

[0159] 100% of both unvaccinated mice and mice vaccinated with the antigen-only (3 μg / HA) vaccine formulation rapidly lost weight, with 7-day survival rates of 0% and 33%, respectively (Figures 7A 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 7A and 7B). Both low-dose (0.12 μg / HA) and high-dose (3 μg / HA) COBRA antigen formulated with R-DOTAP conferred complete protection, demonstrating significant dose-sparing effects (Figure 7A).

[0160] Viral clearance in the lungs after challenge was also assessed. The vaccine formulation containing R-DOTAP completely cleared influenza virus from the lungs within 3 days (Figure 7C), and virus was no longer detectable by day 6 (Figure 7D). In contrast, mice administered antigen alone and unvaccinated mice displayed significant viral loads on both days 3 and 6. Collectively, these studies demonstrated that a recombinant protein vaccine containing R-DOTAP as an immunomodulatory agent induced an immune response capable of protecting mice from viral infection.

[0161] Example 7 Consideration Cationic lipids are excellent delivery vehicles for transporting nucleic acids and proteins / peptides into cells and have been widely used for drug delivery in humans. However, most cationic lipids are inactive and do not activate the immunological signals necessary for an effective immune response to vaccine antigens. Here, we demonstrate that R-DOTAP promotes robust antibody and T cell responses against various viral proteins, which can confer neutralizing activity and protection from viral challenge.

[0162] Both TEM images (Figures 2A-C) and physical property data (Table 1 and Figures 2A-C) showed that R-DOTAP formed uniform, smooth-surfaced spherical structures in the approximately 150 nm size range in sucrose buffer. Mixing antigen with the nanoparticles did not significantly change the size and polydispersity of the formulation (Table 1). Changes in the surface and zeta potential of the nanoparticles after antigen addition were observed.

[0163] [Table 1]

[0164] In this study, we evaluated the ability of R-DOTAP to enhance both humoral and cellular immune responses to large protein antigens using a prototype vaccine formulation containing antigens derived from the respiratory virus influenza. By combining R-DOTAP with COBRA influenza antigens, we assessed the potential of the R-DOTAP-COBRA vaccine to provide an effective universal influenza vaccine by inducing broadly protective immune responses capable of neutralizing multiple influenza virus strains (Figure 5A-5B). Co-formulation of influenza-derived viral protein antigens with R-DOTAP significantly enhanced vaccine immunogenicity, elicited 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] Our results demonstrate that single-component R-DOTAP nanoparticles can fulfill multiple roles essential for generating broad and sustained protective immune responses.

[0166] Ample evidence suggests that CD8 T cells play a critical role in long-term protection against highly variable viruses, such as influenza. While most approved recombinant protein adjuvants effectively induce humoral, Th1-type immune responses, few can induce robust, clinically effective cytotoxic CD8 T cell immune responses in humans. In this study, we demonstrated that R-DOTAP can generate CD8 T cells against internal epitopes of large recombinant protein antigens. These T cells were polyfunctional and possessed an effector phenotype. These T cells were capable of producing multiple cytotoxic cytokines and persisted in vaccinated mice 28 days after the second vaccination, suggesting the establishment of a T cell memory response.

[0167] We observed that vaccines containing R-DOTAP induced both IFN-γ- and IL-4-producing T cells (data not shown) and generated both IgG1 and IgG2a antibody subtypes. No strong bias toward either the Th1 or Th2 subtype was observed. Thus, from an antibody perspective, these results indicate that R-DOTAP induces a balanced Th1 / Th2-type immunity that is optimal for effective vaccine-induced antibody responses. However, among antigen-specific Th1 and CD8 T cells, R-DOTAP promoted the development of high levels of multifunctional cytokine-secreting cells, which have been shown to be optimal for promoting viral clearance.

[0168] In summary, these studies demonstrated that a protein subunit vaccine against influenza based on the R-DOTAP platform induces broadly protective cellular and humoral immune responses and provides significant dose-sparing efficacy against the antigen. The heterogeneity of recombinant proteins that can be formulated and administered using R-DOTAP, as well as the ability to produce multivalent vaccines, suggest that R-DOTAP is an excellent candidate for use in a variety of prophylactic vaccines against infectious diseases. Furthermore, R-DOTAP has proven highly effective in enhancing the efficacy of currently licensed seasonal influenza vaccines, further expanding the potential utility of R-DOTAP. The safety and efficacy profile of R-DOTAP has been successfully established in human clinical trials, paving the way for future trials of an R-DOTAP-based universal influenza vaccine.

[0169] Example 8 Use of R-DOTAP adjuvant formulation for bivalent COBRA H1 / H3 vaccine in a preimmune ferret model In the pre-immune ferret model, ferrets were primed, vaccinated, and boosted with virus before influenza challenge, as described in Figure 8. This model mimics the human response to vaccination by first infecting ferrets with influenza viruses (H1N1-A / Singapore / 6 / 1986- and H3N2-A / Panama / 2007 / 1999).

[0170] H1N1 HAI responses were assessed as shown in Figures 9A-9C. H1 / H3 pre-immune ferrets were vaccinated twice with Y2 / NG2 rHA (15 μg), R-DOTAP alone, or wild-type rHA and R-DOTAP, and HAI titers were measured. H3N2 HAI responses were also assessed as shown in Figures 10A-10C. H1 / H3 pre-immune ferrets were vaccinated twice with Y2 / NG2 rHA (15 μg), R-DOTAP alone, or wild-type rHA and R-DOTAP, and HAI titers were measured.

[0171] As shown in Figures 11A-11C, animals vaccinated with Y2 / NG2 rHA maintained their weight better and had lower nasal wash viral titers.

[0172] This study demonstrated that the COBRA rHA vaccine, adjuvanted with Infectimune® (R-DOTAP), can induce protective HAI antibody responses in pre-immunized ferrets against a panel of viruses from the past decade. It also elicited HAI-reactive antibodies against future drifted virus isolates from 2019 to 2020. Vaccination prevented weight loss and H1N1 virus replication in the lungs of vaccinated animals. In populations with a broader range of pre-immune backgrounds to influenza, such as humans, these vaccines are expected to generate a broader reactive antibody profile through recall of more diverse memory B cell populations.

[0173] List of Arrays SEQ ID NO: 1: NP366-74 ASNENMETM Sequence number 2 NP-311-25 QVYSLIRPNENPAHK SEQ ID NO: 3 Y2-H1N1 HA [ka] SEQ ID NO: 4 NG2-H3N2 HA [ka] SEQ ID NO: 5 J-4 soluble HA [ka] Sequence number 6 TJ-5 [ka] Sequence number 7 N1-I COBRA [ka] SEQ ID NO:8 N2-A COBRA soluble form [ka] Sequence number 9 J1(2013S-2015S)1-566 AA [ka] Sequence number 10 J2(2014-15N through 2015-16N)1-566 AA [ka] SEQ ID NO: 11 J3(2014S-2016S 1-566 AA [ka] Sequence number 12 J41-566 AA (2013S through 2015-16N) [ka] SEQ ID NO: 13 Soluble HA [ka] SEQ ID NO: 14 Soluble HA [ka] SEQ ID NO: 15 Soluble HA sequence J1 H3N2 soluble HA [ka] SEQ ID NO: 16 NG1 soluble HA [ka] SEQ ID NO: 17 NG2 soluble HA [ka] SEQ ID NO: 18 NG3 soluble HA [ka] SEQ ID NO: 19 H1N1 soluble HA sequence Y1 H1N1 soluble HA [ka] SEQ ID NO: 20 Y2 H1N1 soluble HA [ka] SEQ ID NO: 21 Y3 H1N1 soluble HA [ka] SEQ ID NO: 22 Y4 H1N1 soluble HA [ka]

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

Claims

1. a) one or more non-natural recombinant influenza antigens; and b) cationic lipids; 10. A vaccine composition comprising:

2. 2. The vaccine composition of claim 1, wherein the one or more non-natural recombinant influenza antigens comprise a computationally optimized broadly reactive influenza antigen (COBRA) hemagglutinin (HA).

3. 2. The vaccine composition of claim 1, wherein the one or more non-naturally occurring recombinant influenza antigens are recombinant H1N1 and / or H3N2 hemagglutinin influenza proteins.

4. 2. The vaccine composition of claim 1, wherein the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to any of SEQ ID NOs: 3-22, or a combination thereof.

5. 5. The vaccine composition of claim 4, wherein the one or more non-naturally occurring recombinant influenza antigens comprise the amino acid sequence of any of SEQ ID NOs: 3 to 22, or a combination thereof.

6. 5. The vaccine composition of claim 4, wherein the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:3 and / or SEQ ID NO:

4.

7. 2. The vaccine composition of claim 1, wherein the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, a variant thereof, or an analog thereof.

8. 2. The vaccine composition of claim 1, wherein the cationic lipid is R-DOTAP.

9. 10. The vaccine composition of claim 1, wherein the one or more non-naturally occurring recombinant influenza antigens are encapsulated in a liposome comprising a cationic lipid.

10. 10. The vaccine composition of claim 1, wherein the one or more non-natural recombinant influenza antigens are mixed with preformed cationic lipid nanoparticles.

11. 11. The vaccine composition of claim 10, wherein the one or more non-natural recombinant influenza antigens and the preformed cationic lipid nanoparticles are mixed in a 1:1 ratio.

12. 11. The vaccine composition of claim 10, wherein the one or more non-natural recombinant influenza antigens are present as micelles separated from the preformed cationic lipid nanoparticles.

13. 1. A method of inducing an immune response to influenza virus in a subject, comprising: a) one or more non-natural recombinant influenza antigens; and b) cationic lipids; inducing said immune response against influenza virus in said subject by administering to said subject a vaccine composition comprising: A method comprising:

14. 14. The method of claim 13, wherein the one or more non-naturally occurring recombinant influenza antigens are recombinant H1N1 and / or H3N2 hemagglutinin influenza proteins.

15. 14. The method of claim 13, wherein the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to any of SEQ ID NOs: 3-22, or a combination thereof.

16. 14. The method of claim 13, wherein the one or more non-naturally occurring recombinant influenza antigens comprise the amino acid sequence of any of SEQ ID NOs: 3-22, or a combination thereof.

17. 14. The method of claim 13, wherein the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence having 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO:3 and / or SEQ ID NO:

4.

18. 14. The method of claim 13, wherein the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, a variant thereof, or an analog thereof.

19. 14. The method of claim 13, wherein the cationic lipid is R-DOTAP.

20. The immune response is CD8 ＋ Effector T cells, CD4 ＋ The method of claim 13, comprising the induction of effector T cells and memory T cells.

21. CD8 ＋ Effector T cells and CD4 ＋ Induction of effector T cells is achieved by inducing IFNγ and granzyme B-producing CD8 T cells in the subject. ＋ Proliferation of effector T cells and / or IL-4-producing CD4 ＋ 21. The method of claim 20, comprising inducing proliferation of effector T cells.

22. 14. The method of claim 13, wherein the inducing an immune response comprises inducing the production of IgG in the subject.

23. 23. The method of claim 22, wherein the IgG comprises IgG1 and IgG2a.

24. The method of claim 13 , wherein the induction of the immune response comprises inducing the secretion of broadly neutralizing antibodies.

25. 1. A method of preventing or treating influenza infection in a subject, comprising: a) one or more non-natural recombinant influenza antigens; and b) cationic lipids; preventing or treating said influenza infection in said subject by administering to said subject a vaccine composition comprising: A method comprising:

26. 26. The method of claim 25, wherein the one or more non-naturally occurring recombinant influenza antigens are recombinant H1N1 and / or H3N2 hemagglutinin influenza proteins.

27. 26. The method of claim 25, wherein the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to any of SEQ ID NOs: 3-22, or a combination thereof.

28. 26. The method of claim 25, wherein the one or more non-naturally occurring recombinant influenza antigens comprise the amino acid sequence of any of SEQ ID NOs: 3-22, or a combination thereof.

29. 26. The method of claim 25, wherein the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:3 and / or SEQ ID NO:

4.

30. 26. The method of claim 25, wherein the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, a variant thereof, or an analog thereof.

31. 26. The method of claim 25, wherein the cationic lipid is R-DOTAP.

32. 1. A method of enhancing the immunogenicity of an influenza vaccine in a subject, comprising: a) administering said influenza vaccine to said subject; and b) administering a cationic lipid to said subject; enhancing the immunogenicity of the influenza vaccine by A method comprising:

33. 33. The method of claim 32, wherein the influenza vaccine is an inactivated influenza vaccine, an attenuated influenza vaccine, or a recombinant influenza vaccine.

34. 34. The method of claim 33, wherein the influenza vaccine is a monovalent vaccine, a bivalent vaccine, a trivalent vaccine, or a tetravalent vaccine.

35. 33. The method of claim 32, wherein the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, a variant thereof, or an analog thereof.

36. 33. The method of claim 32, wherein the cationic lipid is R-DOTAP.

37. 1. A method for inducing secretion of broadly neutralizing antibodies against influenza virus in a subject, comprising: a) one or more non-natural recombinant influenza antigens; and b) cationic lipids; inducing secretion of the broadly neutralizing antibodies against the influenza virus in the subject by administering to the subject a vaccine composition comprising: A method comprising:

38. 38. The method of claim 37, wherein the one or more non-naturally occurring recombinant influenza antigens are recombinant H1N1 and / or H3N2 hemagglutinin influenza proteins.

39. 38. The method of claim 37, wherein the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to any of SEQ ID NOs: 3-22, or a combination thereof.

40. 38. The method of claim 37, wherein the one or more non-naturally occurring recombinant influenza antigens comprise the amino acid sequence of any of SEQ ID NOs: 3-22, or a combination thereof.

41. 38. The method of claim 37, wherein the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:3 and / or SEQ ID NO:

4.

42. 38. The method of claim 37, wherein the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, a variant thereof, or an analog thereof.

43. 38. The method of claim 37, wherein the cationic lipid is R-DOTAP.

44. 1. A method of inducing a balanced Th1 / Th2 immune response to influenza virus in a subject, comprising: a) one or more non-natural recombinant influenza antigens; and b) cationic lipids; inducing said balanced Th1 / Th2 immune response against said influenza virus in said subject by administering to said subject a vaccine composition comprising: A method comprising:

45. 45. The method of claim 44, wherein the one or more non-naturally occurring recombinant influenza antigens are recombinant H1N1 and / or H3N2 hemagglutinin influenza proteins.

46. 45. The method of claim 44, wherein the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to any of SEQ ID NOs: 3-22, or a combination thereof.

47. 45. The method of claim 44, wherein the one or more non-naturally occurring recombinant influenza antigens comprise the amino acid sequence of any of SEQ ID NOs: 3-22, or a combination thereof.

48. 45. The method of claim 44, wherein the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:3 and / or SEQ ID NO:

4.

49. 45. The method of claim 44, wherein the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, a variant thereof, or an analog thereof.

50. 45. The method of claim 44, wherein the cationic lipid is R-DOTAP.

51. Induction of a Th1 immune response is achieved by inducing CD8+ cells that produce IFNγ and granzyme B in the subject. ＋ Proliferation of effector T cells and / or IL-4-producing CD4 ＋ 45. The method of claim 44, comprising inducing proliferation of effector T cells.

52. IFNγ-producing CD8 ＋ Effector T cells are associated with the production of IgG2a and IL-4-producing CD4 ＋ 52. The method of claim 51, wherein the effector T cells are associated with the production of IgG1.

53. 1. A method of inducing a polyfunctional CD4+ / CD8+ T cell response to influenza virus in a subject, comprising: a) one or more non-natural recombinant influenza antigens; and b) cationic lipids; inducing said polyfunctional CD4+ / CD8+ T cell response against said influenza virus in said subject by administering to said subject an influenza vaccine composition comprising: A method comprising:

54. 54. The method of claim 53, wherein the one or more non-naturally occurring recombinant influenza antigens are recombinant H1N1 and / or H3N2 hemagglutinin influenza proteins.

55. 54. The method of claim 53, wherein the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to any of SEQ ID NOs: 3-22, or a combination thereof.

56. 54. The method of claim 53, wherein the one or more non-naturally occurring recombinant influenza antigens comprise the amino acid sequence of any of SEQ ID NOs: 3-22, or a combination thereof.

57. 54. The method of claim 53, wherein the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:3 and / or SEQ ID NO:

4.

58. 54. The method of claim 53, wherein the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, a variant thereof, or an analog thereof.

59. 54. The method of claim 53, wherein the cationic lipid is R-DOTAP.

60. 54. The method of claim 53, wherein the induction of the polyfunctional CD4+ / CD8+ T cell response comprises inducing the secretion of two or more cytokines.

61. 61. The method of claim 60, wherein the two or more cytokines are selected from the group consisting of IFNγ, granzyme B, and IL-4.

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