Recombinant protein vaccines formulated with enantiospecific cationic lipid R-DOTAP and methods of use thereof

Abstract

Provided herein are vaccine compositions comprising recombinant protein antigens derived from computationally optimized broad reactive influenza antigen (COBRA) proteins and immunomodulators and methods of using the same. In some embodiments, the vaccine composition comprises one or more COBRA proteins, and the immunomodulator is a cationic lipid. The cationic lipid is prepared from R-DOTAP (4, 4 ', 4' '- Methods of use of the vaccine composition include a method of inducing a humoral immune response against influenza virus in a subject, a method of inducing multifunctional CD8 + and CD4 + effector T cells against influenza virus, a method of inducing memory T cells against influenza virus, and a method of inducing multi-functional CD8 + and CD4 + effector T cells against influenza virus. Methods of enhancing immunity against influenza viruses and methods of inducing a balanced Th1 / Th2 immune response against influenza viruses.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 418,381, filed on October 21, 2022, and U.S. Provisional Application No. 63 / 539,066, filed on September 18, 2023. The disclosures of the prior applications are considered part of the disclosure of the present application and are hereby incorporated by reference in their entirety into the disclosure of the present application.

[0003] Incorporation of Sequence Listing

[0004] The material in the accompanying sequence listing is hereby incorporated by reference into this application.The accompanying sequence listing xml file, named ST26.xml, was created on and is 1000 kb. Background Art

[0005] Background Information

[0006] Immunization remains one of the most effective public health measures against viral infections. Increased safety requirements for FDA approval, coupled with the complexities associated with certain viral infections, have prompted the search for a new generation of prophylactic vaccines based on peptides or recombinant proteins. However, most recombinant proteins or peptides are not immunogenic and induce only weak immune responses following administration. Therefore, recombinant protein or peptide vaccines often require adjuvants to stimulate or enhance the immune response to the antigen.

[0007] There are many different adjuvants approved or under investigation for recombinant protein-based vaccines, with different immunomodulatory properties. Many adjuvants promote strong antibody responses, such as alum, squalene, or monophosphoryl lipid A, while other adjuvants, such as CpG or other TLR 7, 8, or 9 agonists, promote stronger Th1 CD4 and CD8 T cell responses. But even these distinctions are somewhat blurred because a single adjuvant can promote different responses through different pathways. For example, squalene-based adjuvants induce CD8 T cell responses through pathways that differ from their antibody-inducing properties. Therefore, it is now recognized that different adjuvants induce different immunological characteristics, which makes them more or less effective against different diseases (e.g., tuberculosis versus influenza). Vaccines against respiratory viruses (e.g., influenza) primarily induce antibody responses, and for influenza, neutralizing antibody titers are the main correlate of immunity. However, natural infection also induces strong CD4 and CD8 T cell responses, which are crucial for the development of lasting immunity. In cases where antigenic drift or shift hinders antibody recognition, CD8 T cell responses to internal proteins are also crucial. Therefore, it is generally believed that the next generation of universal vaccines against respiratory viral pathogens should induce strong neutralizing antibodies and strong CD8 T cell immunity.

[0008] Lipid nanoparticles are some of the most promising delivery vehicles for eukaryotic cells and have been widely used since the late 1980s to deliver nucleic acids into cells, including in human gene therapy clinical trials. Novel mRNA vaccine technologies being used to combat the COVID-19 pandemic also use lipid nanoparticles as delivery vehicles to deliver mRNA into cells. In recent years, cationic lipids have become attractive targets for delivering proteins or peptides for immunotherapy and vaccines. Importantly, cationic lipid-mediated antigen uptake delivers proteins and peptides into the MHC class I and class II processing pathways. Mechanistically, cationic lipid nanoparticles efficiently bind to negatively charged cell membranes in a receptor-independent manner and are rapidly internalized into endosomes in amounts exceeding receptor-mediated uptake. Once inside the endosome, the cationic lipids fuse with the endosomal membrane, delivering some of their contents to the cytoplasm. Therefore, certain cationic lipids are ideally suited as nonviral vectors for the intracellular delivery of peptides, proteins, and inactivated whole viruses into the MHC class I and class II pathways. Recent studies investigating cationic lipids as delivery agents have also revealed that some possess immunostimulatory properties and are able to activate pathways necessary for an effective immune response following vaccination. However, due to the wide variation in the immunostimulatory properties and mechanisms of action of cationic lipids, not all cationic lipids exhibit these properties universally.

[0009] The enantiospecific cationic lipid 1,2-dioleoyl-3-trimethylammoniumpropane (R-DOTAP) has been shown to be particularly robust in inducing CD8 T cell responses to peptide-based vaccines. Further studies have shown that R-DOTAP promotes cellular uptake and cross-presentation of CD8 epitopes of long peptides and promotes the formation of polyfunctional CD8 T cells. R-DOTAP alone has been shown to induce type I interferon in draining LNs, and type I interferon is required for R-DOTAP-mediated induction of antigen-specific CD8 T cells. While it is well established that R-DOTAP promotes robust CD8 T cell responses to peptide-based vaccines, the ability of R-DOTAP to promote CD8 T cell responses to larger recombinant proteins is less clear. While R-DOTAP has also been shown to induce robust antibody responses to the model antigen OVA, the ability of R-DOTAP to promote antibody responses to vaccine-related recombinant proteins has not been studied. Summary of the Invention

[0010] The present invention is based on a groundbreaking discovery: the use of cationic lipids as immunomodulators enhances the immunity induced by influenza recombinant proteins in vaccine compositions.

[0011] In one embodiment, the invention provides a vaccine composition comprising one or more non-naturally occurring recombinant influenza antigens; and a cationic lipid.

[0012] In one aspect, the one or more non-naturally occurring recombinant influenza antigens include a computationally optimized broadly reactive influenza antigen (COBRA) hemagglutinin (HA). In another aspect, the one or more non-naturally occurring recombinant influenza antigens are recombinant H1N1 and / or H3N2 hemagglutinin influenza proteins. In one aspect, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence with at least 80%, 85%, 90% or 95% sequence identity to any one of SEQ ID NO: 3-22 and a combination thereof. In one aspect, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence with at least 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4. In another aspect, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence of any one of SEQ ID NO: 3-22 and a combination thereof. In another aspect, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. In another aspect, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence having at least 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 3 and SEQ ID NO: 4. In one aspect, the one or more non-naturally occurring recombinant influenza antigens comprise the amino acid sequence of SEQ ID NO: 3 and SEQ ID NO: 4. In one aspect, the one or more non-naturally occurring recombinant influenza antigens comprise the amino acid sequence of SEQ ID NO: 3 and SEQ ID NO: 4. In another aspect, the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, variants thereof, or analogs thereof. In one aspect, the cationic lipid is R-DOTAP. In another aspect, the one or more non-naturally occurring recombinant influenza antigens are encapsulated in liposomes comprising cationic lipids. In another aspect, the one or more non-naturally occurring recombinant influenza antigens are mixed with pre-formed cationic lipid nanoparticles. In some aspects, the one or more non-naturally occurring recombinant influenza antigens are mixed with pre-formed cationic lipid nanoparticles in a ratio of 1: 1. In one aspect, the vaccine composition is a universal influenza vaccine.

[0013] In another embodiment, the present invention provides a method of inducing an immune response against influenza virus 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 inducing an immune response against influenza virus in the subject.

[0014] In one aspect, the immune response comprises CD8 + Effector T cells, CD4 + Induction of effector T cells and memory T cells. On the other hand, induction of CD8 + Effector T cells and CD4 + Effector T cells include CD8 T cells that induce the production of IFNγ and granzyme B in subjects + Proliferation of effector T cells and / or IL-4-producing CD4 + In one aspect, the induction of a humoral immune response comprises inducing the production of IgG in the subject. In some aspects, IgG comprises IgG1 and IgG2a. In one aspect, the induction of an immune response comprises inducing the secretion of broadly neutralizing antibodies.

[0015] In another embodiment, the present invention provides a method of preventing or treating 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 influenza infection in the subject.

[0016] 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.

[0017] In one aspect, the influenza vaccine is an inactivated influenza vaccine, an attenuated influenza vaccine, or a recombinant influenza vaccine. In another aspect, the influenza vaccine is a monovalent vaccine, a bivalent vaccine, a trivalent vaccine, or a quadrivalent vaccine.

[0018] In another embodiment, the present invention provides a method of inducing secretion of broadly neutralizing antibodies against influenza virus 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 inducing secretion of broadly neutralizing antibodies against influenza virus in the subject.

[0019] In a further embodiment, the present invention provides a method of inducing a balanced Th1 / Th2 immune response 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 inducing the secretion of broadly neutralizing antibodies against influenza virus in the subject.

[0020] In one aspect, 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 a subject. In some aspects, IFNγ-producing CD8+ effector T cells are associated with the production of IgG2a, while IL-4-producing CD4+ effector T cells are associated with the production of IgG1.

[0021] In another embodiment, the present invention provides a method of inducing a polyfunctional CD4+ / CD8+ T cell response against influenza virus in a subject, 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.

[0022] In one aspect, inducing a multifunctional CD4+ / CD8+ T cell response comprises inducing the secretion of two or more cytokines. In some aspects, the two or more cytokines are selected from IFNγ, granzyme B, and IL-4. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figures 1A-1B The antibody-mediated immune response induced by recombinant influenza proteins formulated with R-DOTAP is demonstrated. Figure 1A is a graph showing total IgG antibody titers obtained with R-DOTAP formulations. Figure 1B is a graph of total IgG antibody titers obtained with Y2-COBRA formulations and sucrose.

[0024] Figures 2A-2C The average particle size of R-DOTAP liposome nanoparticles and R-DOTAP mixed with COBRA Y2 or COBRA-NG2 protein antigens is shown. Figure 2A A graph illustrating particle size distribution. Figure 2B Transmission electron microscopy (TEM) images of R-DOTAP nanoparticles. Figure 2C TEM images of R-DOTAP mixed with a mixture of COBRA-Y2 and COBRA-NG2 resuspended in water are shown.

[0025] Figures 3A-3DT cell responses to R-DOTAP formulations containing nucleoprotein and COBRA HA antigen are demonstrated. Figure 3A It is a graph showing the number of antigen-specific IFN-γ-producing T cells in the spleen in response to a vaccine having influenza nucleoprotein. 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 vaccines with influenza recombinant COBRA sequences. Figure 3C It is a diagram illustrating the specific T cell response to H1N1. Figure 3D is a graph illustrating specific T cell responses to H3N2.

[0026] Figures 4A-4E Antibody-mediated immune responses to COBRA proteins are demonstrated. Figure 4A is a graph showing antigen-specific antibody titers after vaccination with one or two doses. Figure 4B Comparison of sucrose, R-DOTAP, or Addavax TM Graph of anti-Y2 antigen-specific antibody titers 35 and 62 days after vaccination as adjuvant. Figure 4C Comparison of sucrose, R-DOTAP, or Addavax TM Graph of anti-NG2 antigen-specific antibody titers 35 and 62 days after vaccination as adjuvant. Figure 4D Comparison of sucrose, R-DOTAP, or Addavax TM Graph of anti-Y2 antigen-specific IgG1 and IgG2a antibody titers 35 and 62 days after vaccination as adjuvant. Figure 4E Comparison of sucrose, R-DOTAP, or Addavax TM Graph of anti-NG2 antigen-specific IgG1 and IgG2a antibody titers 35 and 62 days after vaccination as adjuvant.

[0027] Figures 5A-5B The ability of bivalent COBRA antigens formulated with R-DOTAP to induce both Th1 and Th2 antibody responses is demonstrated. Figure 5A It is a graph illustrating Th1-specific antibody titers. Figure 5B It is a graph illustrating Th1-specific antibody titers.

[0028] Figures 6A-6C Illustrated are HAI titers at day 35 after vaccination with influenza virus alone or in combination with R-DOTAP nanoparticles. Figure 6A Inactivated influenza vaccine (2011-12 preparation) is a graph of HAI titers when splitting viral hemagglutinin preparations derived from A / California / 07 / 2009X-179A(H1N1). Figure 6B Inactivated influenza vaccine (2011-12 preparation) is a graph of HAI titers when splitting viral hemagglutinin preparations derived from A / Victoria / 210 / 2009X-187 (A / Perth / 16 / 2009-like virus) (H3N2). Figure 6C Inactivated influenza vaccine (2011-12 preparation) is a graph of HAI titers when splitting viral hemagglutinin preparations derived from B / Brisbane / 60 / 2008.

[0029] Figures 7A-7D The effect of the presence of R-DOTAP in the vaccine formulation following vaccination and viral challenge is demonstrated. Figure 7A is a graph illustrating changes in body weight over time after vaccination and infection. Figure 7B is a Kaplan-Mayers plot illustrating survival rates after infection. Figure 7C It is a graph showing the virus titer 3 days after infection. Figure 7D It is a graph showing the virus titer 6 days after infection.

[0030] Figure 8 is a schematic diagram of the ferret model before immunization.

[0031] Figures 9A-9C Results of the H1N1 HAI response are illustrated. Figure 9A is a graph illustrating HAI titers in animals vaccinated with Y2+NG2HA proteins. Figure 9B is a graph illustrating HAI titers in animals vaccinated with Mich / 15+Sing / 16 HA proteins. Figure 9C is shown to collect blood during the experimental timeline to measure Figure 9A and 9B Schematic diagram of the data in .

[0032] Figures 10A-10C Results for the H3N2 HAI response are illustrated. Figure 10A is a graph illustrating HAI titers in animals vaccinated with Y2+NG2 HA proteins. Figure 10B is a graph illustrating HAI titers in animals vaccinated with Mich / 15+Sing / 16 HA proteins. Figure 10C is shown to collect blood during the experimental timeline to measure Figure 10A and 10B Schematic diagram of the data in .

[0033] Figures 11A-11C Further characterization of responses in animals vaccinated with Y2+NG2 HA proteins is described. Figure 11A is a graph illustrating the body weights of animals during the study period. Figure 11B is a graph illustrating viral titers of D3 nasal wash. Figure 11C is shown during the experimental timeline to measure nasal irrigation Figure 11B Schematic diagram of the data in . Summary of the Invention

[0034] The present invention is based on a groundbreaking discovery: the use of cationic lipids as immunomodulators enhances the immunity induced by influenza recombinant antigens in vaccine compositions.

[0035] Before describing the compositions and methods of the present invention, it should be understood that the present invention is not limited to the specific compositions, methods and experimental conditions described, as such compositions, methods and conditions may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the present invention is limited only by the appended claims.

[0036] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "the method" includes one or more methods and / or steps of the type described herein, as will become apparent to those skilled in the art upon reading this disclosure and so forth.

[0037] As used herein, the term "about" in relation to a numerical value is intended to include any additional value that is reasonably close to the numerical value shown. For example, and depending on the context, the value may vary by 5-10%. For example, for a value of about 100, 90 to 110 (or any value between 90 and 110) is intended.

[0038] 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.

[0039] 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 the present invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, it should be understood that modifications and variations are encompassed within the spirit and scope of the present disclosure. Preferred methods and materials are now described.

[0040] Vaccine composition

[0041] In one embodiment, the present invention provides a vaccine composition comprising: one or more non-naturally occurring recombinant influenza antigens; and a cationic lipid.

[0042] As used herein, the term composition is intended to include pharmaceutical compositions, which may also contain other therapeutic agents and can be formulated, for example, by employing conventional pharmaceutically acceptable carriers or diluents and pharmaceutical additives (e.g., excipients, preservatives, etc.) of a type suitable for the desired mode of administration, in accordance with techniques known in the art of pharmaceutical formulation. In certain embodiments, the compositions disclosed herein are formulated with additional agents that promote entry into the desired cells or tissues. Such additional agents include micelles, liposomes, and dendrimers.

[0043] The term "pharmaceutically acceptable" means that the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and will not cause harm to the recipient. For example, the carrier, diluent or excipient or a combination thereof can be administered to a subject together with the conjugate of the present invention without causing any adverse biological effects or interacting in an adverse manner with any other component of the pharmaceutical composition comprising the conjugate.

[0044] The pharmaceutical compositions comprising the peptides or compositions described herein can be administered in any suitable manner, such as parenteral, for example, by subcutaneous, intravenous, intramuscular, intrathecal or intracisternal injection or infusion techniques (e.g., as sterile injectable aqueous or non-aqueous solutions or suspensions), in dosage formulations containing non-toxic, pharmaceutically acceptable carriers or diluents. Depending on the condition being treated, these pharmaceutical compositions can be formulated and administered systemically or topically. The techniques for formulation and administration are well known in the art. Suitable approaches can be, for example, parenteral delivery, including intramuscular, subcutaneous, intramedullary, intrathecal, intracerebroventricular / ventricular, intravenous or intraperitoneal. For injection, the pharmaceutical compositions of the present invention can be formulated in aqueous solutions, preferably in physiologically compatible buffers, such as water, Hanks' solution, Ringer's solution or physiologically buffered saline.

[0045] The compositions of the present invention are "vaccine compositions." As used herein, the term "vaccine" refers to a pharmaceutical formulation (pharmaceutical composition) or product that, upon administration, induces an immune response, particularly a cellular immune response, that recognizes and attacks pathogens or diseased cells (e.g., cancer cells). Vaccines can be used to prevent or treat disease.

[0046] The term "universal influenza vaccine" particularly relates to influenza vaccines and is formulated to provide immune protection against at least two variants of influenza virus. In one aspect, the vaccine composition described herein is a universal influenza vaccine.

[0047] In one aspect, the vaccine compositions described herein provide immune protection against at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 variants of influenza virus. The vaccine compositions described herein provide immune protection against at least 3 variants of influenza virus. The vaccine compositions described herein provide immune protection against at least 4 variants of influenza virus. The vaccine compositions described herein provide immune protection against at least 5 variants of influenza virus. The vaccine compositions described herein provide immune protection against at least 6 variants of influenza virus. The vaccine compositions described herein provide immune protection against at least 7 variants of influenza virus. The vaccine compositions described herein provide immune protection against at least 8 variants of influenza virus. The vaccine compositions described herein provide immune protection against at least 9 variants of influenza virus. The vaccine compositions described herein provide immune protection against at least 10 variants of influenza virus.

[0048] In one aspect, examples of influenza variants include, but are not limited to, H1N1, H3N2, H5N1, and H7N9. In one aspect, the vaccine compositions described herein provide immune protection against at least H1N1 and / or H3N2 hemagglutinin influenza variants.

[0049] The vaccine compositions described herein comprise one or more recombinant influenza antigens.

[0050] In one aspect, the one or more recombinant influenza antigens are recombinant H1N1 and / or H3N2 hemagglutinin influenza proteins.

[0051] 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. Polypeptide as used herein 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 specific polypeptide or peptide is a nucleic acid sequence that is transcribed (with respect to DNA) and translated (with respect to mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by the start codon at the 5' (amino) end and the translation stop codon at the 3' (carboxyl) end. The coding sequence may 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. The transcription termination sequence is typically located at the 3' end of the coding sequence.

[0052] " Antigen " according to the present invention encompasses any substance that triggers an immune response. Specifically, " antigen " refers to any substance that specifically reacts with an antibody or T lymphocyte (T cell), preferably a peptide or protein. According to the present invention, the term " antigen " includes any molecule comprising at least one epitope. Preferably, the antigen in the context of the present invention is a molecule that optionally induces an immune response after processing. According to the present invention, any suitable antigen can be used, which is a candidate for an immune response, wherein the immune response is preferably a cellular immune response. In the context of an embodiment of the present invention, antigens are preferably presented by cells, preferably by antigen-presenting cells, and the cells include diseased cells, particularly cancer cells (in the context of MHC molecules), which result in an immune response against the antigen. Antigen is preferably a product corresponding to or derived from a naturally occurring antigen. Such naturally occurring antigens include tumor antigens.

[0053] In another aspect, the one or more recombinant influenza antigens include a computationally optimized broadly reactive influenza antigen (COBRA) hemagglutinin (HA). The COBRA HA described herein is further described in International Patent Application No. PCT / US2022 / 032799, U.S. Patent No. 9,212,207, and in the scientific publications Allen and Ross (“Bivalent H1 and H3 COBRA Recombinant Hemagglutinin Vaccines Elicit Seroprotectective Antibodies against H1N1 and H3N2 Influenza Viruses from 2009 to 2019”; J. Virology (2022); 96(7)) and Henson et al. (“R-DOTAP Cationic Lipid Nanoparticles Outperform Squalene-Based Adjuvant Systems in Elicitation of CD4 TCells after Recombinant Influenza Hemagglutinin”; Viruses (2023): 15:538), which are incorporated herein by reference in their entirety.

[0054] As used herein, a "non-naturally occurring" peptide or antigen refers to a peptide or antigen that is not found in nature and comprises one or more naturally occurring or non-naturally occurring peptides or antigens combined into a single peptide or antigen.

[0055] In one aspect, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence having at least 80%, 85%, 90% or 95% sequence identity to any one of SEQ ID NOs: 3-22 and combinations thereof. In one aspect, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence having at least 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4. In another aspect, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence of any one of SEQ ID NOs: 3-22 and combinations thereof. In another aspect, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. In another aspect, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence having at least 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 3 and SEQ ID NO: 4. In one aspect, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence of SEQ ID NO: 3 and SEQ ID NO: 4. In one aspect, the one or more non-naturally occurring recombinant influenza antigens comprise the amino acid sequences of SEQ ID NO:3 and SEQ ID NO:4.

[0056] The terms "sequence identity" or "percentage identity" are used interchangeably herein. In order to determine the percentage identity of two polypeptide molecules or two polynucleotide sequences, the sequences are compared to achieve optimal comparison purposes (e.g., a room can be introduced into a first polypeptide or polynucleotide sequence to optimally compare with a second polypeptide or polynucleotide sequence). The amino acids or nucleotides on the corresponding amino acid or nucleotide positions are then compared. When a certain position in the first sequence is occupied by the identical amino acid or nucleotide of the corresponding position in the second sequence, the molecules are identical at that position. The percentage identity between the two sequences depends on the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions (i.e., overlapping positions) x 100). In some embodiments, the length of the reference sequence compared for comparison purposes is at least 80% of the length of the comparison sequence, and in some embodiments, at least 90% or 100%. In one embodiment, the two sequences are identical in length.

[0057] The desired degree of sequence identity ranges from about 80% to 100%, and integer values ​​therebetween. The percent identity between a disclosed sequence and a claimed sequence can be at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9%. Typically, a complete match represents 100% identity over the length of the reference sequence.

[0058] Polypeptides and polynucleotides having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identity to the polypeptides and polynucleotides described herein are encompassed by the present disclosure. For example, a polypeptide can have 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a recombinant COBRA protein sequence described herein.

[0059] 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 in the corresponding portion. A greater degree of homology deviation is permitted if similar amino acids (i.e., conservative amino acid substitutions) are not counted as sequence changes. Examples of conservative substitutions include 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 or leucine.

[0060] In one aspect, the recombinant influenza antigen has a sequence that has at least 80% sequence identity to the sequence of SEQ ID NO: 3. In one aspect, the recombinant influenza antigen has a sequence that has at least 85% sequence identity to the sequence of SEQ ID NO: 3. In one aspect, the recombinant influenza antigen has a sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 3. In one aspect, the recombinant influenza antigen has a sequence that has at least 96% sequence identity to the sequence of SEQ ID NO: 3. In one aspect, the recombinant influenza antigen has a sequence that has at least 97% sequence identity to the sequence of SEQ ID NO: 3. In one aspect, the recombinant influenza antigen has a sequence that has at least 98% sequence identity to the sequence of SEQ ID NO: 3. In one aspect, the recombinant influenza antigen has a sequence that has at least 99% sequence identity to the sequence of SEQ ID NO: 3.

[0061] In one aspect, the recombinant influenza antigen has a sequence that has at least 80% sequence identity to the sequence of SEQ ID NO: 4. In one aspect, the recombinant influenza antigen has a sequence that has at least 85% sequence identity to the sequence of SEQ ID NO: 4. In one aspect, the recombinant influenza antigen has a sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 4. In one aspect, the recombinant influenza antigen has a sequence that has at least 96% sequence identity to the sequence of SEQ ID NO: 4. In one aspect, the recombinant influenza antigen has a sequence that has at least 97% sequence identity to the sequence of SEQ ID NO: 4. In one aspect, the recombinant influenza antigen has a sequence that has at least 98% sequence identity to the sequence of SEQ ID NO: 4. In one aspect, the recombinant influenza antigen has a sequence that has at least 99% sequence identity to the sequence of SEQ ID NO: 4.

[0062] The vaccine compositions described herein comprise cationic lipids.

[0063] Adjuvants are an essential part of subunit vaccines and are added to enhance the immune response to the antigen through immunomodulatory effects. Due to safety issues, very few adjuvants have been approved by regulatory agencies for use in humans. Currently approved subunit vaccine adjuvants for use in humans are very effective in promoting humoral immune responses but less effective in promoting T cell immunity. In this study, a novel pure enantiospecific cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (R-DOTAP) was evaluated as an immunomodulatory agent for subunit vaccines capable of inducing both humoral and cell-mediated immunity. Using recombinant protein antigens derived from a computationally optimized broadly reactive influenza antigen (COBRA) protein, it was demonstrated that R-DOTAP nanoparticles promoted robust cellular and antibody-mediated immune responses in both monovalent and bivalent vaccines. As discussed further below, R-DOTAP-based vaccines induced antigen-specific and multifunctional CD8 + and CD4 + R-DOTAP induced antibody responses that demonstrated balanced Th1 / Th2 immunity, neutralizing activity, and protection against live influenza virus challenge in mice. R-DOTAP also significantly reduced the dose of vaccine antigen. These studies demonstrate that R-DOTAP is an excellent immunostimulant for the production of next-generation subunit vaccines containing multiple recombinant proteins.

[0064] Adjuvants are generally used to regulate or enhance the effect of vaccines by stimulating the immune system to produce a stronger response to vaccines, thereby providing increased immunity to specific diseases. Adjuvants achieve this function by simulating a specific set of evolutionarily conserved molecules (so-called pathogen-associated molecular patterns), including liposomes, lipopolysaccharides, antigenic molecular cages, bacterial cell wall components, and endocytosed nucleic acids, such as RNA, double-stranded RNA, single-stranded DNA, and DNA containing unmethylated CpG dinucleotides. Since the immune system has evolved to recognize these specific antigenic parts, the presence of adjuvants combined with vaccines can enhance the activity of dendritic cells, lymphocytes, and macrophages by simulating natural infection, thereby significantly increasing the innate immune response to antigens.

[0065] The compositions described herein can be formulated with lipid nanoparticles, which act as adjuvants to enhance the presentation of antigens to antigen-presenting cells, thereby increasing the antigen-induced immune response.

[0066] In some aspects described herein, the adjuvant is a cationic lipid. As used herein, the term "cationic lipid" refers to any of a variety of lipid species that have a net positive charge at physiological pH, or have a protonatable group and are positively charged at a pH below the pKa.

[0067] Suitable cationic lipids according to the present disclosure include, but are not limited to: 3-β[4N1N,8-biguanidinospermidine)-carbamoyl]cholesterol (BGSC); 3-β[N,N-biguanidinoethyl-aminoethane)-carbamoyl]cholesterol (BGTC); N,N.1N2N3tetramethyltetrapalmitylspermidine (cellfectin); N-tert-butyl-N'-tetradecyl-3-tetradecyl-aminopropionamidine (CLONfectin); dimethyl dioctadecyl ammonium bromide (DDAB); 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE); 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanium trifluoroacetate (DOSPA); 1,3-dioleoyloxy-2-(6-carboxysperminyl)-propylamide (DOSPER); 4-(2,3-bis-palmitoyloxypropyl)-1-methyl-1H-amidine 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'-trimethylammonium)butanol-s n-glycerol (DOBT) or cholesterol (4'trimethylammonium) butyrate (ChOTB), wherein the trimethylammonium group is linked to the duplex (for DOTB) or cholesterol group (for ChOTB) via a butanol spacer; 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-glycerocholine ester (DOSC); cholesterol hemisuccinate (ChOSC);Lipopolyamines, such as dioctadecylamido glycyl spermine (DOGS) and dipalmitoylphosphatidylethanolpentyl spermine (DPPES), cholesterol-3β-carboxyamido-ethylenetrimethylammonium iodide, 1-dimethylamino-3-trimethylammonium-DL-2-propyl-cholesterol formate iodide, cholesterol-3-O-carboxyamidoethyleneamine, cholesterol-3-β-oxysuccinylamino-ethylenetrimethylammonium iodide, 1-dimethylamino-3-trimethylammonium-DL-2-propyl- Cholesterol-3-β-oxysuccinate iodide, 2-(2-trimethylammonium)-ethylmethylaminoethyl-cholesterol-3-β-oxysuccinate iodide, 3-β-N-(N',N'-dimethylaminoethane)carbamoylcholesterol (DC-chol), and 3-β-N-(polyethyleneimine)carbamoylcholesterol; O,O'-dimyristyl-N-lysylaspartate (DMKE); O,O'-dimyristyl-N-lysylglutamate (D MKD); 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-trimethylammonium propane (DPTAP); 1,2-distearoyl-3-trimethylammonium propane (DSTAP); 1,2-myristoyl-3-trimethylammonium propane (DMTAP); and sodium dodecyl sulfate (SDS). Furthermore, structural variants and derivatives of any of the cationic lipids are contemplated.

[0068] In some aspects, the cationic lipid is selected from DOTAP, DOTMA, DOEPC and combinations thereof. In other aspects, the cationic lipid is DOTAP. In yet other aspects, the cationic lipid is DOTMA. In other aspects, the cationic lipid is DOEPC. In some aspects, the cationic lipid is purified.

[0069] In some embodiments, the cationic lipid is the enantiomer of a cationic lipid. The term "enantiomer" refers to a stereoisomer of a cationic lipid, which is a non-superimposable mirror image of its corresponding stereoisomer, such as an R and S enantiomer. In a plurality of examples, the enantiomer is R-DOTAP or S-DOTAP. In one example, the enantiomer is R-DOTAP. In another example, the enantiomer is S-DOTAP. In some aspects, the enantiomer is purified.

[0070] In one aspect, the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, variants thereof, or analogs thereof.

[0071] In another aspect, the one or more recombinant influenza antigens are encapsulated in liposomes comprising cationic lipids. In another aspect, the one or more recombinant influenza antigens are mixed with preformed cationic lipid nanoparticles.

[0072] In some aspects, the one or more recombinant protein antigens and preformed cationic lipid nanoparticles are mixed in a 1:1 ratio.

[0073] In one aspect, the one or more recombinant influenza antigens are present as micelles separate from the cationic lipid nanoparticles.

[0074] How to use

[0075] In another embodiment, the present invention provides a method of inducing an immune response against influenza virus 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 inducing an immune response against influenza virus in the subject.

[0076] The term "immune response" refers to the overall body response 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.

[0077] The immune system is a system of biological structures and processes that defend an organism against disease. This system is a diffuse, complex network of interacting cells, cellular products, and cell-formed tissues that protect the body from pathogens and other foreign substances, destroy infected and malignant cells, and clear cellular debris. This system 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 part of the adaptive immune system and are crucial for immune surveillance. T cells, or T lymphocytes, are a type of lymphocyte that plays a central role in cell-mediated immunity. There are two main subtypes of T cells: killer T cells and helper T cells. In addition, there are suppressor T cells, which play a role in regulating immune responses. Killer T cells only recognize antigens coupled to class I MHC molecules, while helper T cells only recognize antigens coupled to class II MHC molecules. These two antigen presentation mechanisms reflect the distinct roles of the two T cell types. A third, minor subtype, γδ T cells, recognize intact antigens that are not bound to MHC receptors. In contrast, B cell antigen-specific receptors are antibody molecules on the surface of B cells and recognize pathogens in their entirety without the need for antigen processing. Each B cell lineage expresses a different antibody, so the entire repertoire of B cell antigen receptors represents all the antibodies the body is capable of making.

[0078] "Cellular immune response", "cellular response", "cellular response to an antigen" or similar terms are intended to include cellular responses against cells characterized by presenting an antigen via MHC class I or class II. This cellular response involves cells called T cells or T lymphocytes, which act as "helper cells" or "killer cells". 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 diseased cells, such as cancer cells, thereby preventing the generation of more diseased cells. In a preferred embodiment, the present invention is directed to stimulating an anti-tumor CTL response against tumor cells that express one or more tumor-expressed antigens and preferably present such tumor-expressed antigens via MHC class I.

[0079] In the context of the present invention, the terms "immunoreactive cells", "immune cells" or "immune effector cells" refer to cells that perform effector functions during an immune response. "Immunoreactive cells" are preferably cells that are capable of binding to antigens or presenting antigenic peptides derived from antigens, and mediate an immune response. For example, such cells secrete cytokines and / or chemokines, secrete antibodies, recognize cancer cells, and optionally remove such cells. For example, immunoreactive cells include T cells (cytotoxic T cells, helper T cells, tumor infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages and dendritic cells.

[0080] "Inducing an immune response" may mean that there is no immune response to a specific antigen before induction, but may also mean that there is a certain level of immune response to a specific antigen before induction, and that the immune response is enhanced after induction. Therefore, "inducing an immune response" also includes "enhancing an immune response." Preferably, after inducing an immune response in a subject, the subject is protected from developing a disease, such as influenza, or the disease condition is improved by inducing an immune response. For example, an immune response against influenza antigens can be induced in a subject at risk of influenza virus infection. In this case, inducing an immune response may mean that the subject's disease condition is improved, or that the subject does not develop influenza.

[0081] In one aspect, the immune response includes CD8 + Effector T cells, CD4 + Induction of effector and memory T cells.

[0082] In one aspect, inducing CD8+ and CD4+ effector T cells 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.

[0083] In another aspect, inducing a humoral immune response comprises inducing the production of IgG in the subject. In some aspects, IgG comprises IgG1 and IgG2a.

[0084] In one aspect, inducing an immune response comprises inducing the secretion of broadly neutralizing antibodies.

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

[0086] In another aspect, the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, a variant thereof, or an analog thereof. In one aspect, the cationic lipid is R-DOTAP. In another aspect, the one or more recombinant influenza antigens are encapsulated in liposomes comprising cationic lipids. In another aspect, the one or more recombinant influenza antigens are mixed with preformed cationic lipid nanoparticles. In some aspects, the one or more recombinant influenza antigens are mixed with preformed cationic lipid nanoparticles in a ratio of 1: 1. In one aspect, the one or more recombinant influenza antigens exist as micelles separated from the cationic lipid nanoparticles.

[0087] In another embodiment, a method of preventing or treating influenza infection in a subject comprises 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 influenza infection in the subject.

[0088] In one aspect, the one or more non-naturally occurring recombinant influenza antigens include a computationally optimized broadly reactive influenza antigen (COBRA) hemagglutinin (HA). In another aspect, the one or more non-naturally occurring recombinant influenza antigens are recombinant H1N1 and / or H3N2 hemagglutinin influenza proteins. In one aspect, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence with at least 80%, 85%, 90% or 95% sequence identity to any one of SEQ ID NO: 3-22 and a combination thereof. In one aspect, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence with at least 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4. In another aspect, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence of any one of SEQ ID NO: 3-22 and a combination thereof. In another aspect, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. In another aspect, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence having at least 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 3 and SEQ ID NO: 4. In one aspect, the one or more non-naturally occurring recombinant influenza antigens comprise the amino acid sequence of SEQ ID NO: 3 and SEQ ID NO: 4. In one aspect, the one or more non-naturally occurring recombinant influenza antigens comprise the amino acid sequence of SEQ ID NO: 3 and SEQ ID NO: 4.

[0089] In another aspect, the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, a variant thereof, or an analog thereof. In one aspect, the cationic lipid is R-DOTAP. In another aspect, the one or more recombinant influenza antigens are encapsulated in liposomes comprising cationic lipids. In another aspect, the one or more recombinant influenza antigens are mixed with preformed cationic lipid nanoparticles. In some aspects, the one or more recombinant influenza antigens are mixed with preformed cationic lipid nanoparticles in a ratio of 1: 1. In one aspect, the one or more recombinant influenza antigens exist as micelles separated from the cationic lipid nanoparticles.

[0090] 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.

[0091] In some aspects, administration can be combined with one or more other therapeutic agents. The terms "combination therapy," "in combination with," etc. refer to the simultaneous use of more than one drug or treatment to increase response. For example, the cationic lipids of the present invention can be used in combination with existing influenza vaccines to increase the immune response produced by influenza vaccines alone. The cationic lipids can be used before, simultaneously with, or after the administration of influenza vaccines.

[0092] "Enhanced immunogenicity" means that when an influenza vaccine is administered in combination with the cationic lipid of the present invention, its immunogenicity is higher than that induced by administration of the influenza vaccine alone (eg, without administration of the cationic lipid as an immunomodulator).

[0093] In one aspect, the influenza vaccine is an inactivated influenza vaccine, an attenuated influenza vaccine, or a recombinant influenza vaccine.

[0094] Vaccines typically contain attenuated, inactivated, or killed organisms, or purified products thereof. Several types of vaccines are in use, representing different strategies for attempting to reduce the risk of illness while retaining the ability to induce a beneficial immune response. Influenza vaccines are typically "attenuated," "inactivated," or "subunit" (e.g., recombinant).

[0095] Live, attenuated microorganisms, such as live viruses grown under conditions that have lost their virulence properties or the use of closely related but less dangerous organisms to produce a broad immune response constitute attenuated vaccines. While most attenuated vaccines are viral, some are bacterial in nature.

[0096] Inactivated vaccines consist of "ghosts" of previously virulent microorganisms that have been destroyed by chemicals, heat, or radiation. These have an intact but empty bacterial cell envelope and are considered an intermediate between inactivated and attenuated vaccines. Examples include IPV (polio vaccine), hepatitis A vaccine, rabies vaccine, and most influenza vaccines.

[0097] Instead of introducing the inactivated or attenuated microorganism into the immune system (as in "whole-agent" vaccines), subunit vaccines use fragments of the microorganism to elicit an immune response. Only one viral protein (previously extracted from the serum of chronically infected patients but now produced by recombinantly inserting viral genes into yeast), such as the surface protein, is recombinantly produced and used in the vaccine. The hemagglutinin and neuraminidase subunits of influenza virus are examples of subunit proteins used in recombinant influenza vaccines.

[0098] In another aspect, the influenza vaccine is a monovalent vaccine, a bivalent vaccine, a trivalent vaccine, or a quadrivalent vaccine.

[0099] In one aspect, the one or more non-naturally occurring recombinant influenza antigens include a computationally optimized broadly reactive influenza antigen (COBRA) hemagglutinin (HA). In another aspect, the one or more non-naturally occurring recombinant influenza antigens are recombinant H1N1 and / or H3N2 hemagglutinin influenza proteins. In one aspect, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence with at least 80%, 85%, 90% or 95% sequence identity to any one of SEQ ID NO: 3-22 and a combination thereof. In one aspect, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence with at least 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4. In another aspect, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence of any one of SEQ ID NO: 3-22 and a combination thereof. In another aspect, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. In another aspect, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence having at least 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 3 and SEQ ID NO: 4. In one aspect, the one or more non-naturally occurring recombinant influenza antigens comprise the amino acid sequence of SEQ ID NO: 3 and SEQ ID NO: 4. In one aspect, the one or more non-naturally occurring recombinant influenza antigens comprise the amino acid sequence of SEQ ID NO: 3 and SEQ ID NO: 4.

[0100] In another aspect, the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, a variant thereof, or an analog thereof. In one aspect, the cationic lipid is R-DOTAP. In another aspect, the one or more recombinant influenza antigens are encapsulated in liposomes comprising cationic lipids. In another aspect, the one or more recombinant influenza antigens are mixed with preformed cationic lipid nanoparticles. In some aspects, the one or more recombinant influenza antigens are mixed with preformed cationic lipid nanoparticles in a ratio of 1: 1. In one aspect, the one or more recombinant influenza antigens exist as micelles separated from the cationic lipid nanoparticles.

[0101] 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

[0102] In another embodiment, the present invention provides a method of inducing secretion of broadly neutralizing antibodies against influenza virus 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 inducing secretion of broadly neutralizing antibodies against influenza virus in the subject.

[0103] The development of a universal influenza vaccine that can provide heterosubtypic immunity and provide protection against multiple evolutionary branches has always been a long-term goal for providing protection against influenza infection. To achieve this goal, a variety of strategies are under study, including using multiple subtype antigens to increase coverage, targeting multiple proteins (e.g., including HA and non-HA proteins) as antigens, targeting conserved antigenic regions of influenza antigens, using chimeric proteins containing stems and handles of different subtypes of HA, and using consensus sequence-based methods, such as COBRA sequences containing multiple known mutations in hemagglutinin or neuraminidase. In addition, vaccine technologies that induce CD8 T cells and antibody-mediated immunity are also being actively explored to achieve heterosubtypic protection against influenza. As described herein, a prototype vaccine based on the R-DOTAP platform and HA or nucleoprotein antigens derived from COBRA H1N1 and H3N2 was used, which was able to induce antigen-specific T cell responses, neutralizing antibodies against multiple evolutionary branches of H1N1 and H3N2 strains, and protect mice from the attack of lethal H1N1 strains. Therefore, vaccine formulations containing R-DOTAP and COBRAHA, NA sequences, and nucleoproteins from influenza have great potential to advance the goal of developing a safe and effective universal influenza vaccine.

[0104] The term "broadly neutralizing antibody" or "bNAb," as used herein, refers to neutralizing antibodies that neutralize multiple influenza virus strains. bNAbs are unique in that they target conserved epitopes on the virus, meaning that the virus may mutate but the targeted epitope remains. In contrast, non-bNAbs are specific for a single viral strain with a unique epitope.

[0105] In one aspect, the one or more non-naturally occurring recombinant influenza antigens include a computationally optimized broadly reactive influenza antigen (COBRA) hemagglutinin (HA). In another aspect, the one or more non-naturally occurring recombinant influenza antigens are recombinant H1N1 and / or H3N2 hemagglutinin influenza proteins. In one aspect, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence with at least 80%, 85%, 90% or 95% sequence identity to any one of SEQ ID NO: 3-22 and a combination thereof. In one aspect, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence with at least 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4. In another aspect, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence of any one of SEQ ID NO: 3-22 and a combination thereof. In another aspect, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. In another aspect, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence having at least 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 3 and SEQ ID NO: 4. In one aspect, the one or more non-naturally occurring recombinant influenza antigens comprise the amino acid sequence of SEQ ID NO: 3 and SEQ ID NO: 4. In one aspect, the one or more non-naturally occurring recombinant influenza antigens comprise the amino acid sequence of SEQ ID NO: 3 and SEQ ID NO: 4.

[0106] In another aspect, the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, a variant thereof, or an analog thereof. In one aspect, the cationic lipid is R-DOTAP. In another aspect, the one or more recombinant influenza antigens are encapsulated in liposomes comprising cationic lipids. In another aspect, the one or more recombinant influenza antigens are mixed with preformed cationic lipid nanoparticles. In some aspects, the one or more recombinant influenza antigens are mixed with preformed cationic lipid nanoparticles in a ratio of 1: 1. In one aspect, the one or more recombinant influenza antigens exist as micelles separated from the cationic lipid nanoparticles.

[0107] In a further embodiment, the present invention provides a method of inducing a balanced Th1 / Th2 immune response 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 inducing the secretion of broadly neutralizing antibodies against influenza virus in the subject.

[0108] Based on the cytokine and antibody subclasses induced by vaccines, adjuvants can be broadly classified as Th1, Th2, Th17, and mixed Th1 / Th2 or Th1 / Th17 types. For example, Th1 adjuvants favor IFN-γ production and IgG2a / c antibody subclasses in mouse vaccination. Th2 adjuvants stimulate greater IL-4 production, resulting in a preference for the IgG1 antibody subclass in mice.

[0109] 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.

[0110] In some aspects, IFNγ-producing CD8+ effector T cells are associated with IgG2a production, while IL-4-producing CD4+ effector T cells are associated with IgG1 production.

[0111] In one aspect, administering the vaccine composition to the subject comprises subcutaneous administration or intramuscular administration.

[0112] The term "subject" as used herein refers to any individual or patient on whom the present method is performed. Typically, the subject is a human, but it will be understood by those skilled in the art that the subject can be an animal. Thus, other animals, including vertebrates, such as rodents (including mice, rats, hamsters, and guinea pigs), cats, dogs, rabbits, farm animals (including cattle, horses, goats, sheep, pigs, chickens, etc.), and primates (including monkeys, chimpanzees, orangutans, and gorillas) are included within the definition of a subject.

[0113] The term "administration of" or "administering" is to be understood as 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, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal, oral, sublingual, buccal, rectal, vaginal, nasal, ophthalmic administration, as well as infusion, inhalation, and aerosolization. Preferably, the vaccine compositions described herein are administered by subcutaneous or intramuscular administration.

[0114] In another aspect, administering the vaccine composition comprises administering two doses of the vaccine, and optionally administering a booster.

[0115] In one aspect, the one or more non-naturally occurring recombinant influenza antigens include a computationally optimized broadly reactive influenza antigen (COBRA) hemagglutinin (HA). In another aspect, the one or more non-naturally occurring recombinant influenza antigens are recombinant H1N1 and / or H3N2 hemagglutinin influenza proteins. In one aspect, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence with at least 80%, 85%, 90% or 95% sequence identity to any one of SEQ ID NO: 3-22 and a combination thereof. In one aspect, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence with at least 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4. In another aspect, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence of any one of SEQ ID NO: 3-22 and a combination thereof. In another aspect, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. In another aspect, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence having at least 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 3 and SEQ ID NO: 4. In one aspect, the one or more non-naturally occurring recombinant influenza antigens comprise the amino acid sequence of SEQ ID NO: 3 and SEQ ID NO: 4. In one aspect, the one or more non-naturally occurring recombinant influenza antigens comprise the amino acid sequence of SEQ ID NO: 3 and SEQ ID NO: 4.

[0116] In another aspect, the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, a variant thereof, or an analog thereof. In one aspect, the cationic lipid is R-DOTAP. In another aspect, the one or more recombinant influenza antigens are encapsulated in liposomes comprising cationic lipids. In another aspect, the one or more recombinant influenza antigens are mixed with preformed cationic lipid nanoparticles. In some aspects, the one or more recombinant influenza antigens are mixed with preformed cationic lipid nanoparticles in a ratio of 1: 1. In one aspect, the one or more recombinant influenza antigens exist as micelles separated from the cationic lipid nanoparticles.

[0117] In another embodiment, the present invention provides a method of inducing a polyfunctional CD4+ / CD8+ T cell response against influenza virus in a subject, 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.

[0118] In one aspect, inducing a multifunctional CD4+ / CD8+ T cell response comprises inducing the secretion of two or more cytokines. In some aspects, the two or more cytokines are selected from IFNγ, granzyme B, and IL-4.

[0119] In one aspect, the one or more non-naturally occurring recombinant influenza antigens include a computationally optimized broadly reactive influenza antigen (COBRA) hemagglutinin (HA). In another aspect, the one or more non-naturally occurring recombinant influenza antigens are recombinant H1N1 and / or H3N2 hemagglutinin influenza proteins. In one aspect, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence with at least 80%, 85%, 90% or 95% sequence identity to any one of SEQ ID NO: 3-22 and a combination thereof. In one aspect, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence with at least 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4. In another aspect, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence of any one of SEQ ID NO: 3-22 and a combination thereof. In another aspect, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. In another aspect, the one or more non-naturally occurring recombinant influenza antigens comprise an amino acid sequence having at least 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 3 and SEQ ID NO: 4. In one aspect, the one or more non-naturally occurring recombinant influenza antigens comprise the amino acid sequence of SEQ ID NO: 3 and SEQ ID NO: 4. In one aspect, the one or more non-naturally occurring recombinant influenza antigens comprise the amino acid sequence of SEQ ID NO: 3 and SEQ ID NO: 4.

[0120] In another aspect, the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, a variant thereof, or an analog thereof. In one aspect, the cationic lipid is R-DOTAP. In another aspect, the one or more recombinant influenza antigens are encapsulated in liposomes comprising cationic lipids. In another aspect, the one or more recombinant influenza antigens are mixed with preformed cationic lipid nanoparticles. In some aspects, the one or more recombinant influenza antigens are mixed with preformed cationic lipid nanoparticles in a ratio of 1: 1. In one aspect, the one or more recombinant influenza antigens exist as micelles separated from the cationic lipid nanoparticles.

[0121] Provided below are examples of vaccine combinations for discussion purposes, comprising influenza recombinant antigens and cationic lipids. The following examples are intended to further illustrate embodiments of the present invention, but are not intended to limit the scope of the invention. Although these examples are typical examples that may be used, other procedures, methods, or techniques known to those skilled in the art may also be used alternatively. Example

[0122] Example 1

[0123] Materials and Methods

[0124] 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 Laboratories. All animals were housed under specific pathogen-free conditions at the Department of Laboratory Animal Resources (DLAR) at the University of Kentucky Medical Center or the University of Georgia Animal Research Center. Animal care guidelines of the National Institutes of Health (A2020 02-024-Y1-A5, A201806-018-Y3-A16) were followed, and all animal experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committees at the University of Kentucky (2019-3226) or the University of Georgia. All studies were performed in accordance with the ARRIVE guidelines.

[0125] For influenza challenge studies, A / Brisbane / 02 / 2018 (Bris / 18) was used. For HAI determinations, A / California / 07 / 2009 (Cal / 09), A / Guangdong-Maonan / SWL1536 / 2019 (GD19), A / Singapore / IFNIMH-16-00192016 (Singapore / 16), A / Hong Kong / 4801 / 2014 (Hong Kong / 14), A / Victoria / 210 / 2009X-187 (A / Perth / 16 / 2009-like virus) (H3N2) and B / Brisbane / 60 / 2008 were used. Influenza viruses were obtained from the International Influenza Resource (IRR).

[0126] Reagents and antibodies:

[0127] cGMP-grade R-DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) was provided by Merck & Cie. Evonik produced cGMP-grade R-DOTAP liposomal nanoparticles according to a previously described protocol.

[0128] Influenza COBRA antigens: COBRA HA-Y2 (COBRA-Y2) (H1N1) and COBRA-HA-NG2 (COBRA-NG2) (H3N2) were synthesized at the Vaccine and Immunology Research Center at the University of Georgia. Influenza nucleoprotein from A / Puerto Rico / 8 / 34 / Mount Sinai was obtained from Sino Biologicals, Inc., USA. Vaccine preparations were obtained from the University of Kentucky Health Care Pharmacy.

[0129] Overlapping peptide libraries from influenza A / New York / 383 / 2005 (H3N2) hemagglutinin protein and overlapping peptide libraries from influenza A / California / 07 / 2009 (H1N1) pdm09 were obtained from BEI Resources.

[0130] 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.

[0131] Preparation of R-DOTAP nanoparticles and vaccine formulations:

[0132] cGMP-grade R-DOTAP liposomal nanoparticles were produced by Evonik using a 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 using a steady stream of dry nitrogen and then vacuum dried overnight. The dried R-DOTAP film was then incubated in water for 12 hours to allow it to hydrate. The lipid suspension was then sonicated for 10 minutes and extruded using 400, 200, and 100 nm polycarbonate membrane filters, sequentially, to obtain liposomal nanoparticles of uniform size ( Figures 1A-1B ). Groups of BALB / cJ (n=6-8) mice were immunized with two doses of monovalent COBRA-Y2 formulated with R-DOTAP nanoparticles or sucrose buffer (sucrose) on days 0 and 21. 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). The data represent the (ab) mean ± SEM of the half-maximal titer for each mouse. Comparisons between sucrose or R-DOTAP groups alone were performed using the Student t-test (unpaired two-tailed) **P≤0.05. The nanoparticles were then diluted in 280mM sucrose buffer and stored at -80°C until use. To prepare the vaccine formulation, the concentrated antigen dissolved in PBS buffer was diluted to the desired concentration in 280mM sucrose. Prior to vaccination, the vaccine components were brought to room temperature and then 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 per dose; for intramuscular delivery, 50 μl was used per dose.

[0133] Physical Characterization of Vaccine Formulations:

[0134] 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 4mW 632.8nm laser (angle set to 90°). Dynamic light scattering was used to measure the fluctuation of the scattered light intensity. Distribution analysis and cumulative analysis were performed according to the instructions and using the instrument software to measure the Z mean and polydispersity. Representative particle size distributions are shown in Figure 2. Figures 2A-2C The average particle size, polydispersity and zeta potential measurements are shown in Table 1.

[0135] Enzyme-linked immunosorbent assay (ELISA):

[0136] Blood was collected from euthanized mice by tail vein sampling or cardiac puncture. Serum separation tubes. The separated serum samples were stored at -80°C until analysis. For antibody titer measurement, 96-well plates were coated with 50 μl / well recombinant RBD protein, COBRA-NG2 or COBRA-Y2 protein (at a concentration of 2 μg / ml) at 4°C overnight. After antigen coating, the wells were blocked for 1-2 hours at room temperature with 200 μl / well PBS-T buffer containing 3% skim milk powder and 0.1% Tween-20. After blocking, the buffer was replaced with serum samples diluted in PBS-T buffer containing 1% skim milk powder. After incubation at room temperature for 2 hours, the serum was removed and the wells were washed four times with PBS-T buffer. For detection of protein antibodies, wells were incubated for 1 hour with 100 μl of PBS-T buffer containing 1% skim milk powder and HRP-conjugated anti-mouse IgG (1:5000) (cat#115-035-003; Jackson ImmunoResearch), anti-mouse IgG1 (1:5000) (cat#115-035-205; Jackson ImmunoResearch), anti-mouse IgG2c (1:5000) (Cat#115-035-206; Jackson ImmunoResearch), or anti-mouse IgG2a (1:5000) (Cat#115-035-206; Jackson ImmunoResearch). The wells were then washed four times, and 100 μl of SIGMAFAST OPD (o-phenylenediamine dihydrochloride) chromogenic substrate was added to each well. After incubation for 10 minutes, the reaction was stopped by adding 50 μl of 3 M HCL, and the absorbance at 490 nm (OD490) was measured using a spectraMax M5 microplate reader.

[0137] Enzyme-linked immunospot assay (ELISpot):

[0138] 2.5 x 10 5 Treated splenocytes were stimulated with target T cell epitope peptides or recombinant proteins or no peptide (control) at 37°C for 18-24 hours. After stimulation, the wells were washed with PBS and incubated with biotin-conjugated anti-IFNγ or IL-4 antibodies, followed by incubation with streptavidin-HRP antibodies. To observe antigen-specific IFNγ or IL-4 producing cells, the 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 Ver.6 software. Spot counts were summarized as the median of three replicates. Each sample had unstimulated and PMA / ionomycin control wells to detect background or as a positive control.

[0139] Intracellular cytokine and cell surface staining:

[0140] For intracellular protein analysis, single-cell suspensions of splenocytes were stimulated with the indicated stimulatory antigenic peptides in cRPMI medium supplemented with purified anti-mouse CD28 (2 μg / ml), protein transport inhibitor Brefeldin A (5 μg / ml), and monensin (2.0 μM) at 37°C for 6 hours. After stimulation, the cells were washed with FACS buffer and stained with fluorochrome-conjugated anti-mouse CD3, CD4, CD8, CD44, and CD62L antibodies. The 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, the cells were washed with FACS buffer and immediately analyzed by flow cytometry.

[0141] Vaccination of mice:

[0142] For all injections and implantations, mice were anesthetized with isoflurane. Before subcutaneous or intramuscular injection of the formulation, the injection site was shaved and cleaned with 70% ethanol. For subcutaneous (SC) inoculation, a 100 μl dose was delivered to the unilateral flank of the hind limb; for intramuscular (IM) inoculation, a 50 μl dose was delivered to the hind limb thigh muscle. To prepare R-DOTAP-based vaccine formulations, R-DOTAP nanoparticles (4-6 mg / ml) in 280 mM sucrose buffer were mixed 1:1 with a recombinant protein at a specified concentration resuspended in 280 mM sucrose buffer. For antigen-only vaccine formulations, the recombinant protein was resuspended in 280 mM sucrose buffer at the desired concentration. All vaccination regimens consisted of two doses delivered at intervals of 1-4 weeks.

[0143] Hemagglutination inhibition assay:

[0144] The hemagglutination inhibition (HAI) assay is used to assess functional HA antibodies capable of inhibiting the agglutination of guinea pig erythrocytes for H3N2 viruses and turkey erythrocytes for H1N1 viruses. This protocol was adapted from the World Health Organization (WHO) Laboratory Influenza Surveillance Manual. Guinea pig erythrocytes are commonly used to characterize contemporary influenza A(H3N2) strains, which 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) prior to testing. Briefly, three parts RDE were added to one part serum and incubated overnight at 37°C. RDE was inactivated by incubation at 56°C for 30 minutes.

[0145] RDE-treated sera were diluted in a series of two-fold serial dilutions in a V-bottom microtiter plate. 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 plate was sealed and incubated at room temperature for 30 minutes, followed by the addition of 0.75% guinea pig red blood cells in PBS. Before use, red blood cells (RBCs) were washed twice with PBS, stored at 4°C, and used within 24 hours of preparation. The plate was mixed by gentle stirring, sealed, and the RBCs were allowed to settle at room temperature for 1 hour. The HAI titer was determined by the reciprocal dilution of the last well containing unagglutinated RBCs. Positive and negative serum controls were included for each plate.

[0146] In a separate assay, RDE-treated serum was diluted in a series of two-fold serial dilutions in a V-bottom microtiter plate. An equal volume of each influenza virus (adjusted to approximately 8 hemagglutination units (HAU) / 50 μl) was added to each well. The plate was sealed and incubated with red blood cells in phosphate-buffered saline (PBS) at room temperature for 20 minutes. Before use, the RBCs were washed twice with PBS, stored at 4°C, and used within 24 hours of preparation. The plate was mixed by gentle stirring, sealed, and the RBCs were allowed to settle at room temperature for 30 minutes. The HAI titer was determined by the reciprocal dilution of the last well containing unagglutinated RBCs. For each plate, positive and negative serum controls were included.

[0147] All mice were negative for existing antibodies to human influenza virus (HAI ≤ 1:10) before infection or vaccination, and for this study, seroprotection was defined as an HAI titer > 1:40 and seroconversion was defined as a titer 4-fold higher than baseline, according to the WHO and European Medicines Commission (ECMA) for influenza vaccine evaluation. All mice were naive and seronegative at the time of vaccination, so for this study, seroconversion and seroprotection rates were used interchangeably.

[0148] Mouse challenge experiment:

[0149] For influenza virus challenge studies, DBA / 2J mice (female, 7 to 9 weeks old) were immunized intramuscularly with the indicated vaccine formulations on days 0 and 28. On day 56, they were intranasally challenged with H1N1 A / Brisbane / 02 / 2018 (Bris / 18) influenza virus at 10× LD 50 Dose of 3.6*10 6pfu / mouse was attacked with a 50 μL volume. Animals mock-vaccinated were inoculated intranasally with 50 μL PBS. For all animals attacked with live virus, weight loss and clinical signs (dyspnea, lethargy, hunchback, ruffled hair, no response to stimulation, and severe respiratory distress) were monitored twice daily, morning and evening, for 14 days after infection. Body weight was closely monitored until 14 days after infection. Once mice lost 20% of their original body weight or reached the clinical endpoint, they were humanely euthanized. On the 3rd and 6th days after infection, the lungs of influenza-challenged mice were collected from 3 pre-selected mice in each group for viral titer detection. In short, frozen lungs were processed, and the clarified supernatant containing the virus was added to 90% confluent MDCK cells and incubated for 1 hour. After this step, the cells were washed and supplemented with culture medium containing 1.6% agarose. After incubation at 37°C for 72 hours, the plates were processed, dried, and viral plaques were counted according to the plaque-forming units per gram of lung tissue.

[0150] Equipment, software, and statistical analysis:

[0151] Flow cytometry was performed using a BD FACSDiva TM All flow cytometric data were performed using a BDSymphony A3 flow cytometer with the software. Statistical analyses were performed using GraphPad Prism 9.0 software, version 10.0. Statistical analyses for all other studies were performed using GraphPad Prism 9.0 software, and means were compared using simple Student's T-tests or ANOVA with Tukey's multiple comparison correction. Mantel-Cox tests were used for survival curves.

[0152] Example 2

[0153] Immunogenicity of recombinant influenza proteins formulated with R-DOTAP nanoparticles

[0154] 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, nucleoprotein was formulated with R-DOTAP nanoparticles and B6 mice were immunized with two doses of vaccine; b Binds to CD8 T cell epitope (NP366-74:ASNENMETM, SEQ ID NO:1) and validated IA b T cell immune responses were assessed by binding to a CD4 T cell epitope (NP-311-25: QVYSLIRPNENPAHK, SEQ ID NO: 2).

[0155] In the 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 doses intramuscularly, and T cell responses were measured using an IFN-γ ELISpot assay 7 days after the booster dose.

[0156] To stimulate antigen-specific T cells in the ELISpot assay, either the complete COBRA protein or overlapping peptides from the A / California 07 / 2009 (H1N1) or A / New York / 384 / 05 (H3N2) hemagglutinin were used, which share a consensus sequence with the COBRA sequence and are related to naturally circulating influenza viruses.

[0157] like Figures 3A-3D As shown, the formulation containing R-DOTAP induced strong T cell responses to both nucleoprotein and COBRA HA antigen. Compared with the antigen-only vaccine, the R-DOTAP-based vaccine induced strong CD4 + and CD8 + T cell response ( Figure 3A Likewise, mice vaccinated with the R-DOTAP-Y2NG2 bivalent vaccine showed robust T cell responses to COBRA antigens ( Figures 3B-3D ).and The R-DOTAP adjuvant induced significantly higher T cell immune responses compared to the COBRA adjuvant (an oil emulsion-based adjuvant system). Importantly, T cells generated against COBRA antigens recognized and responded to multiple conserved T cell epitopes ( Figures 3B-3D ).

[0158] Example 3

[0159] Effects of recombinant influenza proteins formulated with R-DOTAP nanoparticles on antigen-specific antibody titers

[0160] To evaluate the antibody-mediated immune response against COBRA protein, serum samples from vaccinated mice were analyzed for antigen-specific antibody titers. It was observed that antibody titers were significantly increased ( Figure 4A The second dose further significantly increased antibody titers compared to the antigen-only group. Combining the two COBRA antigens in a single bivalent vaccine did not cause any formulation issues with R-DOTAP. The addition of NP to the bivalent vaccine was also evaluated and no compatibility or stability limitations were encountered (data not shown).

[0161] With Addavax TM Mice vaccinated with the R-DOTAP-containing formulation showed higher antibody titers measured on days 35 and 62 compared to the adjuvanted formulation ( Figure 4B and 4C ), indicating that R-DOTAP induced the same TM Comparable powerful antibody induction.Next, the dose sparing potential was assessed by immunizing BALB / cJ mice with different doses of COBRA-Y2 (which was formulated with different doses of R-DOTAP nanoparticles), and measuring Y2-specific antibody responses. Mice immunized with 0.35-3.0 μg Y2 antigen formulated with 300 μg R-DOTAP showed a significant increase in the Y2-specific total IgG titer measured 14 days after the booster vaccine (Fig. 1). Animals inoculated with 0.35 μg or 3.0 μg showed similar IgG titers. Similarly, mice inoculated with a vaccine formulation adding 50-300 μg R-DOTAP induced a similar rise in total antibody titers, without any significant difference (Fig. 1) between low and high doses of R-DOTAP.

[0162] Th1-type antibody-mediated immune responses play an important role in protection against viral infection. To further evaluate the immune response induced by R-DOTAP, antibody subclass titers were measured after vaccination. It was observed that R-DOTAP induced class switching, as confirmed 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 ( Figure 4D and 4E ).

[0163] Example 4

[0164] Effects of recombinant influenza proteins formulated with R-DOTAP nanoparticles on the production of neutralizing antibodies

[0165] Next, we characterized the functional ability of the vaccine to elicit antibodies that block the interaction of influenza virus with sialic acid.The hemagglutination inhibition (HAI) assay was used to assess the production of influenza virus neutralizing antibodies against different strains of H1N1 and H3N2 viruses.

[0166] The bivalent vaccine formulated with R-DOTAP (R-DOTAP-Y2NG2) showed similar efficacy to the antigen-only vaccine ( Figures 5A-5B) compared to COBRA Y-2 and COBRA-NG2, respectively, the titer of a variety of drift variants of H1N1 and H3N2 virus strains that share a consensus sequence was significantly enhanced. Even at the lowest antigen dose tested (0.12 ug), robust HAI titers above the 1:40 threshold were observed for all H1N1 viruses. The HAI titer for H3N2 drift variants was lower than that for H1N1 virus, but for the 3 ug dose, it still showed a significant HAI titer above the 1:40 threshold. As expected, it was observed that there was almost no neutralizing activity (HAI titer <1:40) for antibodies induced with only antigen samples or from mice mock-vaccinated. In summary, these results indicate that vaccination with the bivalent COBRA antigen formulated with R-DOTAP induced robust and balanced Th1 and Th2 antibody responses as well as broadly cross-reactive antibodies that can neutralize several H1N1 and H3N2 strain drift variants.

[0167] Example 5

[0168] R-DOTAP enhances the immunogenicity of unadjuvanted seasonal influenza vaccines

[0169] The study evaluated whether R-DOTAP could be used to enhance the immunogenicity of existing human influenza vaccines. As a proof of concept, a trivalent inactivated influenza vaccine was used. (2011-12 formulation), which consists of split viral hemagglutinin preparations from A / California / 07 / 2009X-179A(H1N1), A / Victoria / 210 / 2009X-187(A / Perth / 16 / 2009-like virus)(H3N2), and B / Brisbane / 60 / 2008. The vaccine formulation was prepared by mixing R-DOTAP nanoparticles in a 1:1 ratio with different doses of C57BL / 6J mice were vaccinated with the vaccine (0.1 ml / dose) on days 0 and 21, and the HAI titer of the blood was obtained on day 35.

[0170] Observe that, with only Compared with the inoculated group, the The HAI titers against all virus strains were significantly increased in vaccinated mice ( Figures 6A-6C Importantly, a significant dose-sparing effect was observed in the R-DOTAP vaccine group.

[0171] Example 6

[0172] Recombinant protein formulated with R-DOTAP protects mice against influenza virus challenge

[0173] To determine the protective efficacy of the bivalent influenza vaccine R-DOTAP-Y2NG2 in mice, DBA / 2J mice were vaccinated with a two-dose regimen of formulations containing different doses of COBRA antigen (with or without R-DOTAP) and then vaccinated with A / Brisbane / 2 / 2018(H1N1) (3.6×10 6 Body weight loss was measured as a predictor of protection against challenge.

[0174] 100% of unvaccinated mice and mice vaccinated with antigen-only (3 μg / HA) vaccine formulation rapidly lost weight within 7 days, with survival rates of 0 and 33%, respectively ( Figure 7A and 7B In contrast, less than 5% body weight loss and 100% survival were observed in all groups vaccinated with the bivalent vaccine formulated with R-DOTAP ( Figure 7A and 7B Both low-dose (0.12ug / HA) and high-dose (3ug / HA) COBRA antigens formulated with R-DOTAP provided complete protection, demonstrating a significant dose-saving effect ( Figure 7A ).

[0175] Virus clearance from 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 no virus was detected by day 6 ( Figure 7D In contrast, mice that received antigen alone and unvaccinated mice showed significant viral loads on both days 3 and 6. Together, these studies demonstrate that recombinant protein vaccines containing R-DOTAP as an immunomodulator induce immune responses capable of protecting mice from viral infection.

[0176] Example 7

[0177] discuss

[0178] Cationic lipids are excellent delivery vehicles for transporting nucleic acids and proteins / peptides into cells and have been widely used for human drug delivery. However, most cationic lipids are inert and fail to activate the immune signals necessary for an effective immune response to vaccine antigens. Here, we demonstrate that R-DOTAP promotes robust antibody and T cell responses to multiple viral proteins, providing neutralizing activity and protection from viral attack.

[0179] TEM images ( Figures 2A-2C ) and physical characterization data (Table 1 and Figures 2A-2C) showed that R-DOTAP formed spherical structures with uniform, smooth surfaces in a sucrose buffer, with a size range of approximately 150 nm. Mixing the antigen with the nanoparticles did not significantly alter the size and polydispersity of the formulation (Table 1). Changes in the surface and zeta potential of the nanoparticles were observed after adding the antigen.

[0180] Table 1: Particle size, polydispersity index (PDI), and zeta potential of R-DOTAP formulations

[0181]

[0182]

[0183] In this study, the ability of R-DOTAP to enhance humoral and cellular immune responses to large protein antigens was evaluated using a prototype vaccine formulation containing antigens derived from the respiratory virus influenza virus. By formulating R-DOTAP with COBRA influenza antigens, the potential of the R-DOTAP-COBRA vaccine to provide an effective universal influenza vaccine by inducing broadly protective immune responses that could neutralize multiple influenza virus strains was evaluated. Figures 5A-5B ). They demonstrated that co-formulation of influenza-derived viral protein antigens with R-DOTAP significantly enhanced vaccine immunogenicity, generated robust antigen-specific cellular and antibody-mediated immune responses in mice, mediated significant antigen dose sparing, and protected vaccinated mice from influenza virus challenge.

[0184] The current results demonstrate that single-component R-DOTAP nanoparticles can perform the multiple tasks necessary to generate a broad and long-lasting protective immune response.

[0185] There is ample evidence that CD8 T cells play a crucial role in long-term defense against highly variable viruses such as influenza. While most approved recombinant protein adjuvants effectively induce humoral and Th1-type immune responses, few, if any, are able to induce robust and clinically effective cytotoxic CD8 T cell immune responses in humans. The current study demonstrated that R-DOTAP is able to generate CD8 T cells directed against internal epitopes of large recombinant protein antigens. These T cells were multifunctional and had an effector phenotype. They were able to produce a variety of cytotoxic cytokines and persisted in vaccinated mice 28 days after the second vaccination, indicating the establishment of a T cell memory response.

[0186] It was observed that the vaccine containing R-DOTAP induced T cells that produced both IFN-γ and IL-4 (data not shown), and produced both IgG1 and IgG2a antibody subtypes. No strong preference for Th1 or Th2 subtypes was observed. Therefore, from an antibody perspective, these results indicate that R-DOTAP induced a balanced Th1 / Th2 type immunity that is optimal for an effective vaccine-induced antibody response. However, among antigen-specific Th1 and CD8 T cells, R-DOTAP promoted the production of high levels of multifunctional cytokine-secreting cells, which have been shown to be optimal in promoting viral clearance.

[0187] Taken together, these studies demonstrate that protein subunit vaccines against influenza based on the R-DOTAP platform induce broadly protective cellular and humoral immune responses and provide a significant dose-sparing effect against antigen. The heterogeneity of recombinant proteins that can be formulated and administered using R-DOTAP, coupled with the ability to produce multivalent vaccines, suggests that R-DOTAP is an excellent candidate for use in a variety of preventive vaccines against infectious diseases. Furthermore, R-DOTAP has been shown to be highly effective in enhancing the potency of currently licensed seasonal influenza vaccines, further expanding its potential uses. The safety and efficacy profiles of R-DOTAP have been successfully established in human clinical trials, paving the way for future trials of universal influenza vaccines based on R-DOTAP.

[0188] Example 8

[0189] Use of R-DOTAP adjuvant formulations for bivalent COBRA H1 / H3 vaccines in a pre-immune ferret model

[0190] like Figure 8 As shown, in the pre-immune ferret model, ferrets were primed with virus, vaccinated, and boosted before being challenged with influenza. 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).

[0191] like Figures 9A-9C As shown, H1N1 HAI responses were assessed. Ferrets were vaccinated twice with Y2 / NG2 rHA (15 μg), R-DOTAP alone, or wild-type rHA and R-DOTAP before H1 / H3 immunization, and HAI titers were measured. Figures 10A-10C H3N2 HAI responses were also assessed as shown. Ferrets were vaccinated twice with Y2 / NG2 rHA (15 μg), R-DOTAP alone, or wild-type rHA and R-DOTAP before H1 / H3 immunization, and HAI titers were measured.

[0192] like Figures 11A-11C As shown, body weight was better maintained and nasal wash virus titers were lower in animals vaccinated with Y2 / NG2 rHA.

[0193] This study shows that using The COBRA rHA vaccine with (R-DOTAP) adjuvant is able to elicit protective HAI antibody responses against a variety of viruses from the past decade in pre-immune ferrets. It has also been shown that they elicit HAI reactive antibodies against future drift virus isolates from 2019-2020. Vaccination prevents weight loss and H1N1 virus replication in the lungs of vaccinated animals. In populations with a broader pre-immune background to influenza (such as humans), these vaccines are expected to produce a broader spectrum of reactive antibodies due to the evocation of a more diverse population of memory B cells.

[0194] Sequence Listing:

[0195] SEQ ID NO: 1: NP366-74

[0196] ASNENMETM

[0197] SEQ ID NO:2 NP-311-25

[0198] QVYSLIRPNENPAHK

[0199] SEQ ID NO:3 Y2-H1N1 HA

[0200] MKAILVVLLYTFTTANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETSNSDNGTCYPGDFINYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSQSYINDKGKEVLVLWGIHHPSTTADQQSLYQNADAYVFVGTSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFTMERNAGSGIIISDTPVHDCNTTCQTPEGAINTSLPFQNVHPITIGKCPKYVKSTKLRLATGLRNVPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDKITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRNQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREKIDGVGTGYIPEAPRDGQAYVRKDGEWVLLSTFLGS

[0201] SEQ ID NO:4 NG2-H3N2 HA

[0202] MKTIIALSYILCLVFAQKIPGNDNSTATLCLGHHAVPNGTIVKTITNDRIEVTNATELVQNSSIGEICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFK NESFNWTGVTQNGTSSACIRGSSSSFFSRLNWLTHLNYTYPANLVTMPNNEQFDKLYIWGVHHPGTDKDQIFLYAQSSGRITVSTKRSQQAVIPNIGSRPRIRDIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFK IRSGKSSIMRSDAPIGKCKSECITPNGSIPNDKPFQNVNRITYGACPRYVKQSTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHNVYRDEALNNRFQIKGVEGYIPEAPRDGQAYVRKDGEGEWVLLSTFLGS

[0203] SEQ ID NO:5 J-4 soluble HA

[0204] MKTIIALSYILCLVFAQKIPGNDNSTATLCLGHHAVPNGTIVKTITNDRIEVTNATELVQNSSIGEICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNW TGVTQNGTSSACIRRSNSSFFSRLNWLTHLNYKYPANLVTMPNNEQFDKLYIWGVHHPGTDKDQIFLYAQSSGRITVSTKRSQQAVIPNIGSRPRIRDIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRS DAPIGKCKSECITPNGSIPNDKPFQNVNRITYGACPRYVKQSTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHNVYRDEALNNRFQIKGVEGYIPEAPRDGQAYVRKDGEWLLSTFLGSGLNDIFEAQKIEWHEGHHHHHHH

[0205] SEQ ID NO:6 TJ-5

[0206] MKTIIALSYILCLVFAQKLPGNDNSTATLCLGHHAVPNGTIVKTITNDQIEVTNATELVQSSSTGEICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQNGTSSACIRRSNNSFFSRLNWLTHLNFKYPALNVTMPNNEQFDKLYIWGVHHPGTDKDQIFLYAQASGRITVSTKRSQQAVIPNIGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRITYGACPRYVKQSTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVEGYIPEAPRDGQAYVRKDGEWVLLSTFLGSGLNDIFEAQKIEWHEGHHHHHH

[0207] SEQ ID NO:7 N1-I COBRA

[0208] MPMGSLQPLATLYLLGMLVASVLSAHHHHHHGSGSLVPRGSPSRSIINETADDIVYRLTVIIDDRYESLKNLITLRADRLEMIINDNVSTILASIGSGTGVAGQDVTSVKLAGNSSLCPISGWAIYSKDNSIRIGSKGDVFVIREPFISCSHLECRTFFLTQGALLNDKHSNGTVKDRSPYRTLMSCPIGEAPSPYNSRFESVAWSASACHDGMGWLTIGISGPDNGAVAVLKYNGIITDTIKSWRNNILRTQESECVCVNGSCFTIMTDGPSNGQASYKIFKIEKGKIVKSVELNAPNYHYEECSCYPDTGIVMCVCRDNWHGSNRPWVSFNQNLDYQIGYICSGVFGDNPRPNDGEGSCGPVTVDGANGVKGFSFKYGNGVWIGRTKSNSSRSGFEMIWDPNGWTGTDSDFSVKQDIIAITDWSGYSGSFVQHPELTGLDCIRPCFWVELIRGLPKENTIWTSGSSISFCGVNSDTANWSWPDGAELPFTIDK

[0209] SEQ ID NO:8 N2-A COBRA soluble

[0210] MPMGSLQPLATLYLLGMLVASVLSAHHHHHHGSGSLVPRGSPSRSIINETADDIVYRLTVIIDDRYESLKNLITLRADRLEMIINDNVSTILASIGTGKEICPKLAEYRNWSKPQCKITGFAPFSKDNSIRLSAGGDIWVTREPYVSCDPDKCYQFALGQGTTLNNRHSNDTVHDRTPYRTLLMNELGVPFHLGTKQVCIAWSSSSCHDGKAWLHVCITGDDENATASFIYNGRLVDSIGSWSKKILRTQESECVCINGTCTVVMTDGSASGRADTKILFIEEGKIVHISPLSGSAQHVEECSCYPRYPGVRCVCRDNWKGSNRPIVDINVKDYSIVSSYVCSGLVGDTPRKNDSSSSSHCLNPNNEEGGHGVKGWAFDDGNDVWMGRTISEKLRLGYETFKVIEGWSKPNSKLQINRQVIVERGNRSGYSGIFSVEGKSCINRCFYVELIRGRKQETEVWWTSNSIVVFCGTSGTYGTGSWPDGADINLMPI

[0211] SEQ ID NO:9 J1(2013S-2015S)1-566AA

[0212] MKTIIALSYILCLVFAQKIPGNDNSTATLCLGHHAVPNGTIVKTITNDRIEVTNATELVQNSSIGEICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQNGTSSACIRRSNSSFFSRLNWLTHLNFKYPALNVTMPNNEQFDKLYIWGVHHPGTDKDQIFLYAQSSGRITVSTKRSQQAVIPNIGSRPRIRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCKSECITPNGSIPNDKPFQNVNRITYGACPRYVKQSTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHNVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVALLGFIMWACQKGNIRCNICI

[0213] SEQ ID NO:10 J2(2014-15N thru 2015-16N)1-566AA

[0214] MKTIIALSYILCLVFAQKIPGNDNSTATLCLGHHAVPNGTIVKTITNDRIEVTNATELVQNSSIGEICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQNGTSSACIRRSSSSFFSRLNWLTHLNYKYPALNVTMPNNEQFDKLYIWGVHHPGTDKDQIFLYAQSSGRITVSTKRSQQAVIPNIGSRPRIRDIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCKSECITPNGSIPNDKPFQNVNRITYGACPRYVKQSTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHNVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVALLGFIMWACQKGNIRCNICI

[0215] SEQ ID NO:11 J3(2014S-2016S1-566AA

[0216] MKTIIALSYILCLVFAQKIPGNDNSTATLCLGHHAVPNGTIVKTITNDRIEVTNATELVQNSSIGEICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQNGTSSACIRRSSSSFFSRLNWLTHLNYTYPALNVTMPNNEQFDKLYIWGVHHPGTDKDQIFLYAQSSGRITVSTKRSQQAVIPNIGSRPRIRDIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCKSECITPNGSIPNDKPFQNVNRITYGACPRYVKQSTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHNVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVALLGFIMWACQKGNIRCNICI

[0217] SEQ ID NO:12 J41-566 AA(2013S thru 2015-16N)

[0218] MKTIIALSYILCLVFAQKIPGNDNSTATLCLGHHAVPNGTIVKTITNDRIEVTNATELVQNSSIGEICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESF NWTGVTQNGTSSACIRRSNSSFFSRLNWLTHLNYKYPANLVTMPNNEQFDKLYIWGVHHPGTDKDQIFLYAQSSGRITVSTKRSQQAVIPNIGSRPRIRDIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSI MRSDAPIGKCKSECITPNGSIPNDKPFQNVNRITYGACPRYVKQSTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHNVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLCCVALLGFIMWACQKGNIRCNICI

[0219] SEQ ID NO:13 soluble HA

[0220] MKTIIALSYILCLVFAQKIPGNDNSTATLCLGHHAVPNGTIVKTITNDRIEVTNATELVQNSSIGEICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESF NWTGVTQNGTSSACIRRSNSSFFSRLNWLTHLNFKYPANLVTMPNNEQFDKLYIWGVHHPGTDKDQIFLYAQSSGRITVSTKRSQQAVIPNIGSRPRIRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSI MRSDAPIGKCKSECITPNGSIPNDKPFQNVNRITYGACPRYVKQSTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHNVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLCCVALLGFIMWACQKGNIRCNICI

[0221] SEQ ID NO:14 soluble HA

[0222] MKTIIALSYILCLVFAQKIPGNDNSTATLCLGHHAVPNGTIVKTITNDRIEVTNATELVQNSSIGEICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQNGTSSACIRRSNSSFFSRLNWLTHLNYKYPALNVTMPNNEQFDKLYIWGVHHPGTDKDQIFLYAQSSGRITVSTKRSQQAVIPNIGSRPRIRDIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCKSECITPNGSIPNDKPFQNVNRITYGACPRYVKQSTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHNVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVALLGFIMWACQKGNIRCNICI

[0223] SEQ ID NO:15 Soluble HA sequence J1 H3N2 soluble HA

[0224] MKTIIALSYILCLVFAQKIPGNDNSTATLCLGHHAVPNGTIVKTITNDRIEVTNATELVQNSSIGEICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQNGTSSACIRRSNSSFFSRLNWLTHLNFKYPALNVTMPNNEQFDKLYIWGVHHPGTDKDQIFLYAQSSGRITVSTKRSQQAVIPNIGSRPRIRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCKSECITPNGSIPNDKPFQNVNRITYGACPRYVKQSTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHNVYRDEALNNRFQIKGVEGYIPEAPRDGQAYVRKDGEWVLLSTFLGSGLNDIFEAQKIEWHEGHHHHHH

[0225] SEQ ID NO:16 NG1 Soluble HA

[0226] MKTIIALSYILCLVFAQKIPGNDNSTATLCLGHHAVPNGTIVKTITNDRIEVTNATELVQNSSIGEICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQNGTSSACIRGSSSSFFSRLNWLTHLNYTYPALNVTMPNNEQFDKLYIWGVHHPGTDKDQIFLYAQSSGRITVSTKRSQQAVIPNIGSRPRIRDIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCKSECITPNGSIPNDKPFQNVNRITYGACPRYVKQSTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHNVYRDEALNNRFQIKGVEGYIPEAPRDGQAYVRKDGEWVLLSTFLGSGLNDIFEAQKIEWHEGHHHHHH

[0227] SEQ ID NO:17 NG2 soluble HA

[0228] MKTIIALSYILCLVFAQKIPGNDNSTATLCLGHHAVPNGTIVKTITNDRIEVTNATELVQNSSIGEICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFKNESFNWTGVTQNGTSSACIRGSSSSFFSRLNWLTHLNYTYPALNVTMPNNEQFDKLYIWGVHHPGTDKDQIFLYAQSSGRITVSTKRSQQAVIPNIGSRPRIRDIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCKSECITPNGSIPNDKPFQNVNRITYGACPRYVKQSTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHNVYRDEALNNRFQIKGVEGYIPEAPRDGQAYVRKDGEWVLLSTFLGSGLNDIFEAQKIEWHEGHHHHHH

[0229] SEQ ID NO:18 NG3 Soluble HA

[0230] MKTIIALSYILCLVFAQKIPGNDNSTATLCLGHHAVPNGTIVKTITNDRIEVTNATELVQNSSIGEICDSPHQILDGGNCTLIDALLGDPQCDGFQNKKWDLFVERSRAYSNCYPYDVPDYASLRSLVASSGTLEFKNESFNWTGVTQNGTSSACIRGSSSSFFSRLNWLTHLNYTYPALNVTMPNKEQFDKLYIWGVHHPGTDKDQIFLYAQSSGRITVSTKRSQQAVIPNIGSRPRIRDIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCKSECITPNGSIPNDKPFQNVNRITYGACPRYVKQSTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHNVYRDEALNNRFQIKGVEGYIPEAPRDGQAYVRKDGEWVLLSTFLGSGLNDIFEAQKIEWHEGHHHHHH

[0231] SEQ ID NO:19 H1N1 soluble HA sequence Y1 H1N1 soluble HA

[0232] MKAILVVLLYTFTTANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETSSSDNGTCYPGDFINYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSQSYINDKGKEVLVLWGIHHPSTTADQQSLYQNADAYVFVGTSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFTMERNAGSGIIISDTPVHDCNTTCQTPEGAINTSLPFQNVHPITIGKCPKYVKSTKLRLATGLRNVPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDKITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRNQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREKIDGVKLESTRIYGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGLNDIFEAQKIEWHEGHHHHHH

[0233] SEQ ID NO:20 Y2 H1N1 soluble HA

[0234] MKAILVVLLYTFTTANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETSNSDNGTCYPGDFINYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSQSYINDKGKEVLVLWGIHHPSTTADQQSLYQNADAYVFVGTSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFTMERNAGSGIIISDTPVHDCNTTCQTPEGAINTSLPFQNVHPITIGKCPKYVKSTKLRLATGLRNVPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDKITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRNQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREKIDGVKLESTRIYGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGLNDIFEAQKIEWHEGHHH HHH

[0235] SEQ ID NO:21 Y3 H1N1 soluble HA

[0236] MKAILVVLLYTFTTANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETSNSDNGTCYPGDFINYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLNQSYINDKGKEVLVLWGIHHPSTTADQQSLYQNADAYVFVGTSRYSKKFKPEIATRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFTMERNAGSGIIISDTPVHDCNTTCQTPEGAINTSLPFQNVHPITIGKCPKYVKSTKLRLATGLRNVPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDKITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRNQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREKIDGVKLESTRIYGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGLNDIFEAQKIEWHEGHHH HHH

[0237] SEQ ID NO:22 Y4 H1N1 soluble HA

[0238] MKAILVVLLYTFTTANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTARSWSYIVETSNSDNGTCYPGDFINYEELREQLSSVSSFERFEIFPKTSSWPNHD SNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLNQTYINDKGKEVLVLWGIHHPSTTADQQSLYQNADAYVFVGTSRYSKKFKPEIATRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFTMERNAGSGIIISDTP VHDCNTTCQTPEGAINTSLPFQNVHPITIGKCPKYVKSTKLRLATGLRNVPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDKITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGF LDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRNQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREKIDGVKLESTRIYGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGLNDIFEAQKIEWHEGHHH HHH

[0239] Although the present invention has been described with reference to the above embodiments, it will be understood that modifications and variations are encompassed within the spirit and scope of the present invention. Accordingly, the present invention is limited only by the appended claims.

Claims

1. A vaccine composition comprising: a) one or more non-naturally occurring recombinant influenza antigens; and b) Cationic lipids.

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

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

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 one of SEQ ID NOs: 3-22 or a combination thereof.

6. 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%, 85%, 90% or 95% sequence identity to SEQ ID NO: 3 and / or SEQ ID NO:

4.

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

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

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

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

12. The vaccine composition of claim 10, wherein the one or more non-naturally occurring recombinant influenza antigens are present as micelles separate from the pre-formed cationic lipid nanoparticles.

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

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

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

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

4.

18. 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, variants thereof, or analogs thereof.

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

20. The method of claim 13, wherein the immune response comprises CD8 + Effector T cells, CD4 + Induction of effector and memory T cells.

21. The method according to claim 20, wherein the induction of CD8 + Effector T cells and CD4 + Effector T cells include CD8 T cells that induce the production of IFNγ and granzyme B in subjects + Proliferation of effector T cells and / or IL-4-producing CD4 + Proliferation of effector T cells.

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

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

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

25. A method of preventing or treating 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) cationic lipids, thereby preventing or treating influenza infection in the subject.

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. 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%, 85%, 90% or 95% sequence identity to any one of SEQ ID NOs: 3-22 or a combination thereof.

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

29. 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%, 85%, 90% or 95% sequence identity to SEQ ID NO: 3 and / or SEQ ID NO:

4.

30. 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, variants thereof, or analogs thereof.

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

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

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

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

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

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

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

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

4.

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

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

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. ​​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%, 85%, 90% or 95% sequence identity to any one of SEQ ID NOs: 3-22 or a combination thereof.

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

48. 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%, 85%, 90% or 95% sequence identity to SEQ ID NO: 3 and / or SEQ ID NO:

4.

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

51. The method of claim 44, wherein inducing a Th1 immune response comprises inducing CD8 T cells that produce IFNγ and granzyme B in the subject. + Proliferation of effector T cells and / or IL-4-producing CD4 + Proliferation of effector T cells.

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

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

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. 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%, 85%, 90% or 95% sequence identity to any one of SEQ ID NOs: 3-22 or a combination thereof.

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

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

4.

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

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

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

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