WO2025010117A1 - Influenza b headless ha universal vaccines - Google Patents

Abstract

Disclosed herein are influenza vaccines capable of providing broad cross-protection. Also disclosed are pharmaceutical compositions comprising a nanoparticle disclosed herein and an adjuvant, in some embodiments, it includes vaccine compositions and methods base on a truncated influenza HA protein lacking a head domain. For example, disclosed herein is a polypeptide comprising a truncated influenza HA protein lacking at least a portion of the HA head domain, also referred to herein as a head-removed HA (hrHA).

Description

INFLUENZA B HEADLESS HA UNIVERSAL VACCINES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63/511 , 869, filed July 4, 2023, which is hereby incorporated herein by reference in its entirety. STATEMENT OF GOVERNMENT INTEREST

[0002] This invention was made with Government Support under Grant No. Al 143844 awarded by the National Institutes of Health. The Government has certain rights in the invention.

SEQUENCE LISTING

[0003] This application contains a sequence listing filed in ST.26 format entitled “220702-2960 Sequence Listing” created on May 31 , 2024, having 26,089 bytes. The content of the sequence listing is incorporated herein in its entirety.

BACKGROUND OF THE INVENTION

[0004] The influenza epidemics pose a significant threat to public health. Of them, type B influenza coincided with several severe flu outbreaks. The efficacy of the current seasonal flu vaccine is limited due to the antigenicity changes of circulating strains. Adding appropriate adjuvants to improve immunogenicity and finding effective mucosal vaccines to combat respiratory infection at the portal of virus entry are important strategies to boost protection. SUMMARY OF THE INVENTION

[0005] Disclosed herein are influenza vaccines capable of providing broad crossprotection. Conserved epitopes from influenza virus are promising targets to develop universal vaccines. Notably, the membrane proximal stalk domain of hemagglutinin (HA) is more conserved than the highly variable head domain. Disclosed herein are vaccine compositions and methods based on a truncated influenza HA protein lacking a head domain. For example, disclosed herein is a polypeptide comprising a truncated influenza HA protein lacking at least a portion of the HA head domain, also referred to herein as a head-removed HA (hrHA). Also disclosed is a nanoparticle coated with a hrHA polypeptide. In particular, disclosed herein is a nanoparticle formed by desolvating a nucleoprotein (NP) with a desolvating agent and/or crosslinking a NP protein with a crosslinking agent, and crosslinking a hrHA protein onto the nanoparticle.

[0006] Also disclosed is a nanoparticle formed by desolvating a nucleoprotein (NP) with a desolvating agent and/or crosslinking a NP protein with a crosslinking agent, and crosslinking a fusion protein comprising a matrix protein 2 extracellular (M2e) domain and a neuraminidase (NA) domain.

[0007] Also disclosed are pharmaceutical compositions comprising a polypeptide or nanoparticle disclosed herein and an adjuvant.

[0008] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF FIGURES

[0009] FIGs. 1A to 1 F show recombinant protein construction, nanoparticle fabrication, and characterization. FIG. 1A contains cartoon diagrams of B/hrHA based on B/Brisbane/60/2008 (Victoria lineage) HA protein and full-length nucleoprotein of B/Yamanashi/166/1998 (Yamagata lineage). Dashed lines indicate the sequences replaced with flexible linkers. Arrows indicate the site mutations, K360T, R362Q, and S455C. FIG. 1 B shows Western blot (WB) and Coomassie blue staining (CB) of purified recombinant B/hrHA and B/NP proteins. FIG. 1C is a schematic diagram of double layered nanoparticle fabrication by crosslinking and chemical mechanisms of DTSSP crosslinking as well as biodegradation of nanoparticles in physiological environments. FIG. 1D shows Western blot (WB) and Coomassie blue staining (CB) analysis of fabricated double layered nanoparticles. FIG. 1 E shows size distribution of B/NP nanocore and double layered nanoparticle. FIG. 1 F shows transmission electron microscopy of double layered nanoparticles. Bars represent 200nm in length (left) and 500nm in length (right).

[0010] FIGs. 2A to 2E show nanoparticle internalization by dendritic cells in vitro. FIG. 2A shows immunofluorescence of antigen uptake by JAWS II cells was detected by FITC labeled secondary antibody. The bar scale represents 50 pm. FIGs. 2B and 2C show CD86 expression on JASW II cell surface after antigen stimulation by flow cytometry. MFI, mean fluorescence intensity. FIGs. 2D and 2E show production of proinflammatory cytokines, IL-6 (FIG. 2D) and TNF-a (FIG. 2E), in JAWS II cells treated with antigens at a concentration of 10 pg/ml. Data are presented as mean ± SEM. Statistical significance was analyzed by One-way ANOVA (n=5; *p<0.05; **p<0.01 ; ***p<0.001 ; ns represents no significance).

[0011] FIGs. 3A and 3B show germinal center B cell responses after immunization. FIGs. 3A and 3B show frequencies of B220 and GL-7 positive cell populations from inguinal lymph nodes 7 days after primary vaccination. Data are presented as mean ± SEM. Statistical significance was analyzed by One-way ANOVA (n=5; *p<0.05; ns represents no significance). [0012] FIGs. 4A to 4G show antibody immune responses after immunization in mice. FIG. 4A is a phylogenetic tree of full-length HAs of influenza B strains from Victoria lineage and Yamagata lineage. FIG. 4B is a radar diagram of immune serum breadth binding to HAs from viruses of both Victoria and Yamagata lineage by total IgG ELISA. C to E Serum total IgG (FIG. 4C), lgG1 (FIG. 4D), and lgG2a (FIG. 4E) endpoint titers against purified influenza B viruses, B/Brisbane/60/2008 (Victoria lineage), B/Malaysia/2506/2004 (Victoria lineage) and B/Florida/4/2006 (Yamagata lineage). F and G ADCC activity of pooled inactivated post-boost sera. MDCK cells were infected with B/Malaysia/2506/2004 (Victoria lineage) (FIG. 4F) and B/Florida/4/2006 (Yamagata lineage) (FIG. 4G) mouse challenge viruses at MOI of 5 for singlecycle replication, respectively. Fold induction of the reporter signals from the sera over those from the blank were analyzed. Data are presented as mean ± SEM. Statistical significance was analyzed by One-way ANOVA (n=5; *p<0.05; **p<0.01 ; ns represents no significance).

[0013] FIGs. 5A to 5F show cellular immune responses after boost immunization in mice. A and B Specific cellular immune responses against NP and HAs from two influenza B lineages, respectively. Peptide pool stimulated IL-4- (FIG. 5A) and interferon gamma (IFN-y) (FIG. 5B)-secreting splenocytes were determined using ELISpot assay. FIGs. 5C and 5D show antigen specific IgG plasma cells in bone marrows (FIG. 5C) and spleens (FIG. 5D) by ASC ELISpot assay against viral HAs from both influenza B lineages, respectively. E and F Memory B cells in bone marrows (FIG. 5E) and spleens (FIG. 5F) after polyclonal mitogen stimulation against viral HAs from both influenza B lineages, respectively. Data are presented as mean ± SEM. Statistical significance was analyzed by One-way ANOVA (n=5; *p<0.05; **p<0.01 ; ***p<0.001 ; ns represents no significance).

[0014] FIGs. 6A to 6D show immune protection against homologous and heterologous lineage virus challenge. FIG. 6A and 6B show body weight change and survival rate of immunized mice challenged with 5xLD₅o influenza B/Malaysia/2506/2004 (Victoria lineage) (FIG. 6A) and B/Florida/4/2006 (Yamagata lineage) (FIG. 6B) viruses, respectively 4 weeks after boost immunization. FIGs. 6C and 6D show body weight change and survival rate of pooled immune sera passive transmitted mice against 3xLDso homologous (FIG. 5C) or heterologous (FIG. 5D) lineage virus challenge, respectively. DPI, days post infection. Data represent mean ± SEM. The difference in survival rate was analyzed by using the log-rank test (n=5; *p<0.05; **p<0.01).

[0015] FIGs. 7A to 7E show histology study, viral titers, and inflammations in lungs after challenge. FIG. 7A shows lung tissue histology 5 days after homologous or heterologous lineage virus challenge by H&E staining. An uninfected mouse lung section was used as a negative control. The bar scale presents 100 ^m. FIG. 7B shows viral titers in lungs determined by standard TCID50 assay. FIG. 7C shows lung injury scores after virus infection were determined by leukocyte infiltration degrees. FIGs. 7D and 7E show inflammatory cytokine (IL-6, TNF-a, and IL-12) levels in BALF of virus challenged mice by cytokine ELISA. Data are presented as mean ± SEM. Statistical significance was analyzed by One-way ANOVA (n=5; *p<0.05; **p<0.01 ; ***p<0.001 ; ns represents no significance).

[0016] FIGs. 8A to 8F show IFN-y secreting T cell responses in lungs post infection. Percentage of IFN-y secreting T cell populations were measured in lung tissue homogenates 5 days after 1 *LD₅o B/Malaysia/2506/2004 (Victoria lineage) virus challenge by flow cytometry. FIGs. 8A to 8C show percentage of NP specific IFN-y secreting CD4+ T cells (FIG. 8A and 8B) and CD8+ T cells (FIG. 8A and 8C). FIGs. 8D to 8F show frequency of HA (B/Brisbane/60/2008, Victoria lineage) specific IFN-y secreting CD4+ T cells (FIGs. 8D and 8E) and CD8+ T cells (FIGs. 8D and 8F). Data are presented as mean ± SEM. Statistical significance was analyzed by One-way ANOVA (n=5; *p<0.05; **p<0.01 ; ***p<0.001 ; ns represents no significance).

[0017] FIGs. 9A to 9C show long-term immunity. Serum total IgG endpoint titers against purified B/Brisbane/60/2008 (FIG. 9A), B/Malaysia/2506/2004 (FIG. 9B), and B/Florida/4/2006 (FIG. 9C) by serum binding ELISA 6 months after boost immunization. Data are presented as mean ± SEM. Statistical significance was analyzed by One-way ANOVA (n=5; *p<0.05; **p<0.01 ; ns represents no significance).

[0018] FIGs 10A to 10J show fabrication and characterization of SDAD-crosslinked protein nanoparticles. FIG. 10A is a schematic of the NHS-diazirine crosslinking reactions. FIG. 10B shows size distributions of SDAD protein nanoparticles. FIG. 10C shows nanoparticle characterization by Coomassie blue staining and Western blot. FIG. 10D contains SDAD nanoparticle TEM images. Bone marrow-derived dendritic cells (BMDCs) were stimulated with protein nanoparticles with and without various adjuvant combinations. FIG. 10E shows the expression of CD40, CD80, and CD86 on CD11c+ BMDCs by flow cytometry. FIGs. 10F to FIG. 10J show TNF-a (FIG. 10F), IL-6 (FIG. 10G), IL-12 (FIG. 10H), IL-1J3 (FIG. 101) and IFN-a (FIG. 10J) secretions from BMDCs cultured supernatant after stimulations with protein nanoparticles in combination with different adjuvants. The histograms were presented as mean ± SEM. Statistical significance was analyzed by F-test and two-tailed t-test. *p < 0.05; **p < 0.01 ; ***p < 0.001. FIGs. 10E to 10J display the results of one experiment that is representative of two independent experiments.

[0019] FIGs. 11A to 111 show immune responses induced by intramuscular immunization of different protein nanoparticle vaccines. Sera were collected and analyzed three weeks post-boost immunization. FIG. 11A shows M2e, NA1 , and NA2 specific IgG levels after boost. FIGs. 11 B, 11C, and 11 D show NA2, NA1 , and M2e specific IgG isotypes after boost. Groups of mice were euthanized at week four post the boosting immunization. The spleens and bone marrow were collected and processed into single-cell suspensions for analysis. FIG. 11 E and 11 F show NA-specific IL-4 and IFN-y-secreting cells in spleens. FIGs. 11G, 11 H, and 111 show Aichi-, PR8- and M2e- specific antibody-secreting cells in the bone marrow. The histograms were presented as mean ± SEM. Statistical significance was analyzed by F-test and two-tailed t-test. *p < 0.05; **p < 0.01 ; ***p < 0.001. The results show one experiment with two replicates for each sample. Mice groups were n = 5.

[0020] FIGs. 12A to 12F show protective efficacy against homologous and heterologous influenza viral challenge. Groups of mice were challenged with A/Aichi, A/Philippine, and A/H5N1 influenza viruses at week four post-boosting immunization. Mouse body weight was monitored for 14 days. The body weight changes after A/Aichi (H3N2) (FIG. 12A), A/Philippine (H3N2) (FIG. 12C), and rViet (A/H5N1) (FIG. 12E) virus infections. FIGs. 12B, 12D, and 12F show the survival rates after different viral infections. The data were presented as mean ± SEM. Mice groups were n - 5.

[0021] FIGs. 13A to 13F show adjuvants improve the humoral immune responses and protection efficiency against influenza viral infection after intranasal immunization. Groups of mice were intranasally immunized at a three-week interval with different protein nanoparticle formulations with and without ISCOMs/MPLA or ISCOMs/cGAMP adjuvant combinations. Mouse sera were collected and analyzed three weeks post-primary and boosting immunization. FIG. 13A shows NA2 specific IgG levels after prime and boost. FIG. 13B shows NA2-specific lgG1 and lgG2a levels. FIG. 13C shows M2e-specific IgG levels after prime and boost. FIG. 13D shows M2e-specific lgG1 and lgG2a levels. Groups of immunized mice were challenged with 5x LD₅o of A/Aichi/2/1968 H3N2 influenza virus. FIG. 13E shows body weight changes after viral infection. FIG. 13F shows mice survival rate. The data were presented as mean ± SEM. The Mice groups were n = 5.

[0022] FIGs. 14A to 14D show cellular immune responses after intranasal immunization. Mice were intranasally immunized twice at a three-week interval with protein nanoparticles with or without ISCOMs/MPLA. Spleens and bone marrow were collected one month post the boosting immunization and analyzed by ELISpot assays. FIG. 14A shows IFN-y, IL-2, and IL-4 secreting splenocytes after purified NA2 protein stimulation. FIG. 14B shows NA2-specific antibody-secreting cells in the bone marrow. FIG. 14C shows IFN-y, IL-2, and IL-4 secreting splenocytes after NP peptides stimulation. FIG. 14D shows IFN-y, IL-2, and IL-4 secreting splenocytes after stimulation of A/Aichi virus. The histograms were presented as mean ± SEM. Statistical significance was analyzed by F-test and two-tailed t-test. *p < 0.05; **p < 0.01 ; ***p < 0.001. The results show one experiment with three independent replicates for each sample. Mice groups were n = 3.

[0023] FIGs. 15A to 15G show mucosal immune responses and protection against heterologous influenza viral infection. Mice were intranasally immunized with NP/M2e-NA2 protein nanoparticles with and without ISCOMs/MPLA. The nasal and BALF washes were collected one month post-boosting intranasally immunization to determine the IgA and IgG levels by ELISA. FIG. 15A and 15C show NA2-specific IgA and IgG levels in nasal washes. FIG. 15B and 15D show NA2-specific IgA and IgG levels in BALFs. FIG. 15E shows Aichi-specific IgA and IgG in BALFs. FIG. 15G shows the body weight changes after A/Philippine (H3N2) influenza viral challenge. The data were presented as mean ± SEM. Statistical significance was analyzed by F-test and two-tailed t-test. *p < 0.05; **p < 0.01 ; ***p < 0.001. The Mice groups were n = 3.

[0024] FIGs. 16A to 16K show cell populations in localized pulmonary tissue. The lung tissues were collected and processed one month post the boosting intranasally immunization to determine the inside cell populations. FIG. 16A shows percentages of the CD69+CD103+ population in CD8+CD44+ T cells. FIGs. 16B and 16C shows percentages of CD69+CD103+ and CD69+CD103- populations in CD4+CD44+ T cells. FIG. 16D shows representative gating methods for FIGs. 16A, 16B, and 16C. FIGs. 16E and 16F shows frequencies of CD38+CD69+ and CD38-CD69+ populations in CD19+B220-lgD-lgM- cells. FIG. 16G shows representative gating method for FIGs. 16E and 16F. FIG. 16H shows percentage of CD64+CD24- population in CD11c+CD11b- cells. FIG. 161 shows gating method for results of FIG. 16H. The cells in BALFs were collected for analysis. FIGs. 16J and 16K show percentage of CD4+CD44+ and CD8+CD44+ cells in BALFs. The histograms were presented as mean ± SEM. Statistical significance was analyzed by F-test and two-tailed t-test. *p < 0.05; **p < 0.01 ; ***p < 0.001. The Mice groups were n = 3.

DETAILED DESCRIPTION

[0025] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims. [0026] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0027] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0028] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

[0029] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0030] Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of chemistry, biology, and the like, which are within the skill of the art.

[0031] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

[0032] Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.

Definitions

[0033] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0034] “Optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, the phrase “optionally a signal peptide” means that the signal peptide may or may not be included.

[0035] The term “universal influenza vaccine” refers to vaccine capable of providing cross-protection against at least two, including three, four, five or more, strains or subtypes of influenza.

[0036] The term “individual,” “host,” “subject,” and “patient” are used interchangeably to refer to any individual who is the target of administration, treatment, or vaccination. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human or veterinary patient.

[0037] The term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.

[0038] The term “carrier” means a compound, composition, substance, or structure that, when in combination with a compound or composition, aids or facilitates preparation, storage, administration, delivery, effectiveness, selectivity, or any other feature of the compound or composition for its intended use or purpose. For example, a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject. [0039] The terms “peptide,” “protein,” and “polypeptide” are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another.

[0040] The term “protein domain” refers to a portion of a protein, portions of a protein, or an entire protein showing structural integrity; this determination may be based on amino acid composition of a portion of a protein, portions of a protein, or the entire protein.

[0041] The term “nucleic acid” refers to a natural or synthetic molecule comprising a single nucleotide or two or more nucleotides linked by a phosphate group at the 3’ position of one nucleotide to the 5’ end of another nucleotide. The nucleic acid is not limited by length, and the nucleic acid can include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0042] The term “variant” refers to an amino acid sequence having conservative amino acid substitutions, non-conservative amino acid substitutions (i.e. a degenerate variant), or a peptide having 60%, 65%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%$, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the recited sequence.

[0043] The term “percent (%) sequence identity” is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical with the nucleotides or amino acids in a reference nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST- 2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods. For purposes herein, the % sequence identity of a given nucleotides or amino acids sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given sequence C that has or comprises a certain % sequence identity to, with, or against a given sequence D) is calculated as follows:

[0044] 100 times the fraction W/Z,

[0045] where W is the number of nucleotides or amino acids scored as identical matches by the sequence alignment program in that program’s alignment of C and D, and where Z is the total number of nucleotides or amino acids in D. It will be appreciated that where the length of sequence C is not equal to the length of sequence D, the % sequence identity of C to D will not equal the % sequence identity of D to C. [0046] “Homolog” and “homologous” as used herein refer to sequences that derive from a recent common ancestor and therefore maintain a level of percent sequence identity that is commonly understood in the art.

[0047] “Heterologous” refers to sequences that encode for the same protein (in the case of nucleic acids) or that are demonstrably the same protein (in the case of polypeptides), yet in which such sequences are not derived from a recent common ancestor and maintain a level of percent sequence identity that is less than 100%.

[0048] A “fusion protein” refers to a polypeptide formed by the joining of two or more polypeptides through a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide. The fusion protein can be formed by the chemical coupling of the constituent polypeptides or it can be expressed as a single polypeptide from nucleic acid sequence encoding the single contiguous fusion protein. A single chain fusion protein is a fusion protein having a single contiguous polypeptide backbone. Fusion proteins can be prepared using conventional techniques in molecular biology to join the two genes in frame into a single nucleic acid, and then expressing the nucleic acid in an appropriate host cell under conditions in which the fusion protein is produced.

[0049] A “spacer” as used herein refers to a peptide that joins the proteins of a fusion protein. Generally a spacer has no specific biological activity other than to join the proteins or to preserve some minimum distance or other spatial relationship between them. However, the constituent amino acids of a spacer may be selected to influence some property of the molecule, such as the folding, net charge, or hydrophobicity of the molecule.

[0050] An ’’antigen” refers to a substance that can act as the target of an immune response. As used herein, “antigen” refers to a biological substance, and may consist of a peptide, a polypeptide or protein, a glycoprotein, a saccharide or polysaccharide, a lipid, a nucleic acid, or another biological or bioactive compound, amongst other biological substances.

[0051] The “ectodomain” of a protein refers to the domain of a membrane protein that extends into the extracellular space or is found on the exterior surface of a viral particle.

[0052] The “transmembrane” domain of a protein refers to the domain of a membrane protein that passes through the biological membrane and directly interfaces laterally with the phospholipid bilayer of the membrane.

[0053] The “cytoplasmic” domain, also known as an “intracellular” domain, is the domain of an integral membrane protein that extends into the intracellular space or is found in the interior space of a virus. [0054] A “strain” of influenza as used herein refers to a lineage of the virus with defined and durable antigenic parameters. Each type of influenza (Influenza A, B, C, and D) consists of numerous strains that differ from one another in their antigen sequences.

[0055] A ’’subtype” of influenza A virus refers to the division of influenza A into categories based on the sequence of the viral HA and neuraminidase (NA) proteins. For example, an influenza A virus can have an H1 sequence of its HA protein and an N1 sequence of its NA protein and would therefore be categorized as an H1N1 subtype of influenza. Other subtype examples include H3N2, H5N1 , H7N9, and H9N2.

Nanoparticle

[0056] A nanoparticle is a particle between 1 and 100s of nanometers in size. A nanoparticle can be natural or synthetic. A nanoparticle can be created from biological molecules or from abiological molecules. A nanoparticle has a core containing the material of which the nanoparticle consists. A nanoparticle has a surface that is the interface between the core and the space and/or solution outside of the core. A protein nanoparticle (PNP) is a particle in which the core material consists of protein. PNP can be synthesized in numerous ways. For example, PNP can be synthesized by desolvation of a solution of soluble protein, resulting in precipitation of particulate PNP. PNP can be synthesized by crosslinking proteins to form the particle. PNP can be synthesized by biochemical aggregation based on autonomouns proteinprotein interactions. PNP can be formed by a combination of any of these PNP synthesis methods.

[0057] Disclosed herein is a nanotechnology approach to produce a nanocluster PNPs. In one embodiment, the PNP consists of a protein antigen that can act as a target of an immune response. In one embodiment, the PNP can be used as an active pharmaceutical ingredient in a vaccine. In one embodiment, the PNP is an uncoated particle. In one embodiment, the PNP is coated with another protein antigen on its surface. In one embodiment, the PNP formed by desolvating the core protein with a desolvating agent and/or crosslinking a core protein with a crosslinking agent. Both or either of these procedures can be used to make double-layered nanoparticles. In some cases, the nanoparticle contains a core structure formed by desolvation and a coating formed by crosslinking of a surface antigen. Suitable desolvating agents include, for example, ethanol, acetone, or combinations thereof.

[0058] A humoral immune response is preferentially mounted against proteins, domains, and epitopes that are displayed in a repetitive and/or iterative fashion. Forming a PNP from a protein results in a particle with many repetitive epitopes on the surface of the PNP. In this way, formation of a PNP from a soluble protein results in a substance that is much more highly antigenic for initiating and/or promoting an immune response than the soluble protein alone and yet consists of biological substituents that are identical or nearly-identical to the soluble protein alone. Thus formation of PNP from a soluble protein results in a composition that much more readily promotes humoral immune responses to that protein.

[0059] It is possible to modify a PNP consisting of a core protein by adding a coat of a second protein onto the surface of the PNP. This method leverages the same principle of promoting effective humoral immune responses toward repetitive epitopes yet has the benefit of directing an additional humoral immune response against the second protein that coats the PNP.

[0060] In some embodiments, the core protein comprises one or more influenza virus nucleoprotein (NP) domains. Influenza nucleoprotein (NP) are described, for example, in U.S. Patent No. 9,963,490, which is incorporated by reference in its entirety for the teaching of these proteins and uses as vaccines.

[0061] In some embodiments, the NP sequence has the amino acid sequence MASQGTKRSYEQMETDGERQNATEIRASVGKMIGGIGRFYIQMCTELKLSDYEGRLIQNSLTIE RMVLSAFDERRNKYLEEHPSAGKDPKKTGGPIYRRVNGKWMRELILYDKEEIRRIWRQANNGD DATAGLTHMMIWHSNLNDATYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGVGT MVMELVRMIKRGINDRNFWRGENGRKTRIAYERMCNILKGKFQTAAQKAMMDQVRESRNPGN AEFEDLTFLARSALILRGSVAHKSCLPACVYGPAVASGYDFEREGYSLVGIDPFRLLQNSQVYS LIRPNENPAHKSQLVWMACHSAAFEDLRVLSFIKGTKVLPRGKLSTRGVQIASNENMETMESST LELRSRYWAIRTRSGGNTNQQRASAGQISIQPTFSVQRNLPFDRTTIMAAFNGNTEGRTSDMR TEIIRMMESARPEDVSFQGRGVFELSDEKAASPIVPSFDMSNEGSYFFGDNAEEYDN (SEQ ID NO:24), or a conservative variant thereof having at least about 70%, 80%, or 90% sequence identity to SEQ ID NO:24 (i.e. , one, two, or three conservative amino acid substitutions).

M2e-NA fusion peptide

[0062] In some embodiments, the human M2e sequence comprises the amino acid sequence PIRNEWGSRSN (SEQ ID NO:2), or a conservative variant thereof having at least about 70%, 80%, or 90% sequence identity to SEQ ID NO:2 (i.e., one, two, or three conservative amino acid substitutions). For example, human M2e isolates H1 N1 (A/PR8, A/NC/99) and H3N2 (A/Phil/82) have the amino acid sequence SLLTEVETPIRNEWGSRSNDSSD (SEQ ID NO:3).

[0063] In some embodiments, the swine M2e sequence comprises the amino acid sequence PTRSEWESRSS (SEQ ID NO:4), or a conservative variant thereof having at least 70%, 80%, or 90% sequence identity to SEQ ID NO:4. For example, swine M2e isolates from the 2009 H1 N1 pandemic (A/California/4/2009) have the amino acid sequence SLLTEVETPTRSEWESRSSDSSD (SEQ ID NO:5).

[0064] In some embodiments, the avian M2e sequence (referred to herein as “avian type I”) comprises the amino acid sequence PTRX1X2WESRSS (SEQ ID NO:6), wherein Xi is N, H, or K, wherein X₂ is E or G, or a conservative variant thereof having at least 70%, 80%, or 90% sequence identity to SEQ ID NO:6. For example, avian type I M2e isolates from H5N1 (A/Vietnam/1203/04, A/lndonesia/05, A/mandarin/kr/2010, A/ck/kr/2006) have the amino acid sequence SLLTEVETPTRNEWESRSSDSSD (SEQ ID NO:7). Avian type I M2e isolates from H7N3 (A/dk/Kr/2007), H9N2 (A/ck/Kr/2012) have the amino acid sequence SLLTEVETPTRNGWECRCSDSSD (SEQ ID NO:8). Avian type I M2e isolates from H5N1 (A/ck/Kr/Gimje/2008) have the amino acid sequence SLLTEVETPTRHEWECRCSDSSD (SEQ ID NO:9). Avian type I M2e isolates from H5N1 (A/ck/Vietnam/2011) have the amino acid sequence SLLTEVETPTRKEWECRCSDSSD (SEQ ID NO: 10).

[0065] In some embodiments, the avian M2e sequence (referred to herein as “avian type II”) comprises the amino acid sequence LTRNGWGCRCS (SEQ ID NO:11), or a conservative variant thereof having at least 70%, 80%, or 90% sequence identity to SEQ ID NO:11. For example, avian type II M2e isolates from H5N1 (A/HK/156/97), H9N2 (A/HK/1073/99) have the amino acid sequence SLLTEVETLTRNGWGCRCSDSSD (SEQ ID NO:12).

[0066] In some embodiments, the human M2e sequence comprises the amino acid sequence PIRNEWGSRSN (SEQ ID NO:13), or a conservative variant thereof having at least about 70%, 80%, or 90% sequence identity to SEQ ID NO:13 (i.e., one, two, or three conservative amino acid substitutions). For example, human M2e isolates H1 N1 (A/PR8, A/NC/99) and H3N2 (A/Phil/82) have the amino acid sequence SLLTEVETPIRNEWGSRSNDSSD (SEQ ID NO:14).

[0067] In some embodiments, the NA sequence comprises the amino acid sequence MKFLVNVALVFMVVYISYIYADHHHHHHDDDDKIINETADDIVYRLTVIIDDRYESLKNLITLRADR LEMIINDNVSTILASGGSGGLEHSIHTGNQHQSEPISNTNFLTEKAVASVKLAGNSSLCPINGWA VYSKDNSIRIGSKGDVFVIREPFISCSHLECRTFFLTQGALLNDKHSNGTVKDRSPHRTLMSCPV GEAPSPYNSRFESVAWSASACHDGTSWLTIGISGPDNGAVAVLKYNGIITDTIKSWRNNILRTQ ESECACVNGSCFTVMTDGPSNGQASHKIFKMEKGKVVKSVELDAPNYHYEECSCYPNAGEIT CVCRDNWHGSNRPWVSFNQNLEYQIGYICSGVFGDNPRPNDGTGSCGPVSSNGAYGVKGFS FKYGNGVWIGRTKSTNSRSGFEMIWDPNGWTETDSSFSVKQDIVAITDWSGYSGSFVQHPEL TGLDCIRPCFWVELIRGRPKESTIWTSGSSISFCGVNSDTVGWSWPDGAELPFTIDK (SEQ ID NO:25), or a conservative variant thereof having at least about 70%, 80%, or 90% sequence identity to SEQ ID NO:25 (i.e., one, two, or three conservative amino acid substitutions).

[0068] In some embodiments, the NA sequence comprises the amino acid sequence MKFLVNVALVFMVVYISYIYADHHHHHHDDDDKIINETADDIVYRLTVIIDDRYESLKNLITLRADR LEMIINDNVSTILASGGSGGLETLHFKQYECDSPASNQVMPCEPIIIERNITEIVYLNNTTIDKEKC PKVVEYRNWSKPQCQITGFAPFSKDNSIRLSAGGDIWVTREPYVSCDHGKCYQFALGQGTTLD NKHSNDTIHDRIPHRTLLMNELGVPFHLGTRQVCIAWSSSSCHDGKAWLHVCITGDDKNATAS FIYDGRLVDSIGSWSQNILRTQESECVCINGTCTVVMTDGSASGRADTRILFIEEGKIVHISPLSG SAQHVEECSCYPRYPGVRCICRDNWKGSNRPVVDINMEDYSIDSSYVCSGLVGDTPRNDDRS SNSNCRNPNNERGNQGVKGWAFDNGDDVWMGRTISKDLRSGYETFKVIGGWSTPNSKSQIN RQVIVDSDNRSGYSGIFSVEGKSCINRCFYVELIRGRKQETRVWWTSNSIVVFCGTSGTYGTG SWPDGANINFMPI (SEQ ID NO:26), or a conservative variant thereof having at least about 70%, 80%, or 90% sequence identity to SEQ ID NO:26 (i.e., one, two, or three conservative amino acid substitutions).

[0069] The core and/or fusion protein may further comprise a signal peptide at the N- terminus to facilitate secretion. For example, the core protein may contain a mellitin signal peptide. In some embodiments, the melittin signal peptide has the amino acid sequence MKFLVNVALVFMVVYISYIYADPINMT (SEQ ID NO:15), or a conservative variant thereof having at least 72%, 76%, 80%, 84%, 88%, 92%, or 96% sequence identity to SEQ ID NO: 15. Alternatively, the fusion protein may contain a baculovirus gp64 signal peptide (MVSAIVLYVLLAAAAHSAFA, SEQ ID NO: 16) or a chitinase signal peptide (MPLYKLLNVLWLVAVSNAIP, SEQ ID NO:17) (Wang, B., et al. J Virol 2007 81: 10869-10878), or a conservative variant thereof having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO:16 or SEQ ID NO:17.

[0070] In some embodiments, the fusion protein containing M2e also contains a multimerization domain, such as a tetramerization domain. An example of a suitable tetramerization domain includes a GCN4 (a leucine zipper tetramerization motif found in yeast proteins). For example, the GCN4 domain can have the amino acid sequence GGLKQIEDKLEEILSKLYHIENELARIKKLLGE (SEQ ID NO:18), or a conservative variant thereof having at least 70%, 73%, 76%, 79%, 82%, 85%, 88%, 91%, 94%, or 97% sequence identity to SEQ ID NO:18. In particular embodiments, the M2e fusion protein contains a GCN4 tetramerization domain. Other identified tetramerization domains include tetrabrachion protein, tumor suppressor p53 tetramerization domain, C-terminal 40-residue peptide of the AChE (tryptophan amphiphilic tetramerization (WAT) domain), erythrocyte spectrin tetramerization domain, and Mnt repressor tetramerization domain. In some embodiments, the M2e fusion protein contains one or more of these other identified tetramerization domains.

[0071] In some embodiments, the series of M2e domains are linked to the N-terminus of the multimerization domain. In some embodiments, the series of M2e domains are linked to the C-terminus of the multimerization domain. In some embodiments, the core protein comprises a series of 2 to 8 M2e domains linked to the N-terminus of the multimerization domain and/or a series of 2 to 8 M2e domains linked to the C-terminus of the multimerization domain.

[0072] In some embodiments, the core protein further comprises influenza neuraminidase (NA) protein linked to the multimerization domain. In some embodiments, the series of 2 to 8 M2e domains are linked to the N-terminus of the multimerization domain and/or the NA protein is linked to the C-terminus of the multimerization domain.

[0073] The M2e domains can be linked to each other by a flexible linker. Likewise, in some embodiments, the multimerization domain is linked to the M2e domains, the NA protein, or any combination thereof, by a flexible linker. Suitable flexible linkers can be, for example, a peptide having 3, 4, 5, 6, 7, 8, or 9 amino acid selected from glycine, alanine, and serine. For example, the flexible linker can have the amino acid sequence GGSGGG (SEQ ID NO: 19).

[0074] The disclosed nanoparticle can be used by itself, or it can be coated with another antigen, such as an influenza antigen. This nanoparticle can in some embodiments be coated with the disclosed hrHA polypeptide. In some embodiments, the antigen is coated on the nanoparticle using a crosslinking agent. In some embodiments, the antigen is absorbed onto the nanoparticle surface. In some embodiments, the antigen is absorbed onto the nanoparticle surface followed by covalent crosslinking of the antigen to the nanoparticle surface using a crosslinking agent.

[0075] Crosslinking agents suitable for crosslinking the core protein to produce the nanoparticle, or to coat an antigen on the nanoparticle are known in the art, and include those selected from the group consisting of formaldehyde, formaldehyde derivatives, formalin, glutaraldehyde, glutaraldehyde derivatives, a protein cross-linker, a nucleic acid cross-linker, a protein and nucleic acid cross-linker, primary amine reactive crosslinkers, sulfhydryl reactive crosslinkers, sulfydryl addition or disulfide reduction, carbohydrate reactive crosslinkers, carboxyl reactive crosslinkers, photoreactive crosslinkers, cleavable crosslinkers, AEDP, APG, BASED, BM(PEO)3, BM(PEO)4, BMB, BMDB, BMH, BMOE, BS3, BSOCOES, DFDNB, DMA, DMP, DMS, DPDPB, DSG, DSP, DSS, DST, DTBP, DTME, DTSSP, EGS, HBVS, sulfo- BSOCOES, Sulfo-DST, and Sulfo-EGS.

Headless HA Polypeptide [0076] Disclosed herein is a polypeptide comprising a truncated influenza hemagglutinin (HA) protein lacking a head domain (a head-removed HA (hrHA) polypeptide). The HA protein can be of any subtype and from any strain. The following is the amino acid sequence for the influenza A H3 stalk ectodomain: MKTIIALSYIFCLPLGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSTGK ICNNPHRILDGIDCTLIDALLGDPHCDVFQNETWDLFVERSKAFSNCYPYDVPDYASLRSLVASS GTLEFITEGFTWTGVTQNGGSNACKRGPGSGFFSRLNWLTKSGSTYPVLNVTMPNNDNFDKL YIWGIHHPSTNQEQTSLYVQASGRVTVSTRRSQQTIIPNIGSRPWVRGLSSRISIYWTIVKPGDV LVINSNGNLIAPRGYFKMRTGKSSIMRSDAPIDTCISECITPNGSIPNDKPFQNVNKITYGACPKY VKQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKST QAAIDQINGKLNRVIEKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHT IDLTDSEMNKLFEKTRRQLRENAEEMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRF QIKGVELKSGYK (SEQ ID NO:1). The corresponding amino acid sequences for other HA subtypes are known. Therefore, reference to specific amino acids within SEQ ID NO:1 is also a reference to the corresponding amino acids in the known amino acid sequences for the other subtypes.

[0077] In some embodiments, the disclosed polypeptide lacks at least the HA amino acids corresponding to residues 65 to 320 of SEQ ID NO:1. In some embodiments, the disclosed polypeptide lacks at least the HA amino acids corresponding to residues 62 to 322 of SEQ ID NO:1. In some embodiments, the disclosed polypeptide lacks at least the HA amino acids corresponding to residues 62 to 320 of SEQ ID NO:1. In some embodiments, the disclosed polypeptide lacks at least the HA amino acids corresponding to residues 62 to 321 of SEQ ID NO:1. In some embodiments, the disclosed polypeptide lacks at least the HA amino acids corresponding to residues 63 to 320 of SEQ ID NO:1. In some embodiments, the disclosed polypeptide lacks at least the HA amino acids corresponding to residues 63 to 321 of SEQ ID NO:1. In some embodiments, the disclosed polypeptide lacks at least the HA amino acids corresponding to residues 63 to 322 of SEQ ID NO:1. In some embodiments, the disclosed polypeptide lacks at least the HA amino acids corresponding to residues 64 to 320 of SEQ ID NO:1. In some embodiments, the disclosed polypeptide lacks at least the HA amino acids corresponding to residues 64 to 321 of SEQ ID NO:1. In some embodiments, the disclosed polypeptide lacks at least the HA amino acids corresponding to residues 64 to 322 of SEQ ID NO:1. In some embodiments, the disclosed polypeptide lacks at least the HA amino acids corresponding to residues 65 to 321 of SEQ ID NO:1. In some embodiments, the disclosed polypeptide lacks at least the HA amino acids corresponding to residues 65 to 322 of SEQ ID NO:1.

[0078] Methods and compositions disclosed herein for the sequence of any particular HA are applicable to the sequences of other HA. Methods and compositions from any of the particular HA delineated in this specification may be adapted routinely to another HA for those skilled in the art. Thus, in one embodiment, a sequence for influenza A HA is used for production of hrHA. In one embodiment, a sequence for influenza B HA is used for production of hrHA. In one embodiment, a sequence for influenza A H1 HA is used for production of hrHA. In one embodiment, a sequence for influenza A H1 , H3, H5, H7 or H9 HA is used for production of hrHA.

[0079] In some embodiments, the disclosed polypeptide is a fusion protein. Fusion proteins, also known as chimeric proteins, are proteins created through the joining of two or more genes which originally coded for separate proteins. Translation of this fusion gene results in a single polypeptide with functional properties derived from each of the original proteins. Recombinant fusion proteins can be created artificially by recombinant DNA technology for use in biological research or therapeutics.

[0080] The functionality of fusion proteins is made possible by the fact that many protein functional domains are modular. In other words, the linear portion of a polypeptide which corresponds to a given domain, such as a tyrosine kinase domain, may be removed from the rest of the protein without destroying its intrinsic enzymatic capability. Thus, any of the herein disclosed functional domains can be used to design a fusion protein. For example, the head domain of HA has a function in the binding to proteins on a cell’s surface and in fusion of the viral particle with the cell, while the stalk domain of HA has a structural role in placing the head domain in space away from the viral membrane.

[0081] A recombinant fusion protein is a protein created through genetic engineering of a fusion gene. This typically involves removing the stop codon from a cDNA sequence coding for the first protein, then appending the cDNA sequence of the second protein in frame through ligation or overlap extension PCR. That DNA sequence will then be expressed by a cell as a single protein. The protein can be engineered to include the full sequence of both original proteins, or only a portion of either, or only a portion of both.

[0082] To create an antigenic HA that consists of the conserved stalk domain in the absence of the variable head domain, the head domain is removed from the HA sequence, resulting in hrHA. In order to maintain the overall structural integrity of HA in the absence of the head domain, the head domains in HA are preferably replaced with linkers (or “spacers”) that do not affect the secondary structure of the HA protein. For example, the head domain can be replaced by a linker 3 to 5 amino acids in length that do not form a fixed secondary structure. In some embodiments, the linker comprises 3 to 5 amino acids selected from glycine, alanine, and serine. In particular embodiments, the linker is selected from the group consisting of GGG, GGGG (SEQ ID NO:20), GGGGG (SEQ ID NO:21), and GGGGC (SEQ ID NO:22), GGGSS (SEQ ID NO:23).

[0083] In some embodiments, the hrHA polypeptide comprises the transmembrane and cytoplasmic domains of HA. In some embodiments, the polypeptide lacks the HA transmembrane and/or cytoplasmic domains of HA, and instead the polypeptide comprises a heterologous membrane-anchoring sequence. For example, the heterologous membraneanchoring sequence can be a glycosylphosphatidylinositol (GPI) membrane-anchoring sequence. In some embodiments, the hrHA polypeptide lacks the HA transmembrane and/or cytoplasmic domain and consists only of the ectodomain or some proportion of the ectodomain.

[0084] In some embodiments, the hrHA polypeptide is a stand-alone protein. In some embodiments, the hrHA polypeptide is a stand-alone protein that is not fused to another protein or protein domain. In some embodiments, the hrHA polypeptide is a domain that is fused together with another protein or protein domain to form a fusion protein. In some embodiments, the hrHA polypeptide forms a fusion protein with an oligomerization domain. In some embodiments, the hrHA polypeptide forms a fusion protein with a GCN4 oligomerization domain. In some embodiments, the hrHA polypeptide forms a fusion protein with a GCN4 oligomerization domain that forms trimers. In some embodiments, the hrHA polypeptide forms a fusion protein with a GCN4 oligomerization domain that forms dimers or tetramers.

[0085] Also disclosed is a nanoparticle that is coated with a disclosed hrHA polypeptide. In some cases, the hrHA polypeptide is crosslinked to a polymer nanoparticle surface. In emboidments, the hrHA polypeptide is absorbed onto the nanoparticle surface. In some embodiments, the hrHA polypeptide is absorbed onto the nanoparticle surface and then crosslinked to the nanoparticle surface. In some embodiments, the hrHA polypeptide is encapsulated into the nanoparticle. In particular embodiments, the nanoparticle is formed from a biocompatible polymer. Examples of biocompatible polymers include polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, or polyamines, or combinations thereof. In some cases, the nanoparticle is formed from a polyethylene glycol (PEG), poly(lactide-co-glycolide) (PLGA), polyglycolic acid, poly-beta-hydroxybutyrate, polyacrylic acid ester, or a combination thereof. In embodiments, the nanoparticle is formed from polypeptides. In some embodiments, the nanoparticle is formed by desolvation of polypeptides. In some embodiments, the nanoparticle is formed by desolvation of polypeptides into protein aggregates. In some embodiments, the nanoparticle is formed by desolvation of polypeptides into protein aggregates with defined physicochemical characteristics that are directly determined by the parameters of the treatment methods used for desolvation.

Vaccine Compositions

[0086] Disclosed are vaccine compositions that comprise one or more of the polypeptides or nanoparticles described above. Although not required, the vaccine compositions optionally contain one or more immunostimulants. An immunostimulant refers to essentially any substance that enhances or potentiates an immune response (antibody or cell-mediated) to an exogenous antigen. One preferred type of immunostimulant is an adjuvant.

[0087] Many adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis- or Mycobacterium tuberculosis-derived proteins. The adjuvant may be a submicron oil-in-water emulsion of a metabolizable oil and an emulsifying agent. For example, the adjuvant may comprise MF59™, which is a sub-micron oil-in-water emulsion of a squalene, polyoxyethylene sorbitan monooleate (Tween™ 80) and sorbitan trioleate. The adjuvant may also be a combination of the TLR4 agonist PL (3-O-desacyl-4'- monophosphoryl lipid A) and aluminum salt, e.g., AS04 (GlaxoSmithKline, Philadelphia, Pa.).

[0088] Certain adjuvants are commercially available as, for example, Freund’s Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Rahway, N.J.); AS01, AS02, AS03, and AS04 (GlaxoSmithKline, Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF, interleukin-2, -7, -12, and other like growth factors, may also be used as adjuvants.

[0089] The adjuvant composition can be a composition that induces an antiinflammatory immune response (antibody or cell-mediated). Accordingly, high levels of antiinflammatory cytokines are produced as a result of adjuvant administration. Anti-inflammatory cytokines may include, but are not limited to, interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 10 (IL-10), and transforming growth factor beta (TGF ). [0090] The adjuvant composition can be a composition that induces an inflammatory immune response (antibody or cell-mediate). Accordingly, high levels of inflammatory cytokines are produced as a result of adjuvant administration. Inflammatory cytokines may include, but are not limited to, interleukin 1 alpha (I L-a), interleukin 1 beta (I L-B), interferon gamma (I FNy), and tumor necrosis factor alpha (TNFa). Optionally, an inflammatory response would be mediated by CD4+ T helper cells. Bacterial flagellin has been shown to have adjuvant activity (McSorley et al., J. Immunol. 169:3914-19, 2002). Also disclosed are polypeptide sequences that encode flagellin proteins that can be used in adjuvant compositions.

[0091] Optionally, the adjuvants increase lipopolysaccharide (LPS) responsiveness. Illustrative adjuvants include but are not limited to, monophosphoryl lipid A (MPL), aminoalkyl glucosaminide 4-phosphates (AGPs), including, but not limited to RC-512, RC-522, RC-527, RC-529, RC-544, and RC-560 (Corixa, Hamilton, Mont.).

[0092] In addition, the adjuvant composition can be one that induces an immune response predominantly of the Th1 type. High levels of Th1-type cytokines (e.g., IFN-y, TNFa, IL-2 and IL-12) tend to favor the induction of cell mediated immune responses to an administered antigen. In contrast, high levels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6 and IL- 10) tend to favor the induction of humoral immune responses. Following application of a vaccine as provided herein, a subject will support an immune response that includes Th1- and Th2-type responses. Optionally, the level of Th1-type cytokines will increase to a greater extent than the level of Th2-type cytokines. The levels of these cytokines may be readily assessed using standard assays. Certain adjuvants for eliciting a predominantly Th1-type response include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A, together with an aluminum salt adjuvants are available from Corixa Corporation (Seattle, Wash.). CpG-containing oligonucleotides (in which the CpG dinucleotide is unmethylated) also induce a predominantly Th1 response. Another adjuvant comprises a saponin, such as Quil A, or derivatives thereof, including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham, Mass.); Escin; Digitonin; or Gypsophila or Chenopodium quinoa saponins.

[0093] Additional illustrative adjuvants for use in the disclosed vaccine compositions include Montamide ISA 720 (Seppic, France), SAF (Chiron, Calif., United States), ISCOMS (CSL), MF-59 (Chiron), the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4, available from GlaxoSmithKline, Philadelphia, Pa.), Detox (Enhanzyn™) (Corixa, Hamilton, Mont.), RC-529 (Corixa, Hamilton, Mont.) and other aminoalkyl glucosaminide 4-phosphates (AGPs).

[0094] In some embodiments, the adjuvant is a virosome (e.g. Berna Biotech). In some embodiments, the adjuvant comprises a CpG 1018 and/or CpG 7909 oligonucleotide. In some embodiments, the adjuvant comprises a Imidazoquinoline. In some embodiments, the adjuvant comprises a Polyinosinic:polycytidylic acid (PolykC). In some embodiments, the adjuvant comprises a Pam3Cys. In some embodiments, the adjuvant comprises a ISCOMATRIX adjuvant. In some embodiments, the adjuvant comprises a CAF01 and/or IC31 adjuvant. In some embodiments, the adjuvant comprises a Sigma adjuvant system (MPL from Salmonella Minnesota, synthetic trehalose dicorynomycolate and squalene oil). In some embodiments, the adjuvant comprises TITERMAX (water-in-oil emulsion, consisting of squalene, sorbitan monooleate 80, a block copolymer and microparticulate silica).

[0095] In some embodiments, the adjuvant is incorporated into the VLP in a membrane- anchored form. For example, GM-CSF or a bacterial flagellin protein containing a membrane anchor can be incorporated into the disclosed VLPs.

Pharmaceutical Compositions

[0096] The disclosed vaccines can be used therapeutically in combination with a pharmaceutically acceptable carrier. By “pharmaceutically acceptable” is meant a material that is not biologically or otherwise undesirable, i.e. , the material may be administered to a subject without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.

[0097] The materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (22nd ed.) eds. Loyd V. Allen, Jr., et al., Pharmaceutical Press, 2012. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered. [0098] Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of vaccines to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the vaccine. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like.

[0099] The disclosed vaccines are preferably formulated for delivery via intranasal, intramuscular, subcutaneous, transdermal or sublingual administration.

[0100] Preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

[0101] Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0102] Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.

Combinations

[0103] The disclosed vaccine can be used to supplement existing human vaccines to improve cross protection. Therefore, the disclosed vaccine can further include (or be administered in combination with) a whole inactivated virus, split viral vaccine, live attenuated influenza vaccine, or an influenza virus-like particle (VLP) vaccine. For example, the disclosed vaccine can be combined with a trivalent inactivated vaccine (TIV) (e.g., containing killed A/H1 N1, A/H3N2, and B), trivalent live attenuated influenza vaccine, trivalent split vaccine, trivalent subunit influenza vaccine, trivalent recombinant protein vaccine, or trivalent VLP vaccine. The disclosed vaccine can be combined with a bivalent inactivated vaccine, bivalent live attenuated influenza vaccine, bivalent split vaccine, or bivalent subunit influenza vaccine, bivalent recombinant protein vaccine, or bivalent VLP vaccine. The disclosed vaccine can be combined with a monovalent inactivated vaccine, monovalent live attenuated influenza vaccine, monovalent split vaccine, or monovalent subunit influenza vaccine, monovalent recombinant protein vaccine, or monovalent VLP vaccine. The disclosed vaccine can be combined with a monovalent, bivalent, or trivalent vaccine directed against Influenza A. The disclosed vaccine can be combined with a monovalent, bivalent, or trivalent vaccine directed against Influenza B. The disclosed vaccine can be combined with a monovalent, bivalent, or trivalent vaccine directed against a combination of Influenza A and Influenza B.

[0104] The disclosed vaccine can include a PNP that incorporates sequences from a particular influenza, such as Influenza A or Influenza B. The disclosed vaccine can include a PNP that incorporates sequences from a particular strain of influenza. The disclosed vaccine can include a PNP that incorporates sequences from a subtype of influenza A. The disclosed vaccine can include a PNP that incorporates sequences from a subtype of influenza A HA, such as H1 HA, H3 HA, H5 HA, and others.

[0105] In one embodiment, the vaccine includes a single PNP that incorporates sequences from a particular influenza, such as Influenza A or Influenza B. The disclosed vaccine can include a single PNP that incorporates sequences from a particular strain of influenza. The disclosed vaccine can include a single PNP that incorporates sequences from a subtype of influenza A. The disclosed vaccine can include a single PNP that incorporates sequences from a subtype of influenza A HA, such as H1 HA, H3 HA, H5 HA, and others.

[0106] In one embodiment, the vaccine includes two PNP each of which incorporates sequences from particular influenzas, such as Influenza A or Influenza B. The disclosed vaccine can include two PNP each of which incorporates sequences from a particular strain of influenza. The disclosed vaccine can include two PNP each of which incorporates sequences from a subtype of influenza A. The disclosed vaccine can include two PNP each of which incorporates sequences from a subtype of influenza A HA, such as H1 HA, H3 HA, H5 HA, and others.

[0107] In one embodiment, the vaccine includes three or more PNP each of which incorporates sequences from particular influenzas, such as Influenza A or Influenza B. The disclosed vaccine can include three or more PNP each of which incorporates sequences from a particular strain of influenza. The disclosed vaccine can include three or more PNP each of which incorporates sequences from a subtype of influenza A. The disclosed vaccine can include three or more PNP each of which incorporates sequences from a subtype of influenza A HA, such as H1 HA, H3 HA, H5 HA, and others.

[0108] The disclosed vaccine can further include (or be administered in combination with) one or more of classes of antibiotics, steroids, analgesics, anti-inflammatory agents, anti- histaminic agents, or any combination thereof. Antibiotics include Aminoglycosides, Cephalosporins, Chloramphenicol, Clindamycin, Erythromycins, Fluoroquinolones, Macrolides, Azolides, Metronidazole, Penicillins, Tetracyclines, Trimethoprim-sulfamethoxazole, and Vancomycin. Suitable steroids include andranes, such as testosterone. Narcotic and nonnarcotic analgesics include morphine, codeine, heroin, hydromorphone, levorphanol, meperidine, methadone, oxydone, propoxyphene, fentanyl, methadone, naloxone, buprenorphine, butorphanol, nalbuphine, and pentazocine. Anti-inflammatory agents include alclofenac, alclometasone dipropionate, algestone acetonide, alpha amylase, amcinafal, amcinafide, amfenac sodium, amiprilose hydrochloride, anakinra, anirolac, anitrazafen, apazone, balsalazide disodium, bendazac, benoxaprofen, benzydamine hydrochloride, bromelains, broperamole, budesonide, carprofen, cicloprofen, cintazone, cliprofen, clobetasol propionate, clobetasone butyrate, clopirac, cloticasone propionate, cormethasone acetate, cortodoxone, decanoate, deflazacort, delatestryl, depo-testosterone, desonide, desoximetasone, dexamethasone dipropionate, diclofenac potassium, diclofenac sodium, diflorasone diacetate, diflumidone sodium, diflunisal, difluprednate, diftalone, dimethyl sulfoxide, drocinonide, endrysone, enlimomab, enolicam sodium, epirizole, etodolac, etofenamate, felbinac, fenamole, fenbufen, fenclofenac, fenclorac, fendosal, fenpipalone, fentiazac, flazalone, fluazacort, flufenamic acid, flumizole, flunisolide acetate, flunixin, flunixin meglumine, fluocortin butyl, fluoromethoIone acetate, fluquazone, flurbiprofen, fluretofen, fluticasone propionate, furaprofen, furobufen, halcinonide, halobetasol propionate, halopredone acetate, ibufenac, ibuprofen, ibuprofen aluminum, ibuprofen piconol, ilonidap, indomethacin, indomethacin sodium, indoprofen, indoxole, intrazole, isoflupredone acetate, isoxepac, isoxicam, ketoprofen, lofemizole hydrochloride, lomoxicam, loteprednol etabonate, meclofenamate sodium, meclofenamic acid, meclorisone dibutyrate, mefenamic acid, mesalamine, meseclazone, mesterolone, methandrostenolone, methenolone, methenolone acetate, methylprednisolone suleptanate, momiflumate, nabumetone, nandrolone, naproxen, naproxen sodium, naproxol, nimazone, olsalazine sodium, orgotein, orpanoxin, oxandrolane, oxaprozin, oxyphenbutazone, oxymethoIone, paranyline hydrochloride, pentosan polysulfate sodium, phenbutazone sodium glycerate, pirfenidone, piroxicam, piroxicam cinnamate, piroxicam olamine, pirprofen, prednazate, prifelone, prodolic acid, proquazone, proxazole, proxazole citrate, rimexolone, romazarit, salcolex, salnacedin, salsalate, sanguinarium chloride, seclazone, sermetacin, stanozolol, sudoxicam, sulindac, suprofen, talmetacin, talniflumate, talosalate, tebufelone, tenidap, tenidap sodium, tenoxicam, tesicam, tesimide, testosterone, testosterone blends, tetrydamine, tiopinac, tixocortol pivalate, tolmetin, tolmetin sodium, triclonide, triflumidate, zidometacin, and zomepirac sodium. Anti-histaminic agents include ethanolamines (e.g., diphenhydrmine carbinoxamine), Ethylenediamine (e.g., tripelennamine pyrilamine), Alkylamine (e.g., chlorpheniramine, dexchlorpheniramine, brompheniramine, triprolidine), other antihistamines like astemizole, loratadine, fexofenadine, bropheniramine, clemastine, acetaminophen, pseudoephedrine, triprolidine).

Methods of Vaccinating a Subject

[0109] A method of vaccinating a subject for influenza is disclosed that involves administering the disclosed cross- protective influenza vaccine to a subject in need thereof. The disclosed vaccine may be administered in a number of ways. For example, the disclosed vaccine can be administered intramuscularly, intranasally, or by microneedle in the skin. The compositions may be administered orally, intravenously, subcutaneously, transdermally (e.g., by microneedle), intraperitoneally, ophthalmically, vaginally , rectally, sublingually, or by inhalation.

[0110] Parenteral administration of the composition, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained.

[0111] The exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. For example, effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms disorder are affected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. A typical dosage of the disclosed vaccine used alone might range from about 1 pg/kg to up to 100 mg/kg of body weight or more per vaccination, such as 10 pg/kg to 50 mg/kg, or 50 pg/kg to 10 mg/kg, depending on the factors mentioned above. In addition to dosing by the ratio of mass-of-vaccine to mass-of- patient, standardized vaccine doses for demarcated demographics can also be used. A typical dose for an adult patient may be 1 pg to 10OOpg, or 10pg to 150pg, or 15pg to 135pg per subject. A typical dose for a child patient may be 1 pg to 1000pg, or 10pg to 150pg, or 15pg to 135pg per subject. A typical dose for an elderly patient may be 1 pg to 1000pg, or 10pg to 150pg, or 15pg to 135pg per subject.

[0112] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

EXAMPLES

Example 1 : Layered Protein Nanoparticles Containing Influenza B HA stalk Inducted Sustained Cross-Protection against Viruses Spanning Both Viral Lineages

Introduction

[0113] Influenza virus infection leads to severe contagious respiratory diseases with high mortality and morbidity worldwide. Approximately one-quarter of clinical infection cases are caused by influenza B viruses yearly. Sometimes, they are dominant circulating strains in various influenza seasons, such as the initial 2019-20 US flu season with more than 50% infections. There are two lineages of the influenza B virus, the B/Yamagata/16/88-like lineage (Yamagata-like) and the B/Victoria/02/87-like lineage (Victoria-like), co-circulating in the human population since the 1980s[3], The two lineages are genetically and antigenically distinct based on their hemagglutinin (HA) surface glycoproteins.

[0114] To prevent influenza epidemics each year, seasonal influenza virus vaccines have been developed containing one (the trivalent influenza virus vaccine, TIV) or both (quadrivalent influenza virus vaccine, QIV) lineages of influenza B viruses. However, the vaccines sometimes are less effective with suboptimal protection due to HA antigenic drift, especially on the HA immune-dominant head domain with high plasticity. Thus, influenza vaccines need to be re-formulated and updated frequently. Influenza B HA protein contains major immune-dominant sites in the head domain that can induce strong strain-specific antibody responses in the hosts. The evolution of influenza HA head domains enables the virus to escape the pre-existing immunity by natural infection or vaccination. Conserved antigens have been recognized as ideal vaccine components as these antigens could induce durable immune protection with improved potency and breadth. To overcome these limitations, a universal influenza vaccine containing conserved antigens and providing substantial broadly crossprotection against diverse virus strains is urgently needed.

[0115] The HA stalk domain is immuno-subdominant but more conserved than the variable globular head domain. The immunogenicity of the HA stalk domain has been studied via different approaches. A human clinical study revealed that HA stalk domains from influenza A and B viruses could elicit broadly cross-reactive antibodies by natural infection. Some monoclonal antibodies have been proven to provide substantial protection against homologous and heterologous virus strains. By using chimeric HA (cHA, switching HA head domains to those from different exotic viruses) or mosaic HA (mHA, silencing the immunodominant antigenic sites of head domains) strategies, broadly cross-reactive antibody responses and immune protection were induced against influenza infections. Some stabilized HA stalk domains from influenza A viruses have been constructed via structure-based rational designs and conferred broad protection in laboratory animals. These findings indicate that the HA stalk domain could be a promising antigenic component of a universal influenza vaccine.

[0116] Nucleoprotein (NP) is an internal influenza protein containing conserved T cell epitopes that trigger cross-protection against diverse influenza virus infections. CD8⁺ cytotoxic T-cells (CTL) response is important to protect the host against pathogen infection. Clinical studies have shown that high-level CD8⁺ CTL responses correlate with reduced viral shedding and less severity of infections. Influenza-specific CD8⁺ T lymphocytes have been proven to protect different subtypes by producing cytokines and killing infected cells to control the viral infection. Thus, the antigen-specific CTL response is an important mediator of broadly crossprotection against different influenza viruses. Influenza vaccines based on highly conserved antigens like influenza A M2e and NP have been extensively evaluated on animal models with promising cross-protection against various influenza A infections. Therefore, a universal influenza vaccine that combines conserved antigens with rational design and methodology might elicit broadly immune protection. [0117] Nanoparticles are considered promising platforms for developing new influenza vaccines[30]. Nanoparticles can be taken by antigen-presenting cells (APCs) such as dendritic cells (DCs) and stimulate APC maturation followed by proinflammatory cytokine and chemokine production, stimulating downstream humoral and cellular immune responses. Compared with traditional vaccine formulation, protein nanoparticle vaccines are almost entirely composed of antigens of interest and avoid non-specific responses to carriers. By rational design, some conserved immune-subdominant antigens could be constructed and presented on the nanoparticles retaining their natural biophysical and biochemical features. Studies show that the nanoparticle vaccine candidates like self-assembling ferritin particles, poly (lactic-co-glycolic acid) (PLGA) polymer particles, and inorganic particles presenting the natural structures of conserved antigens could elicit improved antigen-specific immune responses. However, these nanoparticles also have disadvantages, such as weak biocompatibility or off-target immune responses due to their unique structures and compositions.

[0118] Our previous proof-of-concept studies developed a desolvation protein nanoparticle approach to fabricate layered nanoparticles by reducible crosslinkers. Since the components of the nanoparticles are almost pure with a minor amount of crosslinkers for fabrication, the layered nanoparticles have a high density of target antigens and are highly biocompatible. The homobifunctional and water-soluble amine-reactive redox-responsive crosslinker, 3,3'-dithiobis [sulfosuccinimidylpropionate] (DTSSP) has been widely used to successfully cross-link the external shell of various proteins like viral antigens or enzymes. Protein nanoparticles fabricated by DTSSP have been reported stable at a neutral pH and 37°C environment and retained the natural structures as well as functions of loaded proteins. The specific cross-linker is composed of two N-hydroxysulfosuccinimide (sulfo-NHS) moieties capable of reacting with primary amine groups of the lysine side chains via covalent amide bonds. In addition, the disulfide bond within the DTSSP molecule could be cleaved to disrupt crosslinking networks conducting a responsive controlled release of soluble antigens in a reducing environment of the cytosol after the cellular uptake. Our previous results demonstrated the distribution, release, and biodegradation of nanoparticle components in the physiological redox condition, indicating the potential immunogenicity of the layered nanoparticles by controlling the activation of innate immune cells. The safety of protein nanoparticles was also carefully investigated previously by comprehensive analysis in mice after immunizations and no apparent side effects were observed.

[0119] We have crosslinked stabilized influenza A head-removed HA (hrHA) on the M2e nanoparticle cores to fabricate double-layered nanoparticles that have been proven to induce broadly cross-protective antibody responses in animal models. In this study, we constructed structurally stabilized influenza B hrHA (B/hrHA) protein based on HA from B/Brisbane/60/2008 (Victoria lineage) and fabricated layered protein nanoparticles of NP (B/Yamanashi/166/1998, Yamagata lineage) cores and B/hrHA shells. We also investigated the immunogenicity of the nanoparticles, evaluated cross-protection against virus challenges from both lineages, and analyzed the protective mechanisms.

Materials and Methods

[0120] Ethics Statement. This study was carried out in strict accordance with the recommendation in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal studies were approved by the Georgia State University Institutional Animal Care and Use Committee (IACUC) under protocol No. A19012.

[0121] Cell Lines and Viruses. Sf9 insect cells (ATCC CRL-1711), Madin-Darby Canine Kidney cells (MDCK, ATCC CCL-34), and JAWS II murine dendritic cell line (ATCC CRL-11904) were grown in vendor recommended conditions.

[0122] Influenza B virus strains: B/Malaysia/2506/2004 (Victoria lineage) and B/Florida/4/2006 (Yamagata lineage) were passaged in our lab. B/Brisbane/60/2008 (Victoria lineage) (BEI Resources, NR-42005) were obtained from BEI Resources. Viruses were expanded in embryonated chicken eggs described previously and purified by sucrose density gradient centrifugation. HA activity of the viruses was measured using 0.5% turkey red blood cells. Median Tissue Culture Infectious Dose (TCID50) was determined on MDCK cells by the method of Reed and Muench, and protein concentrations were quantified by bicinchoninic acid (BCA) assay according to the manufacturer’s instruction (Thermo Fisher Scientific). The median Lethal Dose (LD50) of the challenge strains B/Malaysia/2506/2004 and B/Florida/4/2006 were determined in mice. Viruses were inactivated following the procedures previously described[5] and the protein concentrations and HA titers were determined as mentioned above.

[0123] Design, Expression, Purification, and Characterization of Recombinant Proteins. According to previous studies, the construction of influenza B/hrHA was based on the coding sequence of influenza B/Brisbane/60/2008 (Victoria lineage) HA protein (GenBank Protein Accession: ANC28539.1). The nucleotide sequence encoding the head domain in the HA1 region from P51 to W339 was replaced with a sequence encoding a flexible 4XGIycine linker (4G) that has been proved not to disrupt the structural folding of the leftover molecule. To block the long helix formation with helix A and C in the low pH condition and stabilize the natural structure in the pre-fusion state, the sequence coding the distal part of HA2 loop B from V422 to D450 was replaced with a flexible no-hydrophobic 3GS linker. To avoid cleavage of the B/hrHA in the physiological condition, two side mutations (K360T and R362Q) were introduced into the cleavage region in HA1 . To further stabilize the B/hrHA structure, the S455C mutation was introduced to form an intra-disulfide bond in HA2. A trimerization GCN4 (TN-GCN4) encoding sequence was fused downstream of the B/hrHA coding sequence connected by a PGS linker sequence and followed by a six-histidine-tag coding sequence for the protein oligomerization and purification. The honeybee melittin secreting signal sequence was employed to facilitate the secreted expression of the recombinant protein. The construction of nucleoprotein (NP) was based on the B/Yamanashi/166/1998 (Yamagata lineage) NP protein (GenBank Protein Accession: ABN50508.1). The coding sequence of NP was in frame with the honeybee melittin SS encoding sequence for secreting expression and a six-histidine-tag coding sequence for purification. Flagellin (FliC) and soluble 4M2e (s4M2e) proteins were constructed and purified previously.

[0124] The encoding genes of recombinant proteins were synthesized and cloned into pFastBac to generate recombinant baculoviruses (rBVs). Recombinant proteins were expressed in Sf9 insect cells (Invitrogen) infected by the rBVs and purified using Ni-NTA resins (Thermo Fisher Scientific) as previously described. Purified recombinant proteins were characterized by SDS-PAGE followed by Coomassie Blue (Bio-Rad) staining and Western blots using anti-His (Invitrogen) and anti-influenza B NP antibodies (Invitrogen). The concentrations of purified recombinant proteins were measured by BCA assay. Polymerization of B/hrHA was determined by BS3 cross-linking at a 2-fold series of dilution from 5mM with 1 pg protein followed by Western blots using an anti-His antibody as described previously.

[0125] Nanoparticle Fabrication and Characterization. A double-layered B/hrHA- NP nanoparticle, termed Nano, was made following the procedure described previously with corresponding modification for different proteins. Briefly, the NP protein in PBS was desolvated by adding a 4-fold volume of absolute ethanol under stirring at 600rpm at room temperature. Desolvated NP nanocores were pelleted by centrifugation at 4°C and resuspended in PBS by sonication at a 40% amplitude on ice. Equal amounts of B/hrHA proteins were added to the NP nanocores for crosslinking using 3,3'-dithiobis [sulfosuccinimidylpropionate] (DTSSP; Thermo Fisher Scientific, Cat. No. 21578) under stirring at room temperature for 1 hr. Centrifugation (15,000 rpm for 30min at 4°C) was followed to pellet nanoparticles. Pellets were resuspended in PBS by sonication at a 40% amplitude on ice.

[0126] Size distribution and surface Zeta- potential of the resulting double-layered nanoparticles were assessed by dynamic light scattering (DLS) with a Malvern Zetasizer Nano ZS (Malvern Instruments). Protein concentration was measured by BCA assay and purity was characterized by SDS-PAGE followed by Coomassie Blue staining. Western blots were performed to determine the composition ratio of different antigens in nanoparticles using GelQuantNET software.

[0127] A transmission electron microscope (TEM) study determined the morphology of nanoparticles. Briefly, a drop (5pl) of nanoparticles suspended in pure water was loaded onto a formvar/carbon-coated TEM grid followed by immediately adding 5pl of 1% phosphotungstic acid (PTA, pH 7.4) to the nanoparticles. After incubation for 1 min, the solution was blotted by blotting papers. Then, the grid was air-dried at room temperature and stored for TEM visualization with a Jeol JEM-100CX II at 100 kV. Digital images were acquired with Apogee Imaging Systems.

[0128] Antigenicity of Recombinant B/hrHA and Nanoparticles. The antigenicity of recombinant B/hrHA and Nano was determined by ELISA binding assay. B/hrHA, Nano, and purified soluble 4M2e (s4M2e, negative control) in two-fold dilution starting from 500ng were used as coating antigens. Binding activity was measured using goat antiserum (BEI Resources, NR-28669) to B/Florida/04/2006 HA at 1:500 dilution and HRP-conjugated anti-goat antibody (Invitrogen).

[0129] Nanoparticle Uptake by JAWS II Cells and Cell Activation. Uptake and internalization of nanoparticles versus their respective soluble recombinant proteins (sProteins) as controls were studied by immunofluorescence imaging in JAWS II cells. JAWS II cells (2x10⁵ cells/well) were seeded in 24-well plates and cultured overnight at 37°C, 5% CO2. Nanoparticles (10 pg) with or without FliC (0.1 pg) were added to the cells. Soluble NP (7.8 pg) and B/hrHA (2.2 pg) proteins were added to the cells, and untreated cells were included as negative controls. After incubation for 16-20 hrs, the cells were washed twice with Dulbecco’s PBS (DPBS) followed by fixation and permeabilization by BD fixation/permeabilization buffer at 4°C for 20min in the dark. The cells were then blocked with 5% BSA in DPBS for 1 hr at room temperature. After being washed with DPBS, the cells were incubated with goat antiserum against B/Florida/04/2006 HA at 1 :500 dilutions for 1 hr at room temperature and stained with FITC-conjugated rabbit anti-goat antibody (1 : 1000) (Invitrogen) for 30min. Cells were fixed with 4% paraformaldehyde and observed by using a Keyence BZ-X710 fluorescence microscope.

[0130] JAWS II cell activation by nanoparticle stimulation was determined by secretion of proinflammatory cytokines (IL-6 and TNF-a) in cell culture supernatants using cytokine ELISA kit (Thermo Fisher Scientific) following the manufacturer’s instructions. Cells were stained with anti-CD86-PE-Cy5 (BD Biosciences) to measure the maturation by flow cytometry as previously described. [0131] Mice Immunization and Virus Challenge. Six to eight-week-old female BALB/c mice were purchased from Jackson Laboratory. Mouse groups (n=5) were immunized intramuscularly (i.m.) with 10 pg Nano, 10 pg B/Nano with 0.1 pg FliC (Nano + FliC), and 10 pg soluble Protein (sProtein; 7.8 pg NP and 2.2 pg B/hrHA) in 50pl PBS twice with a 4-week interval. Mice that received PBS were used as controls.

[0132] Immune sera were collected 3 weeks after each immunization and 6 months after boost immunization for long-term immunity assessment. Inguinal lymph nodes (ILNs) from all the groups were collected 7 days post-prime immunization. Spleens and the bone marrow were collected 3 weeks after boost immunization.

[0133] Mice (n=5) were intranasal (i.n.) challenged with 5xLD₅o influenza B/Malaysia/2506/2004 (Victoria lineage) and B/Florida/4/2006 (Yamagata lineage) viruses in 50pl PBS respectively 4 weeks after the boost immunization. The body weight and survival were monitored daily for 2 weeks post-infection. The weight loss of over 20% was determined as the humane endpoint according to the IACUC protocol. Lung tissues and bronchoalveolar lavage fluid (BALF) were isolated from euthanized mice for viral titer detection, histology study, and proinflammatory response evaluation 5 days after infection.

[0134] For sera passive transmission, 300 pl pooled sera from each immunization group were intraperitoneally (i.p.) injected into naive mice (n=5) one day before viral challenges with 3X LD₅O influenza B/Malaysia/2506/2004 and B/Florida/4/2006 viruses, respectively. The mouse survival and body weight changes after infection were recorded every day for 2 weeks.

[0135] Antibody Responses. Antibody binding titers in immune sera were determined by sera enzyme-linked immunosorbent assay (ELISA) for total IgG, lgG1 , and lgG2a levels by using 96-well ELISA plates (Thermo Fisher Scientific) pre-coated with 5 pg/ml purified influenza B viruses mentioned above or with 2 pg/ml purified HA proteins from B/Ohio/1/2005 (Victoria lineage; BEI Resources, NR-19243), B/Hong Kong/330/2001 (Victoria lineage; BEI Resources, NR-43780), B/Sydney/507/2006 (Yamagata lineage; SinoBiological) and B/Jilin/20/2003 (Yamagata lineage; BEI Resources, NR-19242) respectively at 50 pl/well at 4°C overnight. Endpoint titers were determined as the highest dilution with an OD450 value three times higher than negative control wells.

[0136] Hemagglutination-inhibition (HAI) titers of immune sera were measured as previously described[50]. Briefly, serially diluted sera pretreated with receptor destroying enzyme (Denka Seiken Co., Ltd) at 37°C overnight and then heat-inactivated at 56°C for 30min were incubated with 4 HA unit of purified viruses followed by adding 0.5% turkey red blood cells to each well. The highest dilution able to inhibit virus hemagglutination was determined as the HAI titer.

[0137] For the Micro-neutralization assay, two-fold diluted heat-inactivated immune sera in Dulbecco's Modified Eagle Medium (DMEM) were mixed with 1OOxTCID₅o purified influenza B viruses. After incubation for 2 hrs at 33°C, the mixture was added to MDCK cells (pre-seeded at 2 *10⁴ cell/well in a 96-well plate one day before) with 2 pg/ml TBCK-trypsin and incubated at 33°C for 72 hrs. A standard hemagglutination assay with 0.5% turkey red blood cells was used to detect the virus inhibition and then determined the neutralization titer.

[0138] Antibody-dependent cellular cytotoxicity (ADCC) assay was performed using the ADCC reporter bioassay kit (Promega Life Sciences) as previously described with some modifications. Briefly, the MDCK cells (pre-seeded at 2 xio⁴ cells/well in 96-well plate one day before) were infected with the purified virus at MOI of 5 overnight for single-cycle replication. The cells were washed with PBS the next day and supplemented with serially diluted heat- inactivated immune sera in Roswell Park Memorial Institute (RPMI) medium (Thermo Fisher Scientific). A stable Jurkat cell line expressing mouse FcyRIV (Promega Life Sciences) was added and incubated for 6 hrs at 37°C. Then, cells were equilibrated to room temperature for 15 min, and Bio-Gio luciferase assay substrate (Promega Life Sciences) was added to each well and incubated at room temperature for 5 min. Luminescence was read out on a GloMax (Promega Life Sciences) and fold induction over the baseline was calculated.

[0139] Cellular Immune Responses. Antigen-specific IL-4 or INF-y-secreting cells were measured by the Enzyme-linked immunospot (ELISpot) assay as described before. Three weeks after boost immunization, splenocytes (3x10⁵ cells/well) were isolated and seeded into 96-well filtration plates (Millipore) pre-coated with anti-mouse IL-4 or INF-y capture antibodies (BioLegend). Peptide pools (2 pg/ml) of B/Brisbane/60/2008 HA (BEI Resources, NR-19247), B/Florida/4/2006 NP (BEI Resources, NR-36045), and HA (BEI Resources, NR-18972) were used for stimulation respectively. After incubation at 37°C for 48 hrs, plates were incubated with biotin-conjugated IL-4 or INF-y detection antibodies (BioLegend) followed by the addition of HRP-streptavidin (BioLegend). Results were recorded by Bioreader-6000-E (BIOSYS).

[0140] Antigen-specific antibody-secreting cells (ASCs) were evaluated by the B cell ELISpot assay. Single-cell suspensions were made from spleens and bone marrows collected 3 weeks after boost immunization. The cells were seeded (3x10⁵ cells/well) into 96-well filtration plates (Millipore) pre-coated with purified HA proteins from B/Malaysia/2506/2004 (BEI Resources, NR-51162), B/Brisbane/60/2008 (BEI Resources, NR-19239) and B/Florida/4/2006 (BEI Resources, NR-15169) respectively. After incubation at 37°C overnight, HRP-conjugated anti-mouse IgG was added. Spots were counted with Bioreader-6000-E (BIOSYS).

[0141] Memory B cells were detected as previously described[60], Splenocytes and bone marrow cells (1 *10⁶ cells/ml) were stimulated with 1 pg/ml R848 (Mabtech) and 10 ng/ml recombinant IL-2 (Mabtech) in R-10 media at 37°C, 5% CO2 for 5 days. Cells were harvested and washed with R-10 media and were seeded to the ELISpot plates as mentioned in the above ELISpot assay.

[0142] Flow Cytometry. For germinal center (GC) B cell detection, inguinal lymph nodes (ILNs) were collected 7 days after primary immunization and homogenized lymphocytes were stained with B220-PE and GL7-Pacific Blue.

[0143] For weeks after boost immunization, mice were challenged with I xLDso influenza B/Malaysia/2506/2004 virus. Lungs were collected 5 days after infection and homogenized into single-cell suspensions by Percoll (Sigma Aldrich) gradient centrifugation. Cells were stimulated with 2 pg/ml B/Brisbane/60/2008 HA (BEI Resources, NR-19247) and B/Florida/4/2006 NP (BEI Resources, NR-36045) peptide pools respectively in R-10 medium with 2 pg/ml Golgi stopper (BD Biosciences) as well as purified anti-mouse CD28 (BD Biosciences) and anti-mouse CD49d (BD Biosciences) as co-stimulators. Cells were then stained with surface maker CD3-PE (BD Biosciences), CD8-FITC (BD Biosciences), and CD4-PerCP (BD Biosciences). After fixation and permeabilization, cells were stained with IFN-y-BV711 (BioLegend). Cells were acquired on a BD LSRFortessa flow cytometer (BD Biosciences) and data were analyzed by FlowJo software (FlowJo LLC).

[0144] Lung Viral Titer and Histology. On day 5 after the virus challenge, mice were euthanized, and lung tissues were collected and homogenized in a cold DM EM medium. Supernatants were harvested by centrifugation at 500 ref for 15 min at 4°C. 10-fold diluted lung homogenate supernatants in DMEM were added to MDCK cells (2x10⁴ cells/well) pre-seeded in 96-well plates and cultured for 5 days at 33°C, 5% CO2. A standard HA assay was performed by mixing culture supernatants with 0.5% turkey red blood cells to calculate the viral titers by the Reed-Muench method.

[0145] Lung tissues were fixed in 10% neutral buffered formalin overnight at 4°C. Fixed samples were dehydrated and embedded in paraffin for sectioning. Lung sections were stained with hematoxylin and eosin (H&E). Tissue pathogenesis was recorded and analyzed by a Keyence BZ-X710 microscope for leukocyte infiltration scores. [0146] Inflammatory Cytokine Levels in BALF. BALF was collected 5 days after the virus challenge. Inflammatory cytokines, IL-6, TNF-a, and IL-12, were determined by cytokine ELISA kits following the manufacturer’s instructions (Thermo Fisher Scientific).

[0147] Statistical Analysis. Data were represented by mean ± SEM. One-way ANOVA followed by Tukey’s multiple comparison post-test was used to analyze statistical significance with a P value less than 0.05 considered statistically significant. Log-rank test was used for comparison of survival rate. Data analysis was performed with Graphpad Prism (GraphPad Software).

Results

[0148] Characterization of Recombinant Proteins and Fabricated Nanoparticles. Construction of influenza B head-removed HA (B/hrHA) was based on the HA sequence of B/Brisbane/60/2008 (Victoria lineage). According to the sequence alignment, the stalk domains of influenza B HA proteins are highly conserved among various strains from both lineages. The GCN-4 stabilized trimeric B/hrHA contains the stalk region of HA1 and the ectodomain of HA2 connected by flexible linkers (Fig. 1A, upper). To further stabilize the structure of B/hrHA, site mutations were introduced to prevent the cleavage under the physiological condition (K360T and R362Q) and to form an intra-disulfide bond (S455C) (Fig. 1A, upper)[12, 14], Influenza B nucleoprotein (B/NP) was constructed based on the sequence of B/Yamanashi/166/1998 (Yamagata lineage) NP protein (Fig. 1A, bottom). The recombinant proteins were expressed in insect cells by recombinant baculoviruses (rBVs)-based protein expression. Expressed recombinant proteins were detected by SDS-PAGE followed by Western blotting with right molecular weight bands using His-tag antibody (Fig. 1 B) or B/NP-specific antibody. Purified recombinant proteins were in high purity, showing as a single major band in the Coomassie blue-stained DSD-PAGE gels (Fig. 1B). The polymeric status of purified B/hrHA was determined by bis(sulfosuccinimidyl) suberate (BS3) cross-linking. Major bands with 3- and 2- fold higher molecular weight were observed in Western blots with a higher concentration of cross-linkers compared with that without the crosslinker, indicating the dominant trimeric form of the purified B/hrHA protein.

[0149] The layered nanoparticles (Nano) were generated by ethanol desolvation of B/NP and 3,3'-dithiobis [sulfosuccinimidylpropionate] (DTSSP) crosslinking of B/NP nanocores with B/hrHAs (Fig. 1C). Fabricated nanoparticles are biodegradable in the physiological reducing environment due to the disulfide bond within the DTSSP molecule which confers controlled release of soluble antigens in the cytosol after cellular uptake (Fig. 1 C). Western blotting analysis and Coomassie blue staining showed only two bands, one had 33kDa molecular weight (B/hrHA) and the other with 65kDa molecular weight (B/NP) (Fig. 1 D), indicating that the nanoparticles were composed entirely of the antigens of interest. The ratio of B/hrHA to B/NP in nanoparticles was 1 :3.5 according to the gel staining result (Fig. 1 D). The size of layered nanoparticles was 291 .5 ± 5.424nm, larger than B/NP nanocores that were 165.1 ± 15.72nm (Fig. 1E). The surface charges of both Nano and B/NP cores were negative as measured by ^-potentials of -29.33 ± 0.2603mV and -11.5 ± 0.1 mV, respectively. Morphology of the layered nanoparticles was observed by transmission electron microscopy (TEM), revealing the irregular spherical-shaped particles with an average size of 300nm diameter (Fig. 1F). The antigenicity of purified B/hrHA and fabricated layered nanoparticles were determined by ELISA binding assay recognized by goat antiserum to B/Florida/04/2006 HA. Both B/hrHA and nanoparticles showed substantial binding activities in a dose-dependent fashion compared with purified soluble 4M2e (s4M2e) as the negative control, indicating that purified B/hrHA retained its natural structure and the nanoparticle fabrication with crosslinkers did not affect its stability.

[0150] Nanoparticle Uptake by DCs and DC Maturation. Antigen uptake by APCs such as DCs and consequent DC maturation and proinflammatory cytokine secretion are crucial for the induction of downstream immune responses. It has been validated that effective nanoparticle uptake by DCs could elicit potent immune responses in mice. Here, a murine DC cell line, JAWS II cell, was used to investigate the internalization of nanoparticles and the cell maturation that was demonstrated by proinflammatory cytokine secretion (Fig. 2). Immunofluorescence image showed that the cells contained significantly increased fluorescence intensity after nanoparticle incubation compared with soluble proteins stimulated or untreated cells. Stronger fluorescence was also observed in cells treated with nanoparticles with flagellin (FliC) (Fig. 2A). FliC adjuvanticity has been proven to enhance innate immune cell function and improve vaccines' immunogenicity.

[0151] DCs express various pattern-recognition receptors (PRRs) to sense invasions or risks by binding to pathogen/damage-associated molecular patterns (P/DAMPs) and can be stimulated towards maturation, manifested by upregulated cell surface maturation-marker expression. The maturation of JAWS II cells after nanoparticle treatment was measured by CD86 expression, and analyzed by flow cytometry (Fig. 2B and C). Cells treated with Nano and Nano+FliC had significantly higher CD86 expression than cells treated with soluble proteins or negative control. Thus, the nanoparticles alone or with an adjuvant could effectively stimulate DCs maturation in vitro.

[0152] Matured DCs produce proinflammatory cytokines, which activate downstream humoral and cellular responses[30]. Cytokine ELISA evaluated IL-6 and TNF-<z secretion from cell culture supernatants. Cells stimulated by nanoparticles secreted significantly higher levels of IL-6 (Fig. 2D) and TNF-a (Fig. 2E) compared with soluble protein-treated samples as well as the untreated control group. Therefore, the layered nanoparticles could be internalized by DC cells effectively and stimulated DC maturation with promoted cytokine production.

[0153] Layered Nanoparticle Immunization Elicited Enhanced Germinal Center Responses. Antibodies in serum against foreign antigens increase over time termed affinity maturation in germinal centers (GCs) formed initially within the first week after vaccination or infection. Affinity maturation is realized through continual somatic hypermutation (SHM), clonal proliferation, and selections. GC B cells (B220+GL-7+) from ILNs were investigated on day 7 after the prime immunization. Nanoparticle immunization induced an increased population of GC B cells in ILNs while FliC adjuvanted groups elicited significant numbers of GC B cells with an average of over 2% compared with the soluble protein group or the PBS group (Fig. 3A and 3B). Thus, nanoparticle immunization could induce improved germinal center reactions.

[0154] Nanoparticle Immunization Induced Cross-reactive Antibody Responses via Fc- Mediated Functions. Mice were i.m. immunized twice with nanoparticles (10 pg each mouse) alone or together with FliC (0.1 pg each mouse) as an adjuvant. Mice groups injected with PBS and soluble B/NP and B/hrHA proteins (total protein 10 pg/mouse) with the same ratio as in nanoparticles were controls. Several influenza strains or purified full-length HA proteins from both Victoria and Yamagata lineages were used for antibody-binding activity evaluation (Fig. 4A). Phylogenetic analysis of HA proteins from those viruses revealed limited conservation between the two lineages (Fig. 4A). Immune sera were collected 3 weeks after boost immunization for antibody response detection. FliC-adjuvanted nanoparticle immunization induced significant high levels of total IgG antibody responses against various strains from both lineages (Fig. 4B and 4C). Immune Sera from the nanoparticle immunization group also demonstrated vigorous cross-binding activities (Fig. 4B and 4C). Soluble protein immunization induced relatively weaker antibody-binding to different viral strains except for two Victoria lineage strains, B/Ohio/1/2005 and B/Hong Kong/330/2001 , which showed higher binding but was not significant (Fig. 4B). FliC-adjuvanted nanoparticles also elicited significantly higher cross-reactive IgG 1 and lgG2a antibody titers against viruses from both lineages (Fig. 4D and 4E). The nanoparticle immunization group induced relatively more elevated lgG1 and lgG2a antibody responses to viruses in both lineages (Fig. 4D and 4E), especially to the homologous Victoria lineage strains (Fig. 4E, left and middle) when compared to B/Florida/4/2006 (Yamagata lineage) (Fig. 4E, right). [0155] We performed hemagglutination-inhibition (HAI) and micro-neutralization (MN) assays with RDE-treated heat-inactivated immune sera to explore the antibody functions further. Sera from groups immunized with inactivated viruses with the same procedure were positive controls. We found that immune sera from immunization groups had no detectable HAI titers. In contrast, inactivated viruses immunized groups show high HAI titers to viruses of homologous lineage and lower titers to heterologous lineage. Similar results were observed in microneutralization assays as the immune sera had negligible neutralizing activities compared with the inactivated virus group. This was consistent with previous findings that the cross-protection mechanisms of influenza B HA stalk-specific antibodies were not mediated through hemagglutination inhibition or neutralization of the virus.

[0156] Besides the HAI and neutralization functions, previous studies have shown that HA-specific immune sera without HAI or neutralization activities could provide cross-protection against viral infection through Fc-mediated effector mechanisms such as antibody-dependent cellular cytotoxicity (ADCC). Some monoclonal antibodies (mAbs) were further proven to contribute substantial cross-protection against infection by ADCC. Next, we examined the ADCC activities of pooled heat-inactivated sera from each group against two mouse challenge strains, B/Malaysia/2506/2004 (Victoria lineage) and B/Florida/4/2006 (Yamagata lineage). Nanoparticles alone or with an adjuvant elicited higher ADCC antibody titers to viruses of both lineages in dose-dependent manners compared to the soluble protein or PBS group (Fig. 4F and 4G). Compared with soluble protein and PBS control groups, ADCC titers of nanoparticle immunization groups ranged from 8 to 10-fold inductions to homologous lineage (Fig. 4F) and with 3 to 5-fold induction of activities to heterologous lineage virus (Fig. 4G). In summary, nanoparticle immunization elicited high levels of cross-reactive non-HAI and non-neutralizing antibody responses that engage Fc-mediated effector functions.

[0157] Nanoparticle Immunization Induced Strong and Broad Cellular Immunity. Cellular immunity is crucial to provide broad protection against viral infection by facilitating antibody generation via cytokine production and antigen-specific CTL responses. Early and high cellular immune responses correlate with reduced viral shedding and dampened symptoms. We evaluated the IL-4 and IFN-y-secreting cell frequencies from spleens three weeks after boost immunization. IFN-y-secreting CTL responses have been proved to confer broad protection and IL-4 mediates differentiation and proliferation of B cells facilitating antibody and memory B cell production. Compared to the soluble protein group, significantly increased IL-4-secreting cells were observed in Nano and Nano+FliC groups after stimulation with NP peptide (Fig. 5A, left) and HA peptide pools from both lineages (Fig. 5A, middle and right). Similarly, Nano and Nano+FliC groups also elicited more IFN-y-secreting splenocytes by NP peptide (Fig. 5B, left) and HA peptide pools from both lineages (Fig. 5B, middle and right). Cellular immunity to homologous HA was higher than to heterologous HA (Fig. 5A and 5B, middle and right). The results demonstrated that nanoparticle immunization induced robust cross-reactive T cell responses.

[0158] Since high IL-4-secreting lymphocytes were detected mentioned above and IL-4 facilitates B cells proliferation and differentiation into antibody-secreting plasma cells (ASCs) as well as memory B cell production[30, 50], we measured antigen-specific ASCs and memory B cells from bone marrows and spleens three weeks after the boost immunization by IgG ASCs ELISpot assay to viruses of both Victoria and Yamagata lineages (Fig. 5C and 5D). Compared with the soluble protein group, both Nano and Nano+FliC immunization induced significantly increased IgG ASCs in bone marrows against viruses of both lineages (Fig. 5C). Nano+FliC group had higher numbers of IgG ASCs in spleens against both lineages of viruses (Fig. 5D), but statistical significance was only found in homologous lineage strains (Fig. 5D, left and middle). The nanoparticle alone group also showed high levels, although not significant compared with the soluble protein group, of IgG ASCs in spleens against virus strains of both lineages (Fig. 5D). In contrast, soluble protein induced detectable IgG ASCs but at low levels (Fig. 5C and 5D).

[0159] Memory B cell plays a crucial role as the second line of defense upon infection by rapid reactivation and generating potent broadly protective antibodies after antigen reexposure. We found that both Nano and Nano+FliC immunization significantly induced more cross-reactive memory B cells in bone marrow cells and splenocytes after polyclonal mitogens (R848 and rlL-2) stimulation (Fig. 5E and 5F). In contrast, the memory B cell numbers of the soluble protein group were low (Fig. 5E and 5F). Therefore, the nanoparticles elicited strong cross-relative cellular immunity, and more significant responses were achieved by nanoparticle immunization with adjuvant. Improved quality of cellular immune responses indicated by memory B cell frequency was also found in Nano and Nano+FliC groups.

[0160] Layered Protein Nanoparticles Elicited Cross-protection against Challenges by Viruses of Homologous and Heterologous Lineages. Prophylaxis potency of nanoparticles was measured by challenging mice intranasally (i.n.) with 5xLD_So influenza B/Malaysia/2506/2004 (Victoria lineage) and B/Florida/4/2006 (Yamagata lineage) viruses. Both Nano and Nano+FliC elicited complete protection against viruses of both lineages (Fig. 6A and 6B). Soluble protein immunization provided partial protection against homologous and heterologous strains (Fig. 6A and 6B) with more severe morbidity (Fig. 6B, left). [0161] For serum passive transmission, naive mice intraperitoneally (i.p.) received Nano+FliC immune sera were fully protected against viruses of both lineages (Fig. 6C and 6D). The Nano group immune serum transfer provided complete protection against homologous lineage B/Malaysia/2506/2004 infection and 80% protection against heterologous lineage B/Florida/4/2006 challenge (Fig. 6C and 6D). Both Nano and Nano+FliC serum-transferred groups showed less weight loss after the homologous lineage virus infection (less than 5% weight loss) compared with those against heterologous lineage strains (15% weight loss) (Fig. 6C and 6D, left). By contrast, soluble protein immune sera partially protected mice (20%) from homologous lineage virus infection (Fig. 6C) while no protection against heterologous lineage strains (Fig. 6D). More severe morbidity was observed from heterologous lineage B/Florida/4/2006 challenge groups than homologous lineage B/Malaysia/2506/2004 infection groups (Fig. 6C and 6D, left).

[0162] Histological study of lung tissues 5 days post-infection revealed mild tissue damages in nanoparticle groups, manifested by less inflammation and leukocyte infiltration than the sProtein group (Fig. 7A and 7C). Nanoparticle groups also demonstrated significantly lower lung virus titers than sProtein (>1.5-fold of logTCIDso) (Fig. 7B).

[0163] Besides the histological tissue image, pulmonary immunopathology is also revealed by inflammatory cytokine levels after infection. Thus, we evaluated the inflammatory cytokine (IL-6, TNF-a, and IL-12) levels in BALF 5 days post-infection. Nano and Nano+FliC groups had lower IL-6, TNF-a, and IL-12 cytokines after viral infection than the PBS control group (Fig. 7D and 7E). Soluble protein immunization produced lower proinflammatory cytokines but significantly higher than Nano and Nano+FliC immunization groups (Fig. 7D and 7E).

[0164] These results demonstrated that nanoparticle immunization provides crossprotection against virus infection from both lineages. The immunogenicity of nanoparticles was correlated with less severe pulmonary inflammatory pathogenicity and lower viral loads in tissues. Serum antibody responses contribute to the broad protection of nanoparticle immunization.

[0165] Layered Protein Nanoparticles Induced Strong T Cell Responses in Lung Tissue. Antigen-specific T cells in lung tissue play crucial roles in mediating protective immune responses by clearing pathogens from tissues. Antigen-specific IFN-y secreted CD4+ and CD8+ T cell frequencies were measured in lung lymphocytes stimulated with B/Brisbane/60/2008 HA and B/Florida/4/2006 NP peptide pools, respectively, 5 days after infection. Both Nano and Nano+FliC groups had significantly higher NP-specific IFN-y-secreting CD4+ and CD8+ T cell populations than the sProtein group (Fig. 8A to 8C). Robust NP-specific IFN-y-secreting CD8+ T cells were induced in the Nano group with an average frequency of 24.16% of CD8+ lymphocytes and in the Nano+FliC group with an average 34.14% frequency (Fig. 8A, bottom, and 8C). HA-specific IFN-y-secreting CD4+ T cells were also elicited significantly in Nano and Nano+FliC groups compared to the sProtein group (Fig. 8D, upper and 8E). However, the specific CD8+ T cells were comparable between Nano and Nano+FliC groups (Fig. 8D, bottom, and 8F). The results demonstrated that nanoparticle immunization induced strong antigenspecific T cell responses correlated with protective immunity and pathogen clearance in the lungs after viral infection.

[0166] Layered Protein Nanoparticle Induced Durable Antibody Responses. The capability of long-lasting immune response induction is considered one of the essential features of an ideal vaccine[30]. We evaluated serum antibody levels 6 months after boost immunization. Fig. 9 showed that both the Nano and Nano+FliC groups remained at significantly higher levels of IgG binding to viruses of homologous and heterologous lineages 6 months after vaccination. In contrast, the sProtein group showed reduced antibody levels. The results indicated that the nanoparticles induced durable immune responses.

Discussion

[0167] A critical difference between influenza A and influenza B virus is the lower evolution rates and restricted host reservoirs of influenza B. In some flu seasons, the influenza B virus dominates influenza infection in adults and causes severe conditions in immunocompromised populations such as children and older adults. Thus, a universal influenza B virus vaccine that provides potent broad cross-protection against viruses from Victoria and Yamagata lineages may help to serve as a preventative countermeasure against the emergence of epidemics. Immunity specific to more conserved viral antigens like the immune- subdominant HA stalk region — instead of the more variable immunodominant HA globular head domain — has demonstrated potent and broad protection. Clinical observations have illustrated that the abundance of serum antibodies toward subdominant epitopes of both influenza A and B increased over time after repeated exposure to drifted strains. Seasonal vaccinations, however, do not appear to elicit such antibody responses to vaccine strains. Accumulating evidence has indicated that the HA stalk from influenza A and B viruses in novel vaccine candidates provides broadly protective immunities and has potential as a universal influenza vaccine candidate. Our previous study also demonstrated that influenza A recombinant HA stalk (hrHAs) combined with another conserved antigen, M2e, was highly immunogenic in v/vo[24]. [0168] We constructed influenza B hrHA based on the HA of B/Brisbane/60/2008 (Victoria lineage) by rational structure design. We demonstrated that the purified recombinant protein retained its natural polymeric state and antigenicity. Influenza B Nucleoprotein (NP) from B/Yamanashi/166/1998 (Yamagata lineage) was also developed for our vaccine candidate since the NP proteins among viruses from both lineages are over 98% conserved.

[0169] We generated nanoparticles by adding B/hrHA proteins on the surface of desolvated B/NP nanocores via DTSSP crosslinking. The layered nanoparticles were composed of hrHA and NP in high densities. The antigenicity of the antigens within the nanoparticles was not affected as indicated by specific antibodies’ binding indicating the retained structure and function of antigens after DTSSP crosslinking. When compared with another cross-linking agent, BS³ which is identical to DTSSP, the crosslinking reaction of BS³ is not reversible due to no disulfide bond in the crosslinker molecule (Fig. 1D). Our influenza B nanoparticles were efficiently absorbed and processed by DCs, stimulated DC cell maturation, and activated proinflammatory cytokine production (Fig. 2).

[0170] When we added it to our nanoparticles, Flagellin (FliC) enhanced immune responses in mice. FliC is an adjuvant protein that binds Toll-like receptor 5 (TLR-5) to initiate a subsequent innate signaling cascade in innate immune cells. We found more efficient nanoparticle internalization by JAWS II cells and cytokine production after stimulation with FliC (Fig. 2A and 2D). Nanoparticle immunization with FliC elicited higher levels of antibody responses. Antibody-dependent cellular cytotoxicity (ADCC) function, but not HAI or neutralizing activities, mediated the prophylaxis against viruses of both lineages we observed in our study. These findings were consistent with previous results that non-HAI and non-neutralizing antibodies provided broad immune protections against conserved epitopes of influenza B HA stalk region through Fc-mediated effector mechanisms.

[0171] Cross-reactive T cell responses control infections and correlate with reduced viral shedding, delayed disease progress, and reduced disease severity. Our nanoparticles elicited strong and cross-reactive NP- and HA-specific T cell responses. NP-specific CD8+ and CD4+ T cell responses observed in lungs after viral infection (Fig. 8A to 8C) correlated with reduced viral loads and tissue damage (Fig. 7).

[0172] Upon influenza infection, both CD8+ CTL and CD4+ helper T cells differentiate and proliferate into effector cells, increasing cytokine secretion like IL-4 and IFN-y [50], IL-4 secreting T cells facilitate the proliferation and differentiation of B cells into ASCs. With or without adjuvants, our nanoparticle immunization generated high levels of cross-reactive IL-4 secreting T cell responses, cross- reactive ASCs levels in spleens, and cross-reactive ASCs levels in bone marrow (Fig. 6A to 6D).

[0173] Memory B cells can rapidly reactivate and differentiate into antibody-secreting cells, generate potent and broadly reactive antibodies after antigen re-exposure, and are considered crucial for long-lasting immunity. We observed increased memory B cell responses against divergent antigens after nanoparticle immunization (Fig. 6E, and 6F) and sustained antibody responses six months post-boost immunization (Fig. 9).

[0174] Compared with other nanoparticle platforms like inorganic nanoparticles, selfassembly nanoparticles, polymer nanoparticles, and so on, the layered protein nanoparticles consisting of almost entirely interested antigens had high immunogen loads and avoided potential risks of off-target immune responses or pre-existing immunities to nano core carriers compared with other forms of nanoparticle formulation. Controlled surface protein crosslinking by a degradable redox-responsive crosslinker also ensured stabilities of preserved antigenic structures and modulates antigen release in physiological reducing environments to facilitate subsequent antigen-presenting cell stimulation and maturation as well as to avoid immune tolerance upon antigen binding and internalization. These features enable the layered nanoparticle vaccines to induce robust and durable immune responses. Broadly protective immunity could also be achieved by incorporation of conserved viral antigens in proper design. Immune stimulators could also be co-crosslinked onto nanoparticles to generate multilayered chimeric protein nanoclusters to further improve immunogenicity. Our novel nanotechnology platforms developed several new generations of influenza vaccine candidates composed of different combinations of conserved viral antigens by rational structure design and those vaccines have been proven to have potent and broad immune prophylaxis against viral infection. No apparent adverse effects were observed after immunizations by comprehensive evaluations indicating that the nanoparticle vaccines were safe in vivo.

[0175] In summary, we have successfully fabricated layered protein nanoparticles composed of structure stabilized, conserved influenza B HA stalk region and NP proteins. FliC adjuvanticity efficiently enhanced antigen uptake by DCs, DC maturation, and activation, and further improved the magnitude and breadth of cross immune protections against influenza B Victoria and Yamagata lineages. The layered protein nanoparticle fabricated by a redox- responsive crosslinker (DTSSP) was safe, biocompatible, biodegradable, and highly immunogenic in vivo. Our next aim is to combine the influenza A nanoparticles from our previous study with the influenza B nanoparticles we have fabricated and tested here to create a multi-valent universal influenza nanoparticle vaccine against both influenza A and B. Example 2. ISCOMs/MPLA-adjuvanted SDAD protein nanoparticles induce improved mucosal immune responses and cross-protection in mice

Introduction

[0176] Influenza A virus has been recognized as one of the most threatening respiratory pathogens that could cause acute morbidity and mortality and heavy economic burdens, especially in flu epidemics or occasional flu pandemics. Although vaccination has been proven to be an effective method to prevent or reduce influenza viral infection during annual flu seasons, the selection of vaccine strains depends mainly on circulating viral surveillance and prediction, and mismatched strains could significantly impair vaccine efficiency. Meanwhile, the production of the current quadrivalent influenza vaccine is based on the time-consuming chicken egg or cell culture systems and is not suitable for urgent uses when a pandemic strain is identified. Thus, new vaccine technologies such as mRNA or protein nanoparticle vaccines that are easily manufactured and quality-controlled are promising alternatives for developing a universal influenza vaccine.

[0177] We have focused our studies on different types of protein nanoparticle vaccines against both influenza A and influenza B viral infections. We demonstrated that the doublelayered M2e and full-length hemagglutinin (HA) or HA stalk protein nanoparticles and M2e-NA protein nanoparticles induced immune protection against both homologous and heterologous influenza A viral infections. The nanoparticles fabricated using influenza B virus-derived nucleoprotein (NP) and HA stalk displayed cross-protection against influenza B viruses from two lineages. The fabrication of our previous protein nanoparticles is based on homobifunctional DTSSP crosslinking, more controlled and efficient fabrication methods are worth investigating to improve the nanoparticle formulations. In addition, combining appropriate adjuvants with protein nanoparticle vaccines will be a convenient way to further increase immune responses and protective effectiveness. For example, we found the combination of toll-like receptor 4 (TLR4) ligand- Monophosphoryl lipid A (MPLA) in double-layered NP and neuraminidase (NA) nanoparticles enhanced Th1 immune responses, as well as increased cross-protections against different influenza viral infections.

[0178] Besides aluminum salts, only a few adjuvants have been approved for human usage by the FDA in the past seventy years. Combined adjuvant systems have been evaluated and approved for vaccine usage in humans to exploit the advantages of different adjuvants and enhance a more comprehensive immune response. For example, Adjuvant System 4 (AS04), a combination of MPLA and aluminum salt, was approved in HPV vaccine formulation (Cervarix). A liposomal formulation (Adjuvant system AS01) including MPLA and a synthetic saponin QS21 has been approved for malaria and recombinant zoster vaccine vaccines (RTS and RZV). In the AS04 adjuvant formulation, aluminum stimulates relatively low cellular immunity and induces primarily humoral immune responses. ISCOMs (Immune-stimulating complexes) matrix, described as early as in the 1980s, are cage-like self-assembled nanoparticles containing Quil A, cholesterol, and DOPC, which have been demonstrated to promote both humoral and cellular immune responses, including cytotoxic T cells (CTLs). The ISCOM concept-based Matrix-M adjuvant, composed of saponin extracts, cholesterol, and phospholipids, was applied in the Novavax COVID-19 Vaccine to elicit robust CD4 T cell, a Th1 biased responses in both pre- clinical and clinical studies. In addition, other small molecules like TLR agonists and cGAMP, which function as pathogen-associated molecular patterns (PAMPs) to stimulate innate immune responses, have also been recognized and studied as effective vaccine adjuvants.¹⁹¹ Therefore, exploring appropriate combinations of adjuvants is vital to enhance immune responses and guide immune directions.

[0179] In this study, a heterobifunctional crosslinker, NHS-SS-Diazirine, succinimidyl 2- ((4,4'-azipentanamido) ethyl)-1 ,3'-dithiopropionate, designated as SDAD, was utilized to conjugate the influenza M2e-NA fusion protein on the surface of NP core, and a liposome-based adjuvant containing MPLA and ISCOMs was included as an adjuvant mixture for the novel SDAD protein nanoparticles. The intramuscular immunization of the adjuvanted protein nanoparticles induced significantly improved antigen-specific antibody and cellular immune responses and provided efficient protection against homologous and heterologous influenza viral challenges. The MPLA/ISCOM adjuvant combination also significantly improved the immunogenicity and protection efficiency of protein nanoparticles delivered via the intranasal route. The results emphasize the importance of supplementing appropriate adjuvants to improve the immunogenicity and mucosal immune responses of vaccines in mucosal immunizations.

Results

Generation and characterization of SDAD-crosslinked protein nanoparticles

[0180] We synthesized a novel kind of protein nanoparticles using an SDAD crosslinker containing an amine-reactive N-hydroxysuccinimide (NHS) ester and a photoactivatable diazirine ring at the termini. The diagram for the fabrication process is displayed in Figure 10A. Briefly, through the reactive NHS arm, we conjugated the SDAD onto the surface of the influenza NP protein nanoparticle (NP nanos) that was generated by ethanol desolvation. Following purification of the SDAD-conjugated nanoparticle (NP nanos-SDAD), we further conjugated M2e-NA1 or M2e-NA2 fusion proteins onto the surface of the NP nanos-SDAD by ultraviolet exposure under 350nm. The sizes of the resulting NP/M2e-NA1 (core/shell) and NP/M2e-NA2 nanoparticles were 200 nm to 250 nm by dynamic light scattering (DLS) analysis (Figure 10B). The content of the NP core to the coating proteins in the nanoparticles was analyzed by the Coomassie blue staining and Western blot with ratios ranging from 1.5 to 2.1 (Figure 10C). The nanoparticle size and average ratio of activated NA in total SDAD-conjugated nanoparticles were comparable to the previous DTSSP-conjugated protein nanoparticles. The resultant nanoparticles exhibit a spherical shape by transmission electron microscopy (TEM) (Figure 10D). Unlike self-assembled nanoparticles, the uncontrolled orientation of the shell protein deposited on the nanoparticle cores and the unorganized structures of particle cores themselves constituted the nanoparticles with indistinguishable core/shell structures under TEM. The hetero-bifunctional nature of SDAD prevents crosslinking between particles and between M2e-NA proteins in the coating solution. Unconjugated M2e-NA proteins in the supernatant remain intact and can be used for a subsequent batch of nanoparticle fabrication after pelleting the nanoparticles. Therefore, the SDAD conjugation significantly increases the utilization of the initial proteins, which could be one restricting factor for future industrial applications.

[0181] Dendritic cells (DCs) are important innate immune cells to participate in antigen processing, presentation, and immune regulations. DC maturation and cytokine secretion are indicators of the potential initiation of antigen-specific immune responses. Our previous studies found that protein nanoparticles predominantly induced Th2-biased immune responses versus Th1 cell type. Supplementing appropriate adjuvants with protein nanoparticles could improve the antigen immunogenicity and orchestrate the immune responses. Therefore, we supplemented SDAD protein nanoparticles with different adjuvant combinations and determined the maturation and cytokine secretions of bone marrow-derived dendritic cells (BMDCs) after stimulation by the SDAD nanoparticles with or without ISCOMs, ISCOMs/cGAMP, or ISCOMs/MPI_A adjuvants. As shown in Figure 10E, ISCOMs adjuvanted SDAD nanoparticles significantly induced the DC maturation-marker expression including CD40, CD80 and CD86 with enhanced median fluorescence intensities (MFI) compared with the untreated control, and supplementing cGAMP or MPLA with ISCOMs further increased the expression of CD40, CD80, and CD86 (Figure 10E). Tumor necrosis factor-a (TNF-a) is a well-recognized factor for the maintenance of DC maturation and survival. The cytokines secreted from DCs are important regulators for T helper cell fates. DC-secreted interleukin-12 (IL-12) is a hallmark of inducing the Th1 responses, while IL-6 modulates Th2-oriented immune differentiation. The nanoparticles alone stimulated increased TNF-a expression in supernatant compared with the controls (Figure 10F). Consistent with the surface marker expression, nanoparticles adjuvanted with ISCOMs/cGAMP and ISCOMs/MPLA provoked significantly increased IL-6 and TNF-a generation compared with other groups, while adding ISCOMs alone only slightly increased TNF-a levels (Figure 10G and 10F). ISCOMs and MPLA were reported as the inducers of I L- 1 p and the expression of I L-1 promoted the secretion of IL-12. We observed that the combination of ISCOMs/MPLA uniquely stimulated higher expressions of I L-1 p and IL-12 compared with slightly increased IL-1 p expression by ISCOMs alone or ISCOMs/cGAMP incubations (Figure 101 and 10H), In contrast, ISCOMs/cGAMP preferentially induced IFN-a expression (Figure 10J). The specific induction of IFN-a was related to cGAMP-activated STING-mediated type I IFN production. The activation of BMDCs with SDAD nanoparticle formulations in vitro suggested the potential induction of effective immune responses in vivo.

Immune responses induced by SDAD protein nanoparticle immunizations.

[0182] We evaluated the immunogenicity of adjuvanted SDAD nanoparticles in mice. Different SDAD nanoparticle vaccine formulations included NP/M2e-NA2 nanoparticles (NA2 nano), NP/M2e-NA1 & NP/M2e-NA2 nanoparticles (NA1+NA2 nano), and ISCOMs/MPLA- adjuvanted NA1+NA2 nano (NA1+NA2 nano with Adj). We collected mice sera three weeks after the prime and boost vaccination to analyze antigen-specific antibody responses by ELISA assays. SDAD-crosslinked NP/M2e-NA elicited significantly increased M2e, NA1 , and NA2- specific IgG antibody levels three weeks post the prime and boost immunization (Figure 11A). The boost immunization greatly improved the antigen-specific IgG 1 and lgG2a titers (Figure 11 B-11 D). The combination of MPLA and ISCOMs displayed attractive adjuvant effects with considerably increased M2e, NA1 , and NA2-specific IgG (Figure 11A), lgG1 , and lgG2a antibody (Figure 11 B-11 D) titers after boost immunizations. Notably, compared to the nanoparticle alone group, the addition of MPLA/ISCOMs adjuvants significantly enhanced lgG2a levels with a more balanced lgG2a and lgG1 , which suggested the regulation of immunological profiles by this adjuvant formulation.

[0183] We euthanized the mice four weeks post-boost vaccination and evaluated the cellular immune responses in spleens and bone marrows by ELISpot. As shown in Figures 11 E and 11 F, there were significantly increased and comparable numbers of IL-4 and IFN-y- secreting splenocytes after NA2 stimulation in all nanoparticle-immunized mouse groups in comparison with the naive mice. Supplementing MPLA/ISCOM adjuvants significantly enhanced the virus- (Aichi and PR8) and M2e peptide-specific IgG-secreting cells in bone marrow (Figure 11G-11 I), consistent with the strengthened antibody responses observed in adjuvanted nanoparticles-immunized groups. These results suggested that the MPLA/ISCOMs remarkably improved the immunogenicity of the protein nanoparticles in intramuscular immunization.

Protection against influenza viral infections. [0184] To determine the protective efficacy induced by SDAD protein nanoparticle immunization, different mouse groups were challenged with a dose of 5x LD50 A/Aichi (H3N2), 5* LD₅o of rVet (H5N1), or 3x LD50 of A/Philippine (H3N2) four weeks post-boosting immunization and the body weights were monitored for fourteen days. Although mice receiving the NA2 nano immunization displayed a 100% survival rate (Figures 12B and 12D) during the A/Aichi and A/Philippine influenza viral infections, they lost substantial body weight with symptoms from days 3 to 7 (Figure 12A and 12C). On the other hand, the adjuvanted nanoparticle group had fewer symptoms and recovered quickly during all the viral infections (Figure 12A, 12C, and 12E). After NA homologous A/Aichi and rVet challenges, the mixed NA2 and NA1 nano-immunized mice showed a 90% survival rate which was less than the NA2 nano group (Figure 12B and 12F). These results demonstrated that the MPL and ISCOMs adjuvant combination could enhance the protective efficacy of protein nanoparticle immunization to fight against homologous and heterologous influenza viral infection.

Adjuvants improved the immunogenicity of protein nanoparticles during intranasal vaccination and induced protection against viral infection.

[0185] Besides systemic immunity, the induction of strong mucosal immune responses in local tissues is also crucial for impeding respiratory viral infection. To explore the possibility of protein nanoparticles as mucosal vaccines, we intranasally primed and boosted immunized mice with M2e-NA2 protein nanoparticles with or without the ISCOM/MPLA adjuvant. One group of mice was immunized with ISCOM/cGAMP-adjuvanted protein nanoparticles (Nanos+ISCOM/cGAMP) as a control. Compared to the naive and protein nanoparticle alone (Nanos) groups, mice immunized with adjuvanted protein nanoparticles showed significantly increased NA2 and M2e-specific IgG antibodies three and seven weeks post-vacci nation, respectively (Figure 13A and 13C). ISCOM/cGAMP-adjuvanted protein nanoparticle immunization induced higher antigen-specific antibody levels than ISCOM/MPLA-supplemented nanoparticles (Nanos+ISCOM/MPLA) (Figure 13A and 13C). For NA2- and M2e-specific IgG isotypes, ISCOM/MPLA- or ISCOM/cGAMP-adjuvanted nanoparticle immunization elicited more robust lgG1 antibody levels than lgG2a (Figure 13B and 13D). ISCOM/cGAMP was a more powerful supplement to stimulate increased lgG1 and lgG2a than ISCOM/MPLA adjuvant (Figure 13B and 13D). These results demonstrated that supplementing ISCOM/MPLA or ISCOM/cGAMP adjuvant combinations to protein nanoparticles could significantly improve the immunogenicity of protein nanoparticles during intranasal immunization.

[0186] To determine the protective efficiency against viral infection, mice were challenged with 5x LD50 A/Aichi four weeks post the intranasal boosting immunizations and monitored for fourteen days. During the initial five days post-challenge, the ISCOM/cGAMP- adjuvanted protein nanoparticle immunized group showed more body weight loss than other groups. The body weight recovered from day 6 with a 75% of survival rate (Figure 13E and 13F). In comparison, the protein nanoparticles adjuvanted with ISCOM/MPLA provided complete protection against homologous A/Aichi infection with an average of 12% of body weight loss in the fourteen days (Figure 13E and 13F). The body weight curve of protein nanoparticle-vaccinated mice displayed a similar pattern to the naive group except one mouse survived in this group during the challenge study with a 20% of survival rate (Figure 13E and 13F). These data indicated that supplementing adjuvants in protein nanoparticles during intranasal immunization significantly improved the immunogenicity of antigens and ISCOMs/MPLA adjuvanted protein nanoparticles provided better protection against homologous influenza viral infection.

Intranasal immunization of ISCOMs/MPLA-adjuvanted nanoparticles increased systematic cellular immune responses.

[0187] To further elucidate the immune responses induced by ISCOMs/MPLA adjuvanted nanoparticles, the cytokine-secreting cells in the immune system were analyzed. IL- 2 has been reported as an important cytokine to mediate the differentiation of naive T cells into T helper 1 (Th 1 ) and T helper 2 (Th2) effector cells, while IFN-y and IL-4 are essential cytokines secreted from Th1 and Th2 cells to play major roles in antiviral immunity and humoral immunity, respectively. In this study, groups of mice were intranasally immunized with protein nanoparticles with or without ISCOMs/MPLA to determine the above cytokine secretion in splenocytes one month post-boosting immunization. Mice immunized with ISCOMs/MPLA adjuvanted nanoparticles (Nano with Adj.) have significantly increased numbers of NA2- (Figure 14A), NP peptide- (Figure 14C), and Aichi virus- (Figure 14D) specific IFN-y, IL-2 and IL-4 secreting splenocytes compared with nanoparticles alone (Nanos) or naive groups. The observation of increased IL-4-secreting cells in the spleen after adjuvanted nanoparticle immunization was consistent with the remarkable enhancement of antigen-specific antibody responses in Figure 13. Meanwhile, significantly increased NA2-specific antibody-secreting cells (ASCs) were observed in the spleen and bone marrow cells (Figure 14B). These results suggested that intranasal immunization of ISCOMs/MPLA-adjuvanted protein nanoparticles could elicit strong Th1 and Th2 immune responses.

Mucosal immune responses and protection against heterologous viral infection.

[0188] Besides the induction of potent cellular immune responses in the systemic compartment, an efficient mucosal vaccine should also stimulate the production of secretory IgA (slgA), which is critical in protecting against viruses on mucosal surfaces. Mucosal IgA has been demonstrated to protect against respiratory viruses, including SARS-CoV, MERS-CoV, and influenza. Here, we intranasally immunized mice as previously described and determined the antigen-specific IgG and IgA levels in mucosal fluids, including bronchoalveolar lavage fluid (BALF) and nasal washes one month post the boosting immunization. Significantly increased levels of NA2-specific IgA were detected in the nasal and BALF washes from ISCOMs/MPLA adjuvanted nanoparticles intranasally immunized mice. However, only background signals could be observed from mucosal samples derived from Nanos and naive groups (Figure 15A and 15B). Similarly, there were also greatly enhanced NA2-specific IgG in the nasal and BALF washes from the adjuvanted nanoparticle-immunized group compared with the Nanos and naive groups (Figure 15C and 15D). Meanwhile, we observed improved Aichi virus-specific IgA and IgG antibody responses in the mucosal samples from adjuvanted nanoparticles immunized mice (Figure 15E and 15F), although the signals of virus-specific IgA were lower than the NA2 - specific IgA in Figure 15A. Furthermore, the immunized mice were also challenged with 3x LD50 of A/Philippine one month post the boosting vaccination to evaluate the protective efficiency against heterologous virus infection. As shown in Figure 15G, ISCOMs/MPLA-adjuvanted nanoparticles intranasally immunized mice maintained their initial body weights fourteen days after virus inoculation, while the naive mice and the nanoparticles alone immunized mice indistinguishably lost the body weights below 75% of initial body weights within nine days postinfection. Therefore, the ISCOMs/MPLA adjuvant combination could significantly improve the mucosal antibody immune responses of the protein nanoparticles in intranasal delivery and provide better protection against heterogeneous influenza viral infection.

Recruitment of lymphocytes in localized pulmonary tissue after ISCOMs/MPLA adjuvanted nanoparticles intranasal immunization.

[0189] In addition to the antibody responses in the mucosa, the immune cells in mucosal tissues also play critical roles in protecting against respiratory viral infection. Increasing evidence in recent years has supported the positive functions of lung-resident memory T and B cells in response to bacterial and viral infections. In this study, we found that there were significant increases in percentages of CD8⁺CD44⁺CD69⁺CD103⁺ (Figure 16A), CD4⁺CD44⁺CD69⁺CD103⁺ (Figure 16B), and CD4⁺CD44⁺CD69⁺CD103- (Figure 16C) resident memory T cell populations (T_RM) in lungs from the ISCOMs/MPLA-adjuvanted nanoparticles intranasally immunized mice (Figure 16D). Compared with the Nanos or naive group, ISCOMs/MPLA-adjuvanted nanoparticles elicited increased percentages of CD19⁺B220'lgD’lgM' CD38⁺CD69⁺ (Figure 16E) lung-resident memory B cell populations (BRM) (Figure 16G). Although CD38 is often used to distinguish memory from naive B cell subset in mice, we also observed an induction of CD19⁺B220 gD gM-CD38’CD69⁺ cell population in lungs of the adjuvanted nanoparticle immunized mice (Figure 16F and 16G).

[0190] Alveolar macrophage is another kind of immune cell critical in protecting against influenza A viral infection. Adjuvanted protein nanoparticle immunization stimulated higher percentages of CD11c⁺CD11b’CD64⁺CD24^_ alveolar macrophages in lungs (Figure 16H and 161). The cells from BALF were separated and analyzed for the expression of CD44 on CD4 and CD8 T cells and increased CD4⁺CD44⁺ and CD8⁺CD44⁺ populations were detected (Figure 16J and 16K). As shown in Figure 16, intranasal immunization of the protein nanoparticle alone could not promote the establishment of lung-resident memory cells or the induction of alveolar macrophages in the lung. These data might suggest that ISCOMs/MPLA-adjuvant enhanced antigen uptake and presentation in the respiratory tract leading to the activation of CD4 and CD8 T cells, which would promote the establishment and maintenance of pulmonary TRM and BRM in local tissues.

Discussion

[0191] The nanoparticle size is critical in optimizing the delivery routes and immunogenicity of protein nanoparticle vaccines. However, no agreement exists regarding the optimum nanoparticle size range generating the best immune responses in different circumstances. In our previous studies, we found that our DTSSP-crosslinked protein nanoparticles around 200nm could be efficiently drained to and retained in inguinal LNs and spleens after intramuscular injection and induced strong humoral and cellular immune responses. Similarly, robust immune responses were observed after intramuscular injection of the SDAD-crosslinked protein nanoparticles in this study. However, when applied through the intranasal route, the protein nanoparticles could not induce significant immune reactions, which might be due to the less efficiency of penetrating the mucus surface or uptaking by local antigen-presenting cells (APCs). Thus, it is necessary to include appropriate adjuvant to boost the uptake and process of antigens by APCs during mucosal immunization.

[0192] Supplementing ISCOM/MPLA and ISCOM/cGAMP with nanoparticles could stimulate strong immune responses in intranasal immunization. Various mucosal adjuvants have been reported to boost the antigen-induced immune response, such as bacterial toxins, TLR agonists, cytokines, and chemokines. ISCOMs have been used in different studies as an effective mucosal adjuvant to improve immune response. MPLA has been used with other molecules in different adjuvant systems to boost immune responses. When we formulated these adjuvant combinations with our protein nanoparticle vaccines, we found that ISCOMs/MPLA combination provided better protection against influenza viral infection, although less improvement in antibody responses compared with the ISCOMs/cGAMP combination. ISCOMs and cGAMP have been studied as potent mucosal adjuvants when applied alone or in combinations with other adjuvants, while MPLA takes its advantage as a ligand of TLR4 that is abundantly expressed on the surface of DCs and is essential for DCs activation. cGAMP elicited signaling activities through cytosolic receptors and showed better adjuvanticity when delivered to inside cells. This indicated the ISCOMs/MPLA combination could provide stimulations to DCs more directly and efficiently. Meanwhile, ISCOMs/MPLA-adjuvanted protein nanoparticles induced a more balanced and robust Th1 and Th2 immune response no matter the immunization route. These results indicated the superiority of ISCOMs/MPLA as a potent adjuvant with protein nanoparticles. In parallel with the development of novel universal vaccines, the incorporation of appropriate adjuvants into the mature vaccine formulations will shortly be the quickest method to strengthen the immunogenicity and improve the protective efficiency of original vaccines.

[0193] Although mucosal vaccines have been recognized and proved to be an ideal method to trigger immune responses at the primary infection sites, there is only one licensed influenza mucosal vaccine for human use. The complex properties and structures of the nasal cavity and respiratory tract are naturally challenging obstacles to vaccine delivery. Only the vaccine candidates that penetrate the mucus layer, translocate into antigen-presenting cells and escape antigen clearance will have the chance to stimulate downstream immune responses. In our in vitro studies, we observed that ISCOMs/MPLA adjuvanted protein nanoparticles facilitated BMDCs maturation and stimulated multi-cytokines secretion from BMDCs over ISCOMs alone or ISCOMs/cGAMP. This finding indicated that ISCOMs/MPLA might further stimulate substantial DC maturation and cytokine secretion to help the protein nanoparticle uptake and antigen presentation by antigen-presenting cells, as we have seen that DCs could efficiently internalize the protein nanoparticles in previous studies. The influence of ISCOMs/MPLA on the retention, internalization, and translocation of antigens by immune cells on mucosal surfaces will be further determined.

[0194] Our study provided the first evidence that ISCOMs/MPLA adjuvanted protein nanoparticles could elicit strong mucosal immune responses and the accumulation of lung resident memory cells in the local respiratory tracts. With the understanding of features and functions of lung tissue-resident memory cells in recent years, T_RM and B_RM are realized as attractive targets for vaccine design. The unique effector functions of T_RM cells in restricting the reinfections of various respiratory pathogens at the first exposure sites of mucosal surfaces have been extensively studied during the last decade. Influenza viral infection could elicit lung BRM and plasma cells, and the lung B_RM responded rapidly to localized ASCs following viral challenge. Although site-specific vaccination such as intranasal or pulmonary vaccination could increase the chances for antigen encounter and are more likely to elicit T_RM and B_RM in lungs, ours and most emerging studies demonstrated that intradermal, intramuscular, and intranasal immunizations of mRNA vaccines could induce pulmonary resident memory T cells. Therefore, the heterologous immunizations of systemic prime and mucosal boost will be another new way for next-generation mucosal vaccines.

[0195] In summary, we have developed a novel influenza SDAD protein nanoparticle vaccine and found that the ISCOMs/MPLA adjuvant combination could significantly enhance the immunogenicity and protective effectiveness induced by protein nanoparticles after intramuscular and intranasal immunizations. This work highlights the importance of applying adjuvants in mucosal vaccine formulations. The ISCOMs/MPLA-adjuvanted protein nanoparticles have the potential to be used as mucosal vaccines alone or in combination with other vaccines to improve mucosal immunity and protection in the future.

Materials and Methods

Fabrication and characterization of SDAD cross-linking protein nanoparticles.

[0196] NP core nanoparticles were generated by ethanol desolvation. Briefly, 200 pg of NP protein solution was mixed with four times the volume of ethanol (100%, 1ml’¹ min) while stirring for 20 min. The pellet was resuspended in PBS (300 pl) after centrifugation at 20,000xg for 20 min and sonication with 40%-amp, 3 sec on and 3 sec off. A ten-fold molar excess of SDAD ((NHS-SS-Diazirine) (succinimidyl 2-((4,4'-azipentanamido)ethyl)-1 ,3'-dithiopropionate)), purchased from Thermo Scientific, was added to the NP core nanoparticle solution and stirred for 30 min followed by adding Tris-HCI (20 pl, 1 M, PH 8.0) buffer to quench the reaction for 5 min. The SDAD-conjugated NP core nanoparticles (NP Nano-SDAD) were washed with DPBS and centrifuged at 20,000xg for 20 min to remove any excessive SDAD. M2e-NA1 or M2e-NA2 proteins (200 pg) were mixed with NP Nano-SDAD, incubated, and stirred for 1 hour under the 365 nm UV light (UVP UVL-4 UV Lamp, Analytik Jena US). The nanoparticles were pelleted by centrifugation at 20,000xg for 20min. The pellet was resuspended and sonicated in DPBS (200 pl) for further characterization. The concentration of protein nanoparticles was measured by Micro BCA Protein Assay Kit (Thermo Scientific), and the total yield was calculated as total protein input/ total output *100%. The individual content of NA and NP proteins was characterized by 10% SDS-PAGE followed by coomassie blue staining and western blot. The nanoparticle sizes and morphology were determined by dynamic light scattering analysis (Malvern Zetasizer) and transmission electron microscopy imaging (JEOL 100 CX-II TEM).

Mouse immunization and challenge.

[0197] Groups of mice received primary and boosting immunizations of 10 pg of NP/M2e-NA2 nanoparticles (NA2 nano), mixed NP/M2e-NA2, NP/M2e-NA1 nanoparticles (NA2+NA1 nano), or mixed nanoparticles with ISCOMs/MPLA adjuvants (NA2+NA1 nano with Adj.) by intramuscular injection at three weeks intervals. To generate ISCOMs, a lipid film of 1 ,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC) (Avanti Polar Lipids) and cholesterol (Sigma) obtained by centrifugal drier (Vacufuge Plus, Eppendorf) were solubilized and sonicated in sterile water and then mixed and vortexed with QuilA (InvivoGen). The ISCOMs/MPLA combination comprised MPLA (2 pg, Avanti Polar Lipids) and ISCOMs (16 pg) with a 5:1 :2 ratio of QuilA: cholesterol: DOPC. One group of naive mice was included as controls.

[0198] For the intranasal immunization, mice were intranasally immunized with 30 pl of vaccine formulations including 10 pg of NP/M2e-NA2 nanoparticles (Nanos), NP/M2e-NA2 nanoparticles with ISCOMs/MPLA (Nanos+ISCOMs/MPLA or Nanos+Adj.), or NP/M2e-NA2 nanoparticles with ISCOMs/cGAMP containing ISCOMs (16 pg) and cGAMP (5 pg) (Nanos+ISCOM/cGAMP). cGAMP was purchased from InvivoGen. Four weeks post boosting immunization, mice were intranasally challenged with 5* LDso of A/Aichi/1968 (A/Aichi, H3N2), 3x LD₅o of A/Philippines/1986 (A/Phili, H3N2) or 3x LD50 of reassortant Viet (rViet, H5N1). The body weights were measured, and mice conditions were monitored for fourteen days after infection.

Antigen-specific antibodies

[0199] The sera were collected three weeks post-primary and secondary immunizations, and the antigen-specific antibody levels in the mouse sera were measured by enzyme-linked immunosorbent assay (ELISA). Briefly, the ELISA plates (Nunc, Maxisorp) were coated with 4 pg-¹ ml of NA1 , NA2 purified proteins, M2e peptides (Synpeptide, SLLTEVETPT) or whole inactivated A/Aichi virus overnight and blocked in PBST with 2% BSA. After 1 hour of incubation, 50 pg of sera dilutions were added and incubated for 2 hours at 37°C. Plates were washed and incubated with goat anti-mouse IgG-, lgG1 - or lgG2a-HRP secondary antibodies (SouthernBiotech) for 1 hour, and the plates were read by BioTek Microplate Reader at 450 nm after reacting with TMB and stopping by H₂SO4 (1 M). Mice nasal washes and BALFs were collected one month post-intranasally boosting immunization. The NA2- and Aichi- specific IgA levels were measured using HRP conjugated goat anti-mouse IgA antibody (SouthernBiotech). Bone marrow dendritic cell maturation and cytokine secretion

[0200] Bone marrow dendritic cells (BMDCs) were isolated and cultured at ten million cells per milliliter in the completed RPMI 1640 medium (cRPMI) with GM-CSF (20 ng-¹ ml) as previously described. Briefly, fresh culture medium with GM-CSF was supplemented at day 2 after initial culture and then continually cultured for another three days. The BMDCs were the non-adherent and loosely adherent cells in the culture, which were collected by centrifugation at 250x g for 8 min and then seeded at 1 million cells per well into 24-well plates for the following stimulations.

[0201] Fabricated SDAD protein nanoparticles and the adjuvants were diluted with cRPMI. The BMDCs were stimulated with 4 pg^_1 ml of nanoparticles and nanoparticles with 4 pg- ¹ ml of different adjuvant combinations separately overnight. At 24 hours post simulations, the cell culture supernatant was harvested to analyze cytokine secretion. The cell pellet was resuspended in a staining buffer (PBS with 2% of FBS) and used for the cell staining. As previously described, to determine the expressions of IL-1 p, IL-6, TNF-a, IFN-a, and IL-12 in BM DC-cultured supernatant, the ELISA plates were coated with 4 p^_1 ml of cytokine-specific captured antibodies at 4°C overnight. Fifty microliters of supernatant were added into each well and incubated at 37°C for 2 hours followed by the incubation of individually biotin-conjugated detection antibodies and HRP-conjugated streptavidin. The standard curves for each cytokine were generated respectively. To characterize the BMDCs maturation, we incubated the collected cells with Zombie Aqua™ dye (Zombie Aqua™ Fixable Viability Kit, Biolegend) to distinguish dead/live cells and then stained with anti-mouse CD11c-APC (BD Biosciences), CD40-PE, CD80-FITC, and CD86-APC/Cy7 surface antibodies. After thorough washes, the cells were analyzed by BD LSRFortessa™ Cell Analyzer. The standards and other antibodies used in this assay were purchased from Biolegend.

Cellular immune responses

[0202] Mice were euthanized four weeks after immunization. Single spleen and bone marrow cell suspensions were prepared in cRPMI media for enzyme-linked immunosorbent spot (ELISPOT) assays. The 96-well filter plates (Millipore) were coated overnight with 4 pg/ml of anti-mouse IL-2, IL-4, or IFN-y capture antibodies (Biolegend). Then one million splenocytes were seeded with stimulators containing 4 pg^-1 ml of NA1 and NA2 purified proteins, whole inactivated A/Aichi viruses, or NP peptides (BEI Resources, NR-2611) after blocking. The cells were cultured at 37°C for 48 h and incubated with biotin-conjugated IL-2, IL-4, or IFN-y detection antibodies and HRP-conjugated streptavidin (BioLegend). After KPL True Blue substrate (SeraCare) staining, the colonies were measured by Bioreader-6000-E (BIOSYSTEM). To determine ASCs in spleens and bone marrow, one million cells were seeded into filter plates which were coated with 4 pg-¹ ml of A/Aichi, A/PR8 whole inactivated viruses, M2e peptides or NA2 purified proteins. The antibodies secreted from cells were detected with goat anti-mouse IgG-HRP antibodies. The spots were stained with KPL Trueblue and counted by Bioreader-6000-E.

Flow cytometry

[0203] BALFs and Lung tissues were collected one month after intranasally boosting immunization. The cells in BALFs were isolated by centrifugation at 500x g for 10 min and stained with anti-mouse CD45-PE, CD4-Percp, CD8-FITC, CD44-BV421, CD16/32 (BD Biosciencs) antibodies and Zombie NIR™ dye (Zombie NIR™ Fixable Viability Kit, Biolegend) for T cell population analysis by flow cytometry. Lungs tissues were processed with 1 mg/ml of Collagenase type 4 (Worthington Biochemical) and 30 pg-¹ ml of DNase I (Sigma-Aldrich) in RPMI 1640 media at 37°C for 30 mins followed by grinding through a 70-pm cell strainer and centrifuged at 1500 rpm for 5 min at 4°C. After discarding the supernatant, cells were washed twice with the staining buffer. To analyze the different cell populations, we divided cells into three parts and stained them with different combinations of antibodies as below: (1) anti-mouse CD45-PE, CD4-Percp, CD8-FITC, CD103-APC, CD44-BV421, CD69-PE/Cy7, CD16/32 antibodies, and Zombie NIR™ dye; (2) anti-mouse CD19-APC, B220-AF700, IgD-FITC, IgM- PE/Cy7, CD69-PE, CD38-Pacific Blue, CD16/32 antibodies and Zombie NIR™ dye; (3) antimouse CD45-PE, CD11c-Percp/Cy5.5, CD11b-PE/Cy7, CD64-APC, CD24-BV510 (BD Biosciences), CD103-FITC, CD16/32 antibodies, and Zombie NIR™ dye. The other antibodies used in this assay were purchased from Biolegend. The cells were recorded by BD LSRFortessa™ Cell Analyzer.

Statistical analysis

[0204] The data were displayed by means with the standard error of the mean (SEM). The p values were assigned by calculating F-test and a two-tailed Student’s t-test between two groups, p values less than 0.05 were statistically significant.

[0205] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0206] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A layered nanoparticle formed by

(a) desolvating soluble influenza nucleoprotein (NP) to form a nanoparticle, and

(b) crosslinking a fusion protein onto the nanoparticle with a crosslinking agent to form a layered nanoparticle, wherein the fusion protein comprises a truncated influenza hemagglutin (HA) protein lacking a head domain linked to a multimerization domain.

2. The nanoparticle of claim 1, wherein the multimerization domain comprises a tetramerization domain.

3. The nanoparticle of claim 2, wherein the tetramerization domain comprises a GCN4 or tetrabrachion protein.

4. The nanoparticle of any one of claims 1 to 3, wherein the fusion protein further comprises a signal peptide at the N-terminus.

5. The nanoparticle of claim 4, wherein the signal peptide comprises mellitin signal peptide.

6. The nanoparticle of any one of claims 1 to 5, wherein the desolvating agent comprises ethanol, acetone, or combinations thereof.

7. The nanoparticle of any one of claims 1 to 20, wherein the crosslinking agent is selected from the group consisting of formaldehyde, formaldehyde derivatives, formalin, glutaraldehyde, glutaraldehyde derivatives, a protein cross-linker, a nucleic acid cross-linker, a protein and nucleic acid cross-linker, primary amine reactive crosslinkers, sulfhydryl reactive crosslinkers, sulfydryl addition or disulfide reduction, carbohydrate reactive crosslinkers, carboxyl reactive crosslinkers, photoreactive crosslinkers, cleavable crosslinkers, AEDP, APG, BASED, BM(PEO)3, BM(PEO)4, BMB, BMDB, BMH, BMOE, BS3, BSOCOES, DFDNB, DMA, DMP, DMS, DPDPB, DSG, DSP, DSS, DST, DTBP, DTME, DTSSP, EGS, HBVS, sulfo-BSOCOES, Sulfo-DST, and Sulfo-EGS.

8. The nanoparticle of claim 7, wherein the crosslinking agent is a water-soluble crosslinker that contains amine-reactive NHS-ester ends and whose central disulfide bond can be cleaved with reducing agents.

9. The nanoparticle of claim 7, wherein the crosslinking agent can be cleaved with reducing agents.

10. The nanoparticle of claim 7, wherein the crosslinking agent is DTSSP.

11. The nanoparticle of claim 7, wherein the crosslinking agent is SDAD (NHS-SS-Diazirine, succinimidyl 2-((4,4'-azipentanamido) ethyl)- 1 ,3'-dithiopropionate).

12. The nanoparticle of any one of claims 1 to 11 , where the truncated influenza HA protein lacks at least the amino acids corresponding to residues 65 to 320 of amino acid sequence SEQ ID NO:1.

13. The nanoparticle of any one of claims 1 to 12, wherein the head domain is replaced by a linker 3 to 5 amino acids in length that cannot form a fixed secondary structure.

14. The nanoparticle of claim 13, wherein the linker comprises 3 to 5 amino acids selected from glycine, alanine, and serine.

15. The nanoparticle of claims 1141 , wherein the linker is selected from the group consisting of GGG, GGGG (SEQ ID NO:20), GGGGG (SEQ ID NO:21), and GGGGC (SEQ ID NO:22), GGGSS (SEQ ID NO:23).

16. The nanoparticle of any one of claims 1 to 15, wherein the influenza HA protein is an influenza subtype A HA protein.

17. The nanoparticle of any one of claims 1 to 15, wherein the influenza HA protein is an influenza subtype B HA protein.

18. A vaccine composition comprising the nanoparticle of any one of claims 1 to 17 in a pharmaceutically acceptable excipient.

19. The vaccine composition of claim 18, further comprising an adjuvant.

20. A layered nanoparticle formed by

(a) desolvating soluble influenza nucleoprotein (NP) to form a nanoparticle,

(b) crosslinking an M2e-NA fusion protein onto the nanoparticle with a crosslinking agent to form a layered nanoparticle, wherein the M2e-NA fusion protein comprises an influenza virus matrix protein 2 extracellular (M2e) domains linked to a neuraminidase (NA) domain.

21. The nanoparticle of claim 20, wherein the M2e-NA fusion protein further comprises a signal peptide at the N-terminus.

22. The nanoparticle of claim 21 , wherein the signal peptide comprises mellitin signal peptide.

23. The nanoparticle of any one of claims 20 to 22, wherein the desolvating agent comprises ethanol, acetone, or combinations thereof.

24. The nanoparticle of any one of claims 20 to 23, wherein the crosslinking agent is selected from the group consisting of formaldehyde, formaldehyde derivatives, formalin, glutaraldehyde, glutaraldehyde derivatives, a protein cross-linker, a nucleic acid cross-linker, a protein and nucleic acid cross-linker, primary amine reactive crosslinkers, sulfhydryl reactive crosslinkers, sulfydryl addition or disulfide reduction, carbohydrate reactive crosslinkers, carboxyl reactive crosslinkers, photoreactive crosslinkers, cleavable crosslinkers, AEDP, APG, BASED, BM(PE0)3, BM(PE0)4, BMB, BMDB, BMH, BMOE, BS3, BSOCOES, DFDNB, DMA, DMP, DMS, DPDPB, DSG, DSP, DSS, DST, DTBP, DTME, DTSSP, EGS, HBVS, sulfo- BSOCOES, Sulfo-DST, and Sulfo-EGS.

25. The nanoparticle of claim 24, wherein the crosslinking agent is a water-soluble crosslinker that contains amine-reactive NHS-ester ends and whose central disulfide bond can be cleaved with reducing agents.

26. The nanoparticle of claim 24, wherein the crosslinking agent can be cleaved with reducing agents.

27. The nanoparticle of claim 24, wherein the crosslinking agent is DTSSP.

28. The nanoparticle of claim 24, wherein the crosslinking agent is SDAD (NHS-SS- Diazirine, succinimidyl 2-((4,4'-azipentanamido) ethyl)-1 ,3'-dithiopropionate).