AND METHODS OF USE THEREOF

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to and benefit of U.S. Provisional Patent

Application No. 62/751,691, filed October 28, 2018, the entire contents of which are incorporated herein by reference.

BACKGROUND

[0002] Influenza vims infects millions of people globally during seasonal flu epidemics despite annual vaccination programs and hundreds of millions of dollars spent surveilling outbreaks. In addition to the occasional pandemics, seasonal outbreaks caused by an influenza viral infection of the respiratory system results in about 3-5 million cases of severe illness and 250,000-500,000 deaths worldwide every year. Occasionally, new strains of influenza viruses may be transmitted to humans from animal reservoirs, and thereby result in an influenza pandemic with considerably higher morbidity and mortality than seasonal epidemics. There are four categories of influenza: A, B, C, and D, with A and B being responsible for human infection.

[0003] The influenza vims, A/Brisbane/59/2007 (Bris/07), was the last seasonal H1N1 strain recommended by the World Health Organization prior to the 2009 pandemic. Antibodies elicited by Bris/07 have HAI activity against Bris/07-like and few other seasonal influenza vimses but no binding or HAI activity against the pandemic-like H1N1 strains, such as A/Califomia/07/2009 (CA/09). Similarly, CA/09-elicited antibodies have no HAI activity against Bris/07-like or other H1N 1 seasonal strains. HA immunogens to address the elicitation of antibodies with HAI activity against strains across this seasonal-pandemic 2009 divide are desired.

[0004] Swine influenza is a respiratory disease of pigs caused by type A influenza vims

(SIV) implicated in outbreaks regularly for pigs and rarely for humans. These vimses exhibit high morbidity - but low mortality - and contribute to economic loss for pig producers by decreasing yield. Swine vaccination against SIV with commercially available local/regionally isolated inactivated-whole vims vaccines is partially effective at reducing clinical signs, viral shedding, and transmission. However, as the genetic diversity of the influenza vims increases, and the hemagglutinin (HA) surface protein diverges cross-protection from one strain to another decreases, vaccine effectiveness is negatively affected. Autologous vaccination has often failed to provide satisfactory protection. Vaccination decreases economic loss and can also protect against the emergence of human pandemic strains following the reassortment of human, swine and/or avian Hl hemagglutinin (HA) influenza viruses during co-infection in pigs. The 2009 H1N1 swine flu pandemic was due to such an incident. Influenza A virus subtypes isolated in pigs in the U.S. include, but are not limited to, H1N1, N1N2, and N3N2. There is no current vaccine that protects pigs against human and swine Hl influenza viruses, or even against multiple lineages/clades of swine viruses.

[0005] Influenza A is characterized by its surface antigens hemagglutinin (HA or H) and neuraminidase (NA or N), which undergo continuous genetic changes or genetic shifts, thereby enabling the virus to escape host immune responses. Type A viruses may be subdivided into subtypes based on the combinations of hemagglutinin (HA) and neuraminidase on the surface of the virus. There are 18 different hemagglutinin subtypes (H1-H18) and 11 different neuraminidase subtypes (Nl-Nl 1), which may change and increase, e.g., influenza A (H1N1) or (H3N2). Of the influenza A subtypes, viruses of the Hl, H2, and H3 HA subtypes are known to have modified to circulate in humans. The influenza A virus has high levels of variability in viral strains due to a high propensity for genetic mutations. HA and neuraminidase surface proteins undergo continuous genetic modifications, thereby enabling the virus to avoid host immune responses. Moreover, the HA proteins submit to post- translational modifications by adding glycosaccharides to the consensus N-X-S/T (where X is any amino acid except proline) glycosylation motif. Glycosylation in HA is important for protein folding and stability, and, in some cases, significantly affects receptor binding, cleavage of the precursor HA0 protein, and the virulence and antigenicity of the virus. Furthermore, recent studies have suggested that glycosylation on the HA globular head domain physically shields the antigenic sites, preventing antibody recognition and leading to viral evasion from antibody-mediated neutralization. These findings support the general hypothesis that glycosylation on HA renders the virus resistant to neutralizing antibodies, which can serve as a molecular mechanism by which a newly emerged influenza strain, such as a pandemic virus, may evolve into a seasonal strain among the human population.

[0006] Vaccination is the most effective way to prevent influenza virus infections.

However, the diversity of antigenically distinct isolates is a challenge for vaccine development. Current flu vaccines provide some protection against two A strains and one B strain (trivalent vaccines) or two A and two B strains (quadrivalent vaccines) and can be live attenuated vaccines, inactivated vaccines, or recombinant vaccines. But antigenic drift, due in part to influenza viruses’ low-fidelity polymerases, can reduce vaccine efficacy, and the synergy created by no effective vaccine and a virulent influenza strain has pandemic potential as evidenced by the H1N1 strain that infected approximately 500 million people in 1918. In order to overcome these challenges, an influenza vaccine capable of eliciting a potent, broadly reactive HA-specific antibody response that is protective against both seasonal and potential pandemic influenza strains having undergone genetic drift is desired. An approach for designing these types of broadly reactive antigens useful in potentially effective influenza vaccines was construed, and these antigens were deemed Computationally Optimized Broadly Reactive Antigens (COBRA).

[0007] Influenza vaccines presently available induce antibodies that are not cross- reactive with influenza strains that have undergone genetic drift or with circulating influenza strains. Thus, there is an urgent unmet need for influenza vaccines that are, for example, more broadly protective against seasonal and/or genetically shifted influenza viruses having greater breadth and enhanced potency or effectiveness. Preparing an influenza vaccine efficacious for a particular season or pandemic is difficult, especially since it is nearly impossible to predict which antigenic variants may evolve. Therefore, novel vaccine candidates are needed that will elicit immunity to an expansive spectrum of potential influenza virus variants. Thus, there is a need for broadly reactive, pan-epitopic or universal vaccines against influenza by eliciting immunity to potential variants that would reduce flu-related morbidity and mortality. This disclosure is directed to this and other important issues.

SUMMARY

[0008] As described herein, non-naturally occurring, broadly reactive, pan-epitopic antigens and antigen sequences derived from influenza virus and its subtypes (also called serotypes) are provided. These influenza virus antigens are potent immunogens that can elicit a broadly reactive immune response against different serotypes (subtypes) of influenza virus (e.g., H1N1, H1N2, H1N3, H1N4, H1N5, H1N6, H1N7, H1N8, H1N9, H1N10, H1N11), and, ultimately, against present and future influenza virus strains in a subject. As referred to herein, the influenza virus antigens or antigen sequences that elicit an immune response in a subject are immunogenic antigens or immunogens. These influenza immunogens are termed“broadly reactive” and“pan-epitopic” because they elicit the production of broadly reactive antibodies that are directed against optimized influenza virus hemagglutinin protein comprising epitopes from multiple influenza Hl strains. The hemagglutinin proteins described herein have both sequence similarity and variability, and a diversity of epitopes (antigenic determinants) in their antigens and sequences thereof.

[0009] In some aspects, the non-naturally occurring, pan-epitopic influenza A virus antigen amino acid sequences and the antigens comprising the sequences described herein contain broadly reactive hemagglutinin epitopes that reflect sequence similarities and variabilities of influenza A virus serotypes H1N1, H1N2, H1N3, H1N4, H1N5, H1N6, H1N7, H1N8, H1N9, H1N10, H1N11, or combinations thereof, and/or of past, present and future influenza A virus Hl serotypes. Such antigen sequences and the antigens comprising the sequences are thus called“non-naturally occurring, broadly reactive, pan-epitopic” antigens. The antigens are immunogenic and, when introduced into or administered to a subject, elicit broadly reactive antibodies, such as neutralizing antibodies, against influenza A virus, in particular, influenza A virus antigens, such as the hemagglutinin (HA) polypeptide, in the subject.

[0010] Another aspect may provide influenza A virus antigen sequences that are polynucleotide sequences, for example, polynucleotide sequences that encode the amino acid sequences of the antigens and immunogens described herein. Another aspect provides for a composition comprising these polynucleotide sequences that encode the amino acid sequences of the antigens and immunogens described herein and a pharmaceutically acceptable carrier (e.g., diluent, excipient, preservative). For ease of reference, a“non-naturally occurring, broadly reactive, pan-epitopic” antigen or immunogen of influenza virus described herein is interchangeably referred to as a“broadly reactive antigen or immunogen,” or a“pan-epitopic antigen or immunogen.”

[0011] One aspect of the disclosure provides for a non-naturally occurring, broadly reactive, pan-epitopic influenza A virus antigen that generates an immune response against one or more influenza A virus subtypes.

[0012] Another aspect of the disclosure may be directed to a virus-like particle (VLP) comprising the influenza A virus antigen as described here. [0013] A further aspect provides for a subviral particle (SVP) comprising the influenza

A virus antigen of the disclosure.

[0014] Yet another aspect may be directed to a non-naturally occurring, broadly reactive, pan-epitopic influenza A virus immunogen that generates an immune response against one or more influenza A virus subtypes.

[0015] In a further aspect, the influenza A virus antigen, immunogen, VLP, or SVP may generate an immune response comprises the production of neutralizing antibodies.

[0016] Another aspect provides for the influenza A virus antigen, immunogen, VLP, or

SVP that may generate an immune response comprising the production of T-lymphocytes.

[0017] A further aspect may be directed to a pharmaceutical composition comprising the influenza A virus antigen, immunogen, VLP, or SVP of the disclosure, and a pharmaceutically acceptable carrier (e.g., diluent, excipient, preservative). Yet another aspect provides this pharmaceutical composition comprising the influenza A virus antigen, immunogen, VLP, or SVP of the disclosure, and further comprising an adjuvant (e.g., immune response enhancer, immune response stimulator, inducer of infection protection, Freund’s complete adjuvant, Freund’s incomplete adjuvant, Montanide™ ISA 720 and 51 (SEPPIC Inc., New Jersey), MF59 etc.).

[0018] In a further aspect, an immunogenic composition or vaccine comprising the influenza A virus antigen, immunogen, VLP, or SVP of the disclosure may be provided.

[0019] Another aspect of the disclosure provides for a pharmaceutical composition comprising the immunogenic composition or vaccine of the disclosure and a pharmaceutically acceptable carrier (e.g., diluent, excipient, preservative). Yet another aspect provides this pharmaceutical composition comprising the immunogenic composition or vaccine of the disclosure and further comprising an adjuvant (e.g., immune response enhancer, immune response stimulator, inducer of infection protection).

[0020] One of the aspects of the disclosure may be directed to a method of generating an immune response in a subject, comprising administering to the subject, an effective amount of the influenza A virus antigen, immunogen, VLP, or SVP of the disclosure.

[0021] Another aspect may provide a method of generating an immune response in a subject, comprising administering to the subject, an effective amount of the pharmaceutical composition of the disclosure, including but not limited to, the pharmaceutical compositions comprising the influenza A virus antigen, immunogen, VLP, or SVP of the disclosure and a pharmaceutically acceptable carrier (e.g., diluent, excipient, preservative) or these pharmaceutical compositions further comprising an adjuvant (e.g., immune response enhancer, immune response stimulator, inducer of infection protection). In certain aspects, a method of generating an immune response in a subject comprises administering to the subject, an effective amount of the pharmaceutical composition comprising the immunogenic composition or vaccine of the disclosure and a pharmaceutically acceptable carrier (e.g., diluent, excipient, preservative) or these pharmaceutical compositions further comprising an adjuvant (e.g., immune response enhancer, immune response stimulator, inducer of infection protection).

[0022] Yet a further aspect may be directed to a method of generating an immune response in a subject comprising administering to the subject an effective amount of the immunogenic composition or vaccine of the disclosure.

BRIEF DESCRIPTION OF FIGURES

[0023] FIGs. 1 A-1B showthe amino acid sequences of five representative polypeptides

(hemagglutinin proteins (HA)) of the influenza A virus Hl subtype, (also called serotype), viz, H1N1, H1N2, H1N3, H1N4, H1N5, H1N6, H1N7, H1N8, H1N9, H1N10, and H1N11, herein, which are broadly reactive immunogens that elicit an immune response against influenza virus protein. The hemagglutinin proteins identified as H1 V4, H1 V7, and H1 V8 are 566 amino acids in length; the hemagglutinin proteins of H1 V5 and V6 are 565 amino acids in length. Nucleic acid sequences encoding these polypeptides can be used to generate virus-like particles (VLPs) containing the influenza protein antigens, which are used as immunogens or vaccines to generate neutralizing antibodies in immunized subjects.

[0024] FIGs. 2A-2B provide graphical depictions of hemagglutination inhibition (HAI) serum antibody titers induced by vaccination of mice with VLP vaccines expressing one of three HA antigens isolated from A/California/07/2009 (CA/09) (A), A/New Caledonia/20/l999 (NC/99) (B), or A/Brisbane/59/2007 (Bris/07) (C). HAI titers were determined from collected antisera (day 84 post-vaccination) for each group of mice (n=l 1) vaccinated three times (days 0, 28, and 56) against a panel of 16 H1N1 influenza viruses. Dotted lines indicate the 1 :40 HAI titer. Each bar represents the Geometric Mean Titer (GMT) displayed on a log2 Y-axis with standard error of the mean (SEM). The HAI titers for sera obtained from mice vaccinated with HA antigens isolated from CA/09 and exposed to pandemic H1N1 viruses are depicted (A). The HAI titers observed for sera obtained from mice vaccinated with HA antigens isolated from NC/99 and exposed to seasonal H1N1 viruses are depicted (B). The HAI titers observed for sera obtained from mice vaccinated with HA antigens isolated from Bris/07 and exposed to seasonal H1N1 viruses are depicted (C)

[0025] FIG. 3 illustrates the HA monomer ribbon structure for the consensus seasonal virus (left) with potential glycosylation sites at residues 142 and 144 and the consensus pandemic virus structure (right) with the deletion of lysine at amino acid residue 147 (arrow). The table identifies the amino acid residues of interest in the Cb and Sa loci of the consensus globular heads, where the N-glycosylation motifs are indicated in the boxed area.

[0026] FIG. 4 illustrates the schedule for mouse vaccinations and challenges. BALB/c mice (n=5/group) were vaccinated three times (days 0, 28, and 56) with VLP vaccines expressing one of the three Next Generation V Series HA antigens or the Bris/07 or CA/09 HA antigens. Blood was collected at days 0, 28, 56, and 70. Mice were challenged with CA/09 influenza virus (10 ⁶ PFU) at day 70. Mice were weighed each day post-infection until day 84.

[0027] FIG. 5 graphically depicts hemagglutination inhibition (HAI) serum antibody titers induced by vaccination of mice with VLP vaccines, one of the three Next Generation V Series HA antigens (V4, V5, or V6), or the Bris/07 or CA/09 HA antigens. HAI titers were determined from collected antisera (day 84 post-vaccination) for each group of mice (n=l l) vaccinated three times (days 0, 28, and 56) against a panel of 9 H1N1 influenza viruses. The horizontal dotted line indicates the 1 :40 HAI titer. Each bar represents the Geometric Mean Titer (GMT) displayed on a log2 Y-axis.

[0028] FIG. 6 presents a graph illustrating weight loss following CA/09 H1N1 influenza virus challenge. BALB/c mice (11 mice/group) were vaccinated with VLP vaccines on days 0, 28, and 56 with each vaccine or mock and infected on day 70 with 10 ⁶ PFU of the H1N1 isolate A/California/07/2009 (CA/09) intranasally.

[0029] FIG. 7 shows the amino acid sequences of four representative polypeptides

(hemagglutinin proteins (HA)) of the influenza A virus Hl subtype, (also called serotype), herein, which are broadly reactive immunogens that elicit an immune response against influenza virus protein. The hemagglutinin proteins identified as VIPER 9 (V9), VIPER 10 (V10), VIPER 11 (VI 1), and VIPER 12 (V12) are 565 amino acids in length (SEQ ID NOs:6- 9). Nucleic acid sequences encoding these polypeptides can be used to generate virus-like particles (VLPs) containing the influenza protein antigens, which are used as immunogens or vaccines to generate neutralizing antibodies in immunized subjects.

[0030] FIG. 8 illustrates hemagglutination inhibition assay (HAI or HI) serum antibody titers induced by vaccination of mice with COBRA and wild-type HA virus-like particle (VLP) vaccines. HAI titers were determined for each group of immunologically naive mice (n=l 1) vaccinated two times (week 0, 4, and 8) with either the X6 or Pl COBRA H1N3 VLP vaccines or H1N3 VLP vaccines expressing wild-type HA proteins from influenza A/New Caledonia/20/99 (NC/99), A/Brisbane/59/2007 (Bris/07), or A/Califomi a/07/2009 (CA/09) against a panel of 15 H1N 1 influenza viruses. Values are the Log2 HAI titers of each individual animal from antisera collected on week 12. The dotted lines indicate the 1 :40 HAI titer range. NC/99 VLP (A); Bris/07 VLP (B); CA/09 VLP (C); X6 VLP (D); Pl VLP (E).

[0031] FIG. 9 illustrates the survival to H1N1 influenza virus challenges. H1N1 influenza virus challenge of mice. BALB/c mice (11 mice/group) were vaccinated on days 0, 28, and 56 with each vaccine plus the AF03 adjuvant and infected on day 84 with lxlO ⁶ pfu/ml of the H1N1 influenza virus. Kaplan-Meier survival curves for vaccinated mice challenged with each influenza virus A/Califomia/07/2009 (CA/09) (A) or A/Brisbane/59/2007 (Bris/07) (B). Percent survival per vaccine group is listed in parenthesis. Mice were monitored daily for weight loss over a 14 day observation period. The weight of each mouse was recorded for the entire 14 days.

[0032] FIGs. 10A-10D shows hemagglutination inhibition (HAI) serum antibody titers induced by vaccination of mice with VI, V2, or V3 VLP vaccines. HAI titers were determined for each group of mice (n=l 1) from blood collected at week 12 post-vaccination. Mice were vaccinated with mock, COBRA (X6 or Pl), VIPER (VI, V2, or V3), or wild-type (Bris/07 or CA/09) HA VLP vaccines and the collected sera were tested against a panel of 9 H1N1 influenza viruses (Chile/86; Sing/86; TX/19; Bei/95; NC/99; SE06; Bris/07; CA/09; Mich/l5). Values are the geometric mean titers plus standard errors of the means (SEM) (error bars). The horizontal dotted lines indicate the 1 :40-1 :80 HAI titer range. Bris/07 VLP (A); CA/09 VLP (B); X6 VLP (C); Pl VLP (D); VI (E); V2 VLP (F); V3 VLP (G); Mock (H). All data are reported as absolute mean values ± S.E.M. HAI titters were compared using non-parametric one- way ANOVA test. All statistical analyses were performed using GraphPad Prism 7 software (San Diego, CA, USA) and a p < 0.05 was considered statistically significant (*p < 0.05; **p< 0.01; ***p< 0.001; ****p< 0.0001).

[0033] FIGs. 11A-11C illustrates hemagglutination inhibition (FLAT) serum antibody and survival to H1N1 influenza virus challenge. HAI titers were determined for each group of mice (n=l 1) from blood collected at week 12 post-vaccination. Mice were vaccinated with V4 (A), V5 (B), or V6 (C) HA VLP vaccines and the collected sera were tested against a panel of 9 H1N1 influenza viruses (Chile/86; Sing/86; TX/19; Bei/95; NC/99; SI/06; Bris/07; CA/09; Mich/l5). Values are the geometric mean titers plus standard errors of the means (SEM) (error bars). The dotted lines indicate the 1 :40-l :80 HAI titer range. Kaplan-Meier survival curves for vaccinated mice (Mock, CA/09, Pl, V4, V5, V6, and X6 or Bris/07) challenged with each influenza virus A/Califomia/07/2009 (CA/09) (D) or A/Brisbane/59/2007 (Bris/07) (E). Percent survival per vaccine group is listed in parenthesis. All data are reported as absolute mean values ± S.E.M. HAI titters were compared using non-parametric one- way ANOVA test. All statistical analyses were performed using GraphPad Prism 7 software (San Diego, CA, USA) and a p < 0.05 was considered statistically significant (*p < 0.05; **p< 0.01; ***p< O.OOi; ****p< 0.0001).

[0034] FIG. 12 shows viral lung titers in vaccinated mice. Vaccinated BALB/c mice challenged on week 13 post-vaccination had lungs collected (3 mice/group/time point) on day 3 post-infection from vaccinated mice challenged with A/Califomia/07/2009 (A) or A/Brisbane/59/2007 (B) and percent survival assessed. Viral lung titers are listed as plaque forming units (pfu) per gram of lung tissue. Vaccines used for the vaccination of the mice are listed on the x-axis.

[0035] FIGs. 13A-13D illustrates hemagglutination inhibition (HAI) serum antibody and challenge with H1N1 influenza viruses. HAI titers were determined for each group of mice (n=l l) from blood collected at week 12 post-vaccination. Mice were vaccinated with CA/09 VLP (A); Bris/07 VLP (B); V7 VLP (C); or V8 VLP (D) vaccines and the collected sera were tested against a panel of 9 H1N1 influenza viruses identified on the x-axis. Values are the geometric mean titers plus standard errors of the means (SEM) (error bars). The dotted lines indicate the 1 :40-l :80 HAI titer range. Kaplan-Meier survival curves for vaccinated mice challenged with A/Brisbane/59/2007 (E) or A/Califomia/07/2009 (F). Mice challenged with A/Califomia/07/2009 were monitored daily for weight loss over a l4-day observation period (G). [0036] FIGs. 14A-14D shows hemagglutination inhibition (HAI) serum antibody and challenge with H1N1 influenza viruses. HAI titers were determined for each group of mice (n=l l) from blood collected at week 12 post-vaccination. Mice were vaccinated with Mock (A); Bris/07 VLP (B); CA/09 VLP (C); X6 VLP (D); V9 VLP (E); VI 0 VLP (F); VI 1 VLP (G); or V12 VLP (H) vaccines and the collected sera were tested against a panel of 9 H1N1 influenza viruses identified on the x-axis. The dotted lines indicate the 1 :40-1 :80 HAI titer range. All data are reported as absolute mean values ± S.E.M. HAI titters were compared using non-parametric one- way ANOVA test. All statistical analyses were performed using GraphPad Prism 7 software (San Diego, CA, USA) and a p < 0.05 was considered statistically significant (*p < 0.05; **p< 0.01; ***p< 0.001; 0.0001).

[0037] FIG. 15 illustrates survival to H1N1 influenza virus challenge. B ALB/c mice

(11 mice/group) were vaccinated on days 0, 28, and 56 with each vaccine plus the AF03 adjuvant and infected on week 13 with lxlO ⁶ pfu/ml of the A/Califomia/07/2009 H1N1 influenza virus. Kaplan-Meier survival curves for vaccinated mice (identified in the legend) challenged with A/Califomia/07/2009 (CA/09) (A). Percent survival per vaccine group is listed in parenthesis. Viral lung titers are listed as plaque forming units (pfu) per gram of lung tissue on the y-axis (B). Vaccines used for vaccination of mice are listed on the x-axis. Vaccinated mice were challenged with CA/09 as described here. All data are reported as absolute mean values ± S.E.M. HAI titters were compared using non-parametric one- way ANOVA test. All statistical analyses were performed using GraphPad Prism 7 software (San Diego, CA, USA) and a p < 0.05 was considered statistically significant (*p < 0.05; **p< 0.01;

***p< 0.001; ****p< 0.0001).

[0038] FIG. 16 illustrates a schematic of an infection schedule. Ferrets were infected intranasally (10 ⁶ PFU/ml) with A/Singapore/6/l986 H1N1 influenza viruses. Ferrets were bled at days 14 and 81 post-infection. At day 84, all ferrets were mock vaccinated or vaccinated with one of 6 recombinant HA (rHA) vaccines plus ADDAVAX adjuvant. Blood was collected at days 98 (two weeks post-vaccination) and day 125 (6 weeks post-vaccination). Ferrets were infected with the H1N1 influenza vims A/C A/07/09 (10 ⁶ PFU/ml) at day 132. The ferrets were observed for 2 weeks post-challenge for clinical signs of infection. Nasal washes were collected at day 1, 3, and 5 post-infection, and the experiment was terminated at day 166.

[0039] FIGs. 17A-17D. show hemagglutination inhibition (HAI) serum antibody and challenge with H1N1 influenza viruses. HAI titers were determined for each group of pre- immune ferrets (n=4) from blood collected at day 125 post-vaccination. Pre-immune ferrets were vaccinated with Mock (A); CA/09 rHA (B); Pl rHA (C); X6 rHA (D); V3 rHA (E); V6 rHA (F); or V12 rHA (G) vaccines, and the collected sera were tested against a panel of 9 H1N1 influenza viruses identified on the x-axis. Values are the geometric mean titers plus standard errors of the means (error bars). The dotted lines indicated the 1 :40-1 :80 HAI titer range. All data are reported as absolute mean values ± SEM. HAI titers were compared using non-parametric one-way ANOVA test. All statistical analyses were performed using GraphPad Prism 7 software (San Diego, CA, EISA) and a p < 0.05 was considered statistically significant (*p < 0.05; **p< 0.01; ***p< 0.001; ****p< 0.0001).

[0040] FIG. 18 illustrates a challenge with A/California/07/2009 H1N1 influenza virus.

Vaccinated pre-immune ferrets (4 ferrets/group) were infected on day 132 with lxlO ⁶ pfu/ml of the A/California/07/2009 H1N1 influenza virus. The percent original weight per vaccine group by days post-infection is illustrated (A). The viral nasal wash titers (pfu/ml) (Y-axis) for each individual ferret collected on days 1, 3, and 5 post-infection (X-axis) (B) for V3, V6, V12, Pl, CA/09, X6, and Mock (left to right, respectively). All data are reported as absolute mean values ± SEM. HAI titers were compared using non-parametric one-way ANOVA test. All statistical analyses were performed using GraphPad Prism 7 software (San Diego, CA, EISA) and a p < 0.05 was considered statistically significant (*p < 0.05).

[0041] FIGs. 19A-19C illustrates the results of a Focal Reduction Assay (FRA).

Vaccinated, pre-immune ferrets (n=4) were vaccinated at day 0 and 84, with rHA vaccines identified in the legend. At day 125, sera were collected and tested in a focal reduction assay against 5 H1N1 influenza viruses isolated between 1983 and 2009. The collected sere were assayed against Chile/83 (A); Sing/86 (B); NC/99 (C); Bris/07 (D); CA/09 (E). For each virus, the virus concentration was standardized to 1.2 x 10 ⁴ FFU/ml (Focus forming units) (corresponding to 600 FFU/50 pl which is the volume of virus added to each plate). A monolayer of MDCK SIAT cells (2.5 - 3 x 10 ⁵ cells/ml) (100 pL/well in 96-well plate) was plated the day before the assay was run. Cells were 95-100% confluent at the time of the assay. To determine the number of foci detected as a percent infected cells were normalized to 100%. The dotted lines represent the 50% inhibition and the 80% inhibition by sera compared to virus only control wells.

[0042] FIG. 20 shows a schematic depicting the COBRA Pl H1N1 HA trimerized proteins. Each COBRA HA structure presented is generated using the 3D-JIGSAW algorithm and renderings were provided using PyMol for Macintosh platform (MacPyMol). The Sa site (shown in cyan) and Sb site (shown in pink) are adjacent, and the Cb site (shown in dark blue) is presented lower on the structure. The glycans at residues 142 and 144 of the Sa site are specified (highlighted in green), as is the lysine at residue 147 (147K; highlighted in purple) which is adjacent to the glycan at residue 144 of the Sa site.

[0043] FIG. 21 shows a phylogenetic tree of Hl HA sequences of interest. The swine

Hl influenza viruses are separated into three distinct lineages. The Eurasian lineage (grey), the Classical lineage (Alpha clade = blue; Beta clade = Magenta; Gamma clade = pink; Pandemic clade = Red), and the Human Seasonal-like lineage (green). Black sequences are human Hl . Challenge viruses were chosen from two separate lineages: Classical (A/C A/07/09) and Human Seasonal -like (A/SW/NC/l 52702/15). Distance of the tree equals amino acids substitutions per site.

[0044] FIGs. 22A-22B provide a study design for testing COBRA vaccine effectiveness in mice. FIG. 22A (A) provides a COBRA-Based Approach for designing COBRA vaccines using HA reference sequences from Global Initiative on Sharing All Influenza Data (GISAID) or an online flu genome database. FIG. 22A (B) shows the design of HA Vaccine Groups, i.e., SW1, SW2, SW3, SW4, Pl, X3, X6, A/SW/NC/l 52702/15, A/CA/07/09, and Mock PBS. FIG. 22B (C) shows a schematic for the preparation of a vaccine, where a VLP was constructed with the HA of interest, an N3 subtype neuraminidase (NA), and HIV Gag protein. The VLP was mixed 1 : 1 with an oil-in-water adjuvant for the preparation of the vaccine. FIG. 22B (D) shows a depiction of a mouse challenge protocol.

[0045] FIG. 23 shows SW COBRA (A) and Hu and Pl COBRA(B) survival curves, respectively, for A/SW/NC/l 52702/15 or NC/15 (Human seasonal-like) challenge. For the SW COBRA Survival curves (FIG. 23 (A)): Sw/NC/l5 and SW-l had 100% survival; SW-2 and Sw-4 vaccinated mice died (Day 6); SW-3 was similar to PBS control, which plateaued around Day 6 or Day 7. For the Human and Pl COBRA Survival curves (FIG. 23 (B)): X3 and Sw/NC/l5 had 100% survival; Pl mice died (Day4); X6 and PBS plateaued around Day 6 or Day 7 at about 50% survival; CA/09 plateaued around 75% on Day 6. FIG. 23(C) and FIG. 23(D) show SW COBRA and Hu and Pl COBRA Survival curves, respectively, for A/CA/07/09 or CA/09 (classical) challenge. For SW COBRA Survival curves (FIG. 23 (C)), CA/09, SW-l, SW-2, and SW-4 had 100% survival; PBS plateaued at less than 25% survival (Day6); SW-3 plateaued at about 50% survival (Day6). For Hu and Pl COBRA Survival curves (FIG. 23 (D)): CA/09 and Pl had 100% survival; X3 and X6 plateaued to between 50% and 75% survival (Day 6); and PBS plateaued to less than 25% survival (Day 6).

[0046] FIGs. 24A-24B show SW COBRA (FIG. 24 A (A)) and Hu and Pl COBRA

(FIG. 24B (B)) weight loss curves, respectively, for NC/15 (Human seasonal -like) challenge. SW COBRA (FIGs. 24B (C)) and Hu and Pl COBRA (FIG. 24B (D)) weight loss curves, respectively, are shown for CA/09 challenge.

[0047] FIG. 25 shows SW COBRA and Hu and Pl COBRA challenged by NC/15 (A) and FIG. 25 (B), respectively, or challenged by CA/09 (C) and (D), respectively. The vaccines tested are identified on the x-axis

[0048] FIGs. 26A-26E show Hemagglutination Activity Inhibition (HAI) titers (Log 2) from sera collected from mice vaccinated with COBRA-based HA vaccines or wild-type HA- based vaccines: SW1, SW2, SW3, SW4, Pl, X3, X6, CA/09, NC/15 and challenged by the viruses identified on the x-axis.

[0049] FIGs. 27A-27B show the amino acid sequences of four representative polypeptides (hemagglutinin proteins (HA)) of the swine influenza virus. The hemagglutinin proteins identified as SWINE COBRA/SWl (SEQ ID NO: 35), SWINE COBRA/SW2 (SEQ ID NO:36), and SWINE COBRA/SW4 (SEQ ID NO:38) are 566 amino acids in length; the hemagglutinin proteins of SWINE COBRA/SW3 (SEQ ID NO:37) is 565 amino acids in length. Nucleic acid sequences encoding these polypeptides can be used to generate virus-like particles (VLPs) containing the influenza protein antigens, which are used as immunogens or vaccines to generate neutralizing antibodies in immunized subjects.

DETAILED DESCRIPTION

[0050] Influenza viruses are a significant cause of morbidity and mortality in humans, particularly the elderly, infants, and children, and in other species. Potentially fatal, influenza infections cost individuals, employers, and governments billions of dollars in lost wages, lost revenue, and lost productivity annually. The lack of long-term vaccines requires annual inoculations against three or four viral strains that may, or may not, be relevant to the season’s influenza strain.

[0051] In order to overcome the antigenic variability and improve the protective efficacy of influenza vaccines, the development of Computationally Optimized Broadly Reactive Antigens (COBRA) for hemagglutinin (HA) as immunogens that elicit antibodies with HAI activity against both historical seasonal, as well as H1N1 influenza viruses isolated from humans and swine was developed. Two candidate COBRA HA vaccines, Pl and X6, elicited antibodies with differential patterns of HAI activity against a panel of seasonal and pandemic-like H1N1 viruses. The COBRA X6 elicited antibodies that recognized primarily seasonal-like viruses. The COBRA Pl HA elicited antibodies that had HAI activity against both pandemic-like and some seasonal -like H1N1 viruses, but notably not the vaccine strains from 2006 or 2007

[0052] Featured herein are synthetic (non-naturally occurring), immunogenic antigens, e.g., protein and glycoprotein antigens, derived from the influenza (“flu”) hemagglutinin (HA) protein of the Hl strain of influenza A virus, that elicit a potent, broadly reactive, and long- lasting immune response in a subject, e.g., a human subject. Such immunogenic antigens are also referred to as“immunogens” herein. Immunogenic compositions, including but not limited to vaccines, comprising

[0053] Provided are immunogens that protect against disease or symptoms caused by influenza virus subtypes, the influenza A Hl strain, or seasonal influenza Hl strains, spanning several years, including drifted strains not yet in existence. In one embodiment, fully synthetic protein antigens are featured, such as for example, influenza A Hl virus HA protein antigens. Such Hl HA antigens are synthetic proteins not found in nature, yet they retain all of the functions of a natural Hl HA viral protein and are immunogenic, i.e., they can elicit an immune response, in particular, a broadly reactive immune response in the form of neutralizing antibodies, reactive T lymphocytes, or combinations thereof, following administration or delivery to, or introduction into, a subject. Also provided in embodiments of the disclosure are immunogenic compositions, e.g., vaccines, comprising the synthetic influenza A Hl virus protein antigens, or nucleic acid sequences encoding the antigens.

[0054] An Hl HA amino acid sequence, a protein antigen thereof or a fragment of the

Hl HA protein antigen thereof having such a sequence, a portion or a fragment of such a sequence, are useful as an immunogen, or in an immunogenic composition, e.g., a vaccine, that elicits a broadly reactive immune response in a subject (e.g., human, non-human primate, canine, feline, rodent) to whom the immunogenic composition, or vaccine, is administered. The Hl virus immunogens comprise antigenic determinants that represent different“antigenic spaces” that are derived from the sequences of many Hl virus strains analyzed based on seasonal periods of time (either overlapping or non-overlapping seasonal time periods, e.g., calendar time periods), geographical location, or combinations of both. Such overlapping or non-overlapping time periods may encompass different intervals of time, for example, 5 months, 6 months, 7 months, eight months, nine months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 10 years or more, including time intervals therebetween.

[0055] Detailed embodiments are disclosed herein; however, it is to be understood that the disclosed embodiments are merely illustrative and may be embodied in various forms. In addition, each of the examples given in connection with the various embodiments of the disclosure is intended to be illustrative, and not restrictive.

Definitions:

[0056] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention pertains or relates. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et ah, Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics , 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287- 9); Kendrew et al. (eds.); The Encyclopedia of Molecular Biology , published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); Molecular Biology and Biotechnology: a Comprehensive Desk Reference, Robert A. Meyers (ed.), published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0057] As used herein,“a” or“an” shall mean one or more. As used herein when used in conjunction with the word“comprising,” the words“a” or“an” mean one or more than one. As used herein“another” means at least a second or more.

[0058] By“adjuvant” is meant a substance or vehicle that non-specifically enhances the immune response to an antigen. Adjuvants may include a suspension of minerals (e.g., alum, aluminum hydroxide, aluminum phosphate) on which antigen is adsorbed; or water-in- oil emulsion in which antigen solution is emulsified in mineral oil (e.g., Freund’s incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (e.g., Freund’s complete adjuvant) to further enhance antigenicity. Immunostimulatory oligonucleotides (such as those including, for example, a CpG motif) can also be used as adjuvants (see, e.g., U.S. Patent Nos. 6, 194,388; 6,207,646; 6,214,806; 6,218,371; 6,239, 116; 6,339,068; 6,406,705; 6,429, 199). Adjuvants also include biological molecules, such as costimulatory molecules. Exemplary biological adjuvants include, without limitation, interleukin-l (IL-2), the protein memory T- cell attractant“Regulated on Activation, Normal T Expressed and Secreted” (RANTES), granulocyte-macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-a), interferon-gamma (IFN-g), granulocyte-colony stimulation factor (G-CSF), lymphocyte function-associated antigen 3 (LFA-3, also called CD58), cluster of differentiation antigen 72 (CD72), (a negative regulator of B cell responsiveness), peripheral membrane protein, B7-1 (B7-1, also called CD80), peripheral membrane protein, B7-2 (B7-2, also called CD86), the TNF ligand superfamily member 4 ligand (OX40L), the type 2 transmembrane glycoprotein receptor belonging to the TNF superfamily (4-1BBL), or the like.

[0059] By“administer” is meant giving, supplying, or dispensing a composition, agent, therapeutic, or the like, or combinations thereof, to a subject, or applying or bringing the composition, agent, therapeutic, or the like, or combinations thereof, into contact with a subject. Administering or administration may be accomplished by any of a number of routes, such as, for example, without limitation, topical, oral, subcutaneous, intramuscular, intraperitoneal, intravenous (IV), (injection), intrathecal, intramuscular, dermal, intradermal, intracranial, inhalation, rectal, intravaginal, intraocular, and/or the like.

[0060] By“agent” is meant any substance (e.g., a small molecule chemical compound, an antibody, a nucleic acid molecule, a peptide, a polypeptide, fragments thereof).

[0061] By“ alteration” is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 5% or greater percent change in expression levels (e.g., a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%).

[0062] By“ ameliorate” is meant to improve a condition, which, for example, may occur by decreasing (e.g., reducing, diminishing, suppressing, attenuating, arresting, stabilizing) the development or progression of a disease or pathological condition. [0063] By“analog” is meant a molecule that is not identical, but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog’s function relative to the naturally-occurring polypeptide. Such biochemical modifications could, for example, without limitation, increase the analog’s protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid.

[0064] By“antibody” is meant an immunoglobulin (Ig) molecule produced by B lymphoid cells and having a specific amino acid sequence. Antibodies are evoked or elicited in subjects (e.g., humans, animals, mammals) following exposure to a specific antigen (or immunogen). A subject capable of generating antibodies or immunoglobulin (i.e., an immune response) directed against a specific antigen or immunogen is said to be immunocompetent. Antibodies are characterized by reacting specifically with (e.g., binding to) an antigen or immunogen in some demonstrable way, antibody and antigen or immunogen each being defined in terms of the other.

[0065] “Eliciting an antibody response” refers to the ability of an antigen, immunogen, or other molecule to induce the production of antibodies. Antibodies are of different classes (e.g., IgM, IgG, IgA, IgE, IgD) and subtypes or subclasses (e.g., IgGl, IgG2, IgG2a, IgG2b, IgG3, IgG4). An antibody or immunoglobulin response elicited in a subject can neutralize a pathogenic (e.g., infectious, disease-causing) agent by binding to epitopes (e.g., antigenic determinants) on the agent and blocking or inhibiting the activity of the agent, and/or by forming a binding complex with the agent that is cleared from the system of the subject (e.g., via the liver).

[0066] As used herein,“broadly reactive” means that an immune response is elicited against a viral protein (e.g., a virus antigen, virus antigen sequence, virus protein, virus protein sequence) in a subject that is sufficient to inhibit (e.g., block, impede, neutralize, prevent) infection of a broad range of related influenza viruses (e.g., influenza virus subtypes H1N1, H1N2, H1N3, H1N4, H1N5, H1N6, H1N7 _, H1N8, H1N9, H1N10, H1N11, other influenza virus subtypes).

[0067] By“antigen” is meant a substance (e.g., compound, composition) that may stimulate the production of antibodies, an immune response, or a T-cell response in a subject, including compositions that are injected or absorbed into a subject. An antigen reacts with the products of specific humoral or cellular immunity, including, for example, those induced by heterologous immunogens. In some embodiments of the disclosure, the antigen is an influenza viral protein. Other embodiments provide an antigen that elicits or stimulates an immune response in a subject termed an“immunogen.”

[0068] The term“antigenic drift” refers to a mechanism for variation in organisms or microorganisms such as viruses that involves the accumulation of mutations within the genes that code for antibody-binding sites (also called antigenic determinants or epitopes). This process results in a new strain of virus or virus particles that is not inhibited or blocked as effectively by antibodies that were originally generated against the antigens of virus strains prior to mutation, thus allowing the virus to spread more easily throughout a partially immune population. By way of example, antigenic drift may occur in influenza virus subtypes, e.g., H1N1, H1N2, H1N3, H1N4, H1N5, H1N6, H1N7 _, H1N8, H1N9, H1N10, H1N11

[0069] In the context of a live virus, the term“attenuated” reflects a virus that is attenuated if its ability to infect a cell or subject and/or its ability to produce disease is reduced (for example, diminished, abrogated, eliminated) compared to the ability of a wild-type virus to produce disease in the subject. Typically, an attenuated virus retains at least some capacity to elicit an immune response following administration to an immunocompetent subject. In some cases, an attenuated virus can elicit a protective immune response without causing any signs or symptoms of infection. In some embodiments, the ability of an attenuated virus to cause disease or pathology in a subject is reduced at least or equal to 5% (e.g., 10%, 25%, 50%, 75%, 80%, 85%, 90%, 95%) or greater, relative to the ability of a wild-type virus to cause disease or pathology in the subject.

[0070] A“codon-optimized” nucleic acid (or polynucleotide) refers to a nucleic acid sequence that has been altered such that the codons are optimal for expression in a particular system (such as, e.g., a particular species, group of species). For example, a nucleic acid sequence can be optimized for expression in mammalian cells. Codon optimization does not alter the amino acid sequence of the encoded protein.

[0071] In this disclosure,“comprises,”“comprising,”“containing,� �“having,” and the like can have the meaning ascribed to them in U.S. Patent law and can mean“includes,” “including,” and the like;“consisting essentially of’ or“consists essentially of’ and likewise has the meaning ascribed in U.S. Patent law, where the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, and excludes prior art embodiments.

[0072] “Detect” refers to identifying the presence, absence, or amount of a substance

(e.g., analyte, compound, agent) to be detected. By“detectable label” is meant an agent that, when linked to a molecule of interest, renders the latter detectable (e.g., via spectroscopic, photochemical, biochemical, immunochemical, chemical means). Nonlimiting examples of useful detectable labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, haptens

[0073] By“disease” is meant any condition, disorder, pathology, or the like that damages or interferes with the normal function of a cell, tissue, organ, or the like. In this disclosure, examples of diseases include those caused by influenza virus infection and the symptoms and adverse effects that are caused by infection of the body with the influenza virus. Influenza virus causes influenza, more commonly known as“the flu.” Symptoms of the flu can be mild to severe and include high fever, runny nose, sore throat, muscle pain, headache, cough, and fatigue. Severe flu, especially in susceptible individuals such as children and the elderly, can be fatal.

[0074] By“ effective amount” is meant the amount of an active therapeutic agent (e.g., composition, compound, biologic (e.g., a vaccine, peptide, polypeptide, polynucleotide)) required to ameliorate, reduce, improve, abrogate, diminish, eliminate, or the like, the symptoms and/or effects of a disease, condition, or pathology in a subject suffering from the symptoms and/or effects of a disease, condition, or pathology relative to an untreated subject. The effective amount of an immunogen or a composition comprising an immunogen, as used to practice the methods of therapeutic treatment of disease, condition, or pathology caused by the influenza virus, varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an “effective” amount.

[0075] A“therapeutically effective amount” refers to a quantity of a specified agent sufficient to achieve a desired effect in a subject being treated with that agent. For example, this may be the amount of an influenza virus immunogen or vaccine useful for eliciting an immune response in a subject and/or for preventing infection by influenza virus. Ideally, in the context of the present disclosure, a therapeutically effective amount of an influenza virus vaccine or an anti -influenza immunogenic composition is an amount sufficient to increase resistance to, prevent, ameliorate, reduce, and/or treat infection caused by influenza virus in a subject without causing a substantial cytotoxic effect in the subject. The effective amount of an influenza vaccine or immunogenic composition useful for increasing resistance to, preventing, ameliorating, reducing, and/or treating infection in a subject depends on, for example, the subject being treated, the manner of administration of the therapeutic composition and other factors, as noted supra.

[0076] By“fragment” is meant a portion of a polypeptide or nucleic acid molecule.

This portion may contain at least 10% (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%) of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids. A portion or fragment of a polypeptide may be a peptide. In the case of an antibody or immunoglobulin fragment, the fragment typically binds to the target antigen.

[0077] By“fusion protein” is meant a protein generated by expression of a nucleic acid

(polynucleotide) sequence engineered from nucleic acid sequences encoding at least a portion of two different (heterologous) proteins or peptides. To create a fusion protein, the nucleic acid sequences must be in the same open reading frame and contain no internal stop codons. For example, a fusion protein includes influenza virus protein fused to a heterologous protein.

[0078] By“genetic vaccine” is meant an immunogenic composition comprising a polynucleotide encoding an antigen.

[0079] By“influenza virus polypeptide” is meant an amino acid sequence that is at least 85% (e.g., 90%, 95%, 97%, 99%) identical to an amino acid sequence of an influenza virus antigen or an immunogenic fragment thereof, as set forth in, for example, FIGs. 1 A-1B (SEQ ID NOs: l5), FIG. 7 (SEQ ID NOs:6-9), and TABLE 1 (SEQ ID NOs: 10-27), capable of inducing an immune response in an immunized subject. In some embodiments, an influenza virus polypeptide comprises or consists of a HlNl, H1N2, H1N3, H1N4, H1N5, H1N6, H1N7, H1N8, H1N9, H1N10, or H1N11 sequence or a fragment thereof. TABLE 1 provides fragments of an influenza virus antigen, including for example, the Cb, Sa, and Sb antigenic sites of Hl hemagglutinin (HA) antigens. [0080] By“influenza virus polynucleotide” is meant a nucleic acid molecule encoding an influenza virus polypeptide (antigen or antigen protein).

[0081] The terms“geographical location or geographical region” refers to preselected divisions of geographical areas of the earth, for example, by continent or other preselected territory or subdivision (e.g., the Middle East, which spans more than one continent). Examples of different geographical regions include countries (e.g., Turkey, Egypt, Iraq, Azerbaijan, China, United States); continents (e.g., Asia, Europe, North America, South America, Oceania, Africa); recognized geopolitical subdivisions (such as the Middle East); or hemispheres of the world (e.g., Northern, Southern, Eastern, or Western hemispheres).

[0082] “Hybridization” means hydrogen bonding, which may be Watson-Crick,

Hoogsteen, or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, in DNA, adenine and thymine, and cytosine and guanine, are, respectively, complementary nucleobases that pair through the formation of hydrogen bonds.

[0083] By“immunogen” is meant a compound, composition, or substance which is capable, under appropriate conditions, of eliciting or stimulating an immune response, such as the production of antibodies, and/or a T-cell response, in an animal, including compositions that are injected or absorbed into an animal. As used herein, an“immunogenic composition” is a composition comprising an immunogen (such as an influenza virus polypeptide) or a vaccine comprising an influenza virus polypeptide). As will be appreciated by the skilled person in the art, if administered to a subject in need prior to the subject’s contracting disease or experiencing full-blown disease, an immunogenic composition can be prophylactic and result in the subject’s eliciting an immune response, e.g., a neutralizing antibody and/or cellular immune response, to protect against disease, or to prevent more severe disease or condition, and/or the symptoms thereof. If administered to a subject in need following the subject’s contracting disease, an immunogenic composition can be therapeutic and result in the subject’s eliciting an immune response, e.g., a neutralizing antibody and/or cellular immune response, to treat the disease, e.g., by reducing, diminishing, abrogating, ameliorating, or eliminating the disease, and/or the symptoms thereof. In some embodiments, the immune response is a B cell response, which results in the production of antibodies, e.g., neutralizing antibodies, directed against the immunogen or immunogenic composition comprising the antigen or antigen sequence. In a manner similar to the foregoing, in some embodiments, an immunogenic composition or vaccine can be prophylactic. In some embodiments, an immunogenic composition or vaccine can be therapeutic. In some embodiments, the disease isinfluenza, characterized by high fever, headache, muscle aches, fatigue, runny nose, cough, sore throat, or a combination thereof

[0084] The term “immune response” is meant any response mediated by an immunoresponsive cell. In one example of an immune response, leukocytes are recruited to carry out a variety of different specific functions in response to exposure to an antigen (e.g., a foreign entity). Immune responses are multifactorial processes that differ depending on the type of cells involved. Immune responses include cell-mediated responses (e.g., T cell responses), humoral responses (B cell/antibody responses), innate responses and combinations thereof.

[0085] By“immunogenic composition” is meant a composition comprising an antigen, antigen sequence, or immunogen, wherein the composition elicits an immune response in an immunized subject.

[0086] The term“immunize” (or immunization) refers to rendering a subject protected from a disease, infectious disease, or pathology, or the symptoms thereof, caused by influenza virus, such as by vaccination.

[0087] The terms“isolated,”“purified,” or“biologically pure” refer to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation. A“purified” or“biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid, protein, or peptide is purified if it is substantially free of cellular material, debris, non- relevant viral material, or culture medium when produced by recombinant DNA techniques, or of chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using standard purification methods and analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term“purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified. The term“isolated” also embraces recombinant nucleic acids, proteins or viruses, as well as chemically synthesized nucleic acids or peptides.

[0088] By“isolated polynucleotide” is meant a nucleic acid (e.g., a DNA molecule) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

[0089] By an“isolated polypeptide” is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 40%, by weight (e.g., at least 50%, by weight, at least 60%, by weight) free from the proteins and naturally-occurring organic molecules with which it is naturally associated. In certain embodiments, an isolated polypeptide preparation is at least 75% (e.g. 90%, 99%), by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. An isolated polypeptide may be obtained, for example, by extraction from a natural source; by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any standard, appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis. An isolated polypeptide can refer to broadly active virus immunogen polypeptide generated by the methods described herein.

[0090] By“linker” is meant one or more amino acids that serve as a spacer between two polypeptides or peptides of a fusion protein.

[0091] By“marker” is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease, condition, pathology, or disorder.

[0092] As used herein,“obtaining” as in“obtaining an agent” includes synthesizing, isolating, purchasing, or otherwise acquiring the agent. [0093] The term“operably linked” refers to nucleic acid sequences as used herein. By way of example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects (allows) the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, are in the same open reading frame.

[0094] The nucleotide sequence encoding an influenza virus protein (antigen protein) generated by the described methods can be optimized for expression in mammalian cells via codon-optimization and RNA optimization (such as to increase RNA stability) using procedures and techniques practiced in the art.

[0095] A broadly reactive, pan-epitopic immunogen, such as an influenza virus protein, for eliciting an immune response in a subject possesses a collective set of strongly immunogenic epitopes (also called antigenic determinants). An influenza virus protein described herein is a“pan-epitopic” immunogen that is suitable for use as a vaccine and elicits a broadly reactive immune response, e.g., a neutralizing antibody response, against a plurality of influenza virus subtypes that express proteins on the viral surface (e.g., hemagglutinin (HA) protein), when introduced into a host subject, for example, a human subject infected with influenza virus. The immunogenic antigen (or vaccine) is advantageous for providing an anti influenza virus immunogen (or a vaccine) that elicits a broadly active immune response against influenza virus antigens with antigenic variability and similarity, and treats or protects against infection and disease caused by more than one influenza virus type.

[0096] By“open reading frame (ORF)” is meant a series of nucleotide triplets (codons) that code for amino acids without any termination codons. These sequences are usually translatable into a peptide or polypeptide.

[0097] As used herein, an influenza virus“outbreak” refers to a collection of virus subtypes, strains, or isolates from within a geographical location (e.g., within a single country) in a given time period (e.g., in a year or span of years).

[0098] The term“pharmaceutically acceptable vehicle” refers to conventional carriers

(e.g., vehicles, diluents, Keyhole Limpet Hemocyanin (KLH), Concholepas Concholepas Hemocyanin (CCH), Bovine Serum Albumin (BSA), Ovalbumin (OVA)) and excipients that are physiologically and pharmaceutically acceptable for use, particularly in mammalian, e.g., human, subjects. Such pharmaceutically acceptable vehicles are known to the skilled practitioner in the pertinent art and can be readily found in Remington's Pharmaceutical Sciences , by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975) and its updated editions, which describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic or immunogenic compositions, such as one or more influenza virus vaccines, and additional pharmaceutical agents. In general, the nature of a pharmaceutically acceptable carrier depends on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids/liquids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers may include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate, which typically stabilize and/or increase the half-life of a composition or drug. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

[0099] By“plasmid” is meant a circular nucleic acid molecule capable of autonomous replication in a host cell.

[0100] By“polypeptide” (or protein) is meant a polymer in which the monomers comprise amino acid residues that are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used. The terms“polypeptide” or“protein” as used herein are intended to encompass any amino acid sequence and include modified sequences such as glycoproteins. The term“polypeptide” is specifically intended to cover naturally occurring proteins, as well as those which are recombinantly or synthetically produced. The term“residue” or“amino acid residue” also refers to an amino acid that is incorporated into a protein, polypeptide, or peptide.

[0101] Conservative amino acid substitutions are those substitutions that, when made, least interfere with the properties of the original protein, that is, the structure and especially the function of the protein is conserved and is not significantly changed by such substitutions. Examples of conservative amino acid substitutions are known in the art, e.g., as set forth in, for example, U.S. Publication No. 2015/0030628. Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site; and/or (c) the bulk of the side chain

[0102] The substitutions that are generally expected to produce the greatest changes in protein properties are non-conservative, for instance, changes in which (a) a hydrophilic residue, for example, seryl or threonyl, is substituted for (or by) a hydrophobic residue, for example, leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, for example, lysyl, arginyl, or histadyl, is substituted for (or by) an electronegative residue, for example, glutamyl or aspartyl; or (d) a residue having a bulky side chain, for example, phenylalanine, is substituted for (or by) one not having a side chain, for example, glycine.

[0103] By“promoter” is meant an array of nucleic acid control sequences, which direct transcription of a nucleic acid. A promoter includes necessary nucleic acid sequences near the start site of transcription. A promoter also optionally includes distal enhancer or repressor sequence elements. A“constitutive promoter” is a promoter that is continuously active and is not subject to regulation by external signals or molecules. In contrast, the activity of an “inducible promoter” is regulated by an external signal or molecule (for example, a transcription factor). By way of example, a promoter may be a CMV promoter.

[0104] As will be appreciated by the skilled practitioner in the art, the term“purified” does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide, protein, virus, or other active compound is one that is isolated in whole or in part from naturally associated proteins and other contaminants. In certain embodiments, the term“substantially purified” refers to a peptide, protein, virus or other active compound that has been isolated from a cell, cell culture medium, or other crude preparation and subjected to routine methods, such as fractionation, chromatography, or electrophoresis, to remove various components of the initial preparation, such as proteins, cellular debris, and other components.

[0105] A“recombinant” nucleic acid, protein or virus is one that has a sequence that is not naturally occurring or that has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. Such an artificial combination is often accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques. A“non-naturally occurring” nucleic acid, protein, or virus is one that may be made via recombinant technology, artificial manipulation, or genetic or molecular biological engineering procedures and techniques, such as those commonly practiced in the art.

[0106] By“ reduces” is meant a negative alteration of at least 5%, 10%, 25%, 30%,

40%, 50%, 75%, 80%, 85%, 90%, 95%, 98%, or 100%.

[0107] By“reference” is meant a standard or control condition.

[0108] A“reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least 16 amino acids (e.g., 20, 25,35, 40, 50, 100). For nucleic acids, the length of the reference nucleic acid sequence will generally be at least 50 nucleotides (e.g., 60, 75, 100, 300) or any integer thereabout or therebetween.

[0109] By“specifically binds” is meant a compound or antibody that recognizes and binds a polypeptide, such as a virus polypeptide, peptide, or vaccine product, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide, such as a virus polypeptide or peptide.

[0110] Nucleic acid molecules useful in the methods described herein include any nucleic acid molecule that encodes a polypeptide as described, or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having“substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By“hybridize” is meant pairing to form a double- stranded molecule between complementary polynucleotide sequences (e.g., a gene), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger, (1987), Methods Enzy mol., 152:399; Kimmel, A. R., (1987), Methods Enzymol. 152:507).

[0111] By way of example, stringent salt concentration may ordinarily be less than 750 mM NaCl and 75 mM trisodium citrate (e.g., 500 mM NaCl and 50 mM trisodium citrate, 250 mM NaCl and 25 mM trisodium citrate). Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide or the like, while high stringency hybridization can be obtained in the presence of at least 35% formamide (e.g., 40%, 45%, 50%). Stringent temperature conditions will ordinarily include temperatures of at least 30°C (e.g., 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 42°C). Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In certain embodiments, hybridization will occur at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS (e.g., 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 pg/ml denatured single stranded DNA,42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 pg/ml single stranded DNA). Useful variations on these conditions will be apparent to those skilled in the art.

[0112] For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will be less than 30 mM NaCl and 3 mM trisodium citrate (e.g., 15 mM NaCl and 1.5 mM trisodium citrate). Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least 25°C (e.g., 42°C, 68°C). In a certain embodiment, wash steps will occur at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS (e.g., 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS, 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS). Additional variations on these conditions will be apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis {Science 196: 180, 1977); Grunstein and Hogness ( Proc . Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. {Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel {Guide to Molecular Cloning Techniques , 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0113] By“substantially identical” is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity or greater to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). For example, such a sequence may be 60% or greater (e.g., 80%, 85%, 90%, 95%, 98%, 99%) identical at the amino acid level or nucleic acid to the sequence used for comparison. [0114] “Sequence identity” refers to the similarity between amino acid or nucleic acid sequences that is expressed in terms of the similarity between the sequences. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the sequences are. Homologs or variants of a given gene or protein will possess a relatively high degree of sequence identity when aligned using standard methods. Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e ³ and e ¹⁰⁰ indicating a closely related sequence. In addition, other programs and alignment algorithms are described in, for example, Smith and Waterman, 1981, Adv. Appl. Math. 2:482; Needleman and Wunsch, 1970, J Mol. Biol. 48:443; Pearson and Lipman, 1988 , Proc. Natl. Acad. Sci. U.S.A. 85:2444; Higgins and Sharp, 1988, Gene 73 :237-244; Higgins and Sharp, 1989, CABIOS 5: 151-153; Corpet et al., 1988, Nucleic Acids Research 16: 10881-10890; Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. U.S.A. 85:2444; and Altschul et al., 1994, Nature Genet. 6: 119-129. The NCBI Basic Local Alignment Search Tool (BLAST™) (Altschul et al. 1990, J. Mol. Biol. 215:403-410) is readily available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx.

[0115] By“sequon” is meant a sequence of consecutive amino acids (typically a tri peptide sequence of Asn-Xaa-Ser/Thr, where Xaa is any amino acid except for proline) in a protein for N-glycosylation. For example, the sequon could serve as the attachment site to a polysaccharide, oftentimes, an N-liked-glycan, where the polysaccharide is linked to the protein via the nitrogen atom in the side chain of asparagine. This N-glycosylation rule is generally based on the GlcNAc(l-N) linkage type. Instead of Serine or Threonine, the +2 position (i.e., the second residue after Asparagine) may be Cysteine or Valine. An extended sequon may have consecutive amino acids, for example, Asp/Glu-Xaa-Asn-Xaa-Ser/Thr.

[0116] By“subject” is meant an animal, e.g., a mammal, including, but not limited to, a human, a non-human primate, or a non-human mammal, such as a bovine, equine, canine, ovine, or feline mammal, or a sheep, goat, llama, camel, or a rodent (e.g., rat, mouse, gerbil, hamster). In a nonlimiting example, a subject is one who is infected with an influenza virus, or who is at risk of infection by such virus, or who is susceptible to such infection. In some aspects as described herein, the subject is a human subject, such as a patient.

[0117] As used herein, all ranges of numeric values provided herein include the endpoints and all possible values disclosed between the disclosed values. These ranges are understood to be shorthand for all of the values within the range, inclusive of the first and last stated values. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,

12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or greater, consecutively, such as to 100 or greater.

[0118] As used herein, the terms“treat,”“treating,”“treatment,” and the like refer to reducing, diminishing, decreasing, abrogating, ameliorating, or eliminating, a disease, condition, disorder, or pathology, and/or symptoms associated therewith. While not intending to be limiting,“treating” typically relates to a therapeutic intervention that occurs after a disease, condition, disorder, or pathology, and/or symptoms associated therewith, have begun to develop to reduce the severity of the disease, and the associated signs and symptoms. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disease, condition, disorder, pathology, or the symptoms associated therewith, be completely eliminated.

[0119] As used herein, the terms“prevent,”“preventing,”“prevention,”“prop hylactic treatment” and the like, refer to inhibiting or blocking a disease state, or the full development of a disease in a subject, or reducing the probability of developing a disease, disorder or condition in a subject, who does not have, but is at risk of developing, or is susceptible to developing, a disease, disorder, or condition. [0120] As referred to herein, a“transformed” or“transfected” cell is a cell into which a nucleic acid molecule or polynucleotide sequence has been introduced by molecular biology techniques. As used herein, the term“transfection” encompasses all techniques by which a nucleic acid molecule or polynucleotide may be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked nucleic acid (DNA or RNA) by electroporation, lipofection, and particle gun acceleration.

[0121] By“vaccine” is meant a preparation of immunogenic material (e.g., protein or nucleic acid; vaccine) capable of stimulating (eliciting) an immune response, administered to a subject to treat a disease, condition, or pathology, or to prevent a disease, condition, or pathology, such as an infectious disease (caused by influenza virus infection, for example). The immunogenic material may include, for example, attenuated or killed microorganisms (such as attenuated viruses), or antigenic proteins, peptides or DNA derived from such microorganisms. Vaccines may elicit a prophylactic (preventative) immune response in the subject; they may also elicit a therapeutic response immune response in a subject. As mentioned above, methods of vaccine administration vary according to the vaccine, and can include routes or means, such as inoculation (intravenous or subcutaneous injection), ingestion, inhalation, or other forms of administration. Inoculations can be delivered by any number of routes, including parenteral, such as intravenous, subcutaneous or intramuscular. Vaccines may also be administered with an adjuvant to boost the immune response.

[0122] As used herein, a“vector” refers to a nucleic acid (polynucleotide) molecule into which foreign nucleic acid can be inserted without disrupting the ability of the vector to replicate in and/or integrate into a host cell. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. An insertional vector is capable of inserting itself into a host nucleic acid. A vector can also include one or more selectable marker genes and other genetic elements. An expression vector is a vector that contains the necessary regulatory sequences to allow transcription and translation of inserted gene or genes in a host cell. In some embodiments of the present disclosure, the vector encodes influenza virus protein. In some embodiments, the vector is the pTR600 expression vector (U.S. Patent Application Publication No. 2002/0106798; Ross et ah, 2000, Nat Immunol. 1(2): 102-103; and Green et ah, 2001, Vaccine 20:242-248).

[0123] By“virus-like particle (VLP)” is meant virus particles, as disclosed herein, influenza virus VLPs, made up of one of more viral structural proteins, but lacking the viral genome. Because VLPs lack a viral genome, they are non-infectious and yield safer and potentially more-economical vaccines and vaccine products. In addition, VLPs can often be produced by heterologous expression and can be easily purified. Most VLPs comprise at least a viral core protein that drives budding and release of particles from a host cell. Influenza virus VLPs can be produced by transfection of host cells with plasmids encoding proteins derived from influenza virus. After incubation of the transfected cells for an appropriate time to allow for protein expression (such as for approximately 72 hours), VLPs can be isolated from cell culture supernatants. By way of example, a protocol for purifying or isolating influenza VLPs from cell supernatants can involve low speed centrifugation (to remove cell debris), vacuum filtration and ultracentrifugation of the VLPs through 20% glycerol.

[0124] Unless specifically stated or obvious from context, as used herein, the terms“a”,

“an”, and“the” are understood to be singular or plural. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description.

[0125] Unless specifically stated or obvious from context, as used herein, itis understood that numerical values described herein are within a range of normal tolerance in the art, for example within 1 (e.g., 1.5, 2, 2.5, 3) standard deviations of the mean. For example, the numerical value may be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are within a range of normal tolerance in the art.

[0126] The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of some embodiments for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0127] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0128] All terms used herein are intended to have their ordinary meaning in the art unless otherwise provided. All concentrations are in terms of percentage by weight of the specified component relative to the entire weight of the topical composition, unless otherwise defined. Influenza Vims

[0129] The influenza vims is an enveloped, single-stranded, negative-sense RNA vims belonging to the Orhomyxoviridae family. Influenza vimses are categorized into four genera (A, B, C, or D), of which A and B subtypes vimses are responsible for most human influenza infections. Influenza A is a highly contagious respiratory illness, while influenza B, although similar to influenza A, infects only humans. Influenza C infections cause mild respiratory illness, and influenza subtype D infects only cattle.

[0130] The influenza A vims genome resides in the viral core and consists of approximately 13,500 nucleotides in eight ribonucleoprotein (RNP) segments that encode at least 17 proteins. The influenza A vims includes the stmctural proteins hemagglutinin (HA), neuraminidase (NA). Influenza vims also comprises six additional internal genes, which give rise to eight different proteins, including polymerase genes PB1, PB2 and PA, matrix proteins Ml and M2, nucleoprotein (NP), and non-stmctural proteins NS 1 and NS2 (See, e.g., Horimoto et al., 2001, Clin Microbiol Rev. 14(1): 129-149). A matrix of Ml stmctural protein encapsulates the viral core, and glycoproteins hemagglutinin (HA) and neuraminidase (NA) overlaying, or studding, this Ml matrix facilitate viral adhesion to host cells and host immune system avoidance. Transport of a vims into the cell and delivery of RNPs to the nucleus depends on each of these stmctural proteins.

[0131] There are 18 known HA proteins and 11 known NA proteins, with each influenza A vims subtype expressing only one type of HA protein (e.g., Hl) and one type of NA protein (e.g., Nl). Each monomer comprises an HA1 and HA2 region linked by a disulfide bridge. The HA1 domain is responsible for binding to sialic acid. The C-terminus of the HA2 domain anchors HA to the viral membrane, and a region near the N-terminus of the HA2 domain comprises a fusion peptide. When outside the cell or in a high pH environment, the fusion peptide is concealed in a hydrophobic pocket of HA. Once internalized and residing in a low pH endosome, the fusion peptide is released the HA1 and HA2 domains dissociate. The fusion peptide inserts into the endosomal membrane thereby drawing the viral and endosomal membranes into close proximity, and the HA2 domain undergoes conformational change that causes the viral envelope and the endosome’ s membrane to fuse.

[0132] HA is a viral surface glycoprotein comprising three identical monomers. HA comprises approximately 560 amino acids (e.g., 565 amino acids, 566 amino acids) and represents 25% of the total virus protein. As described herein, HA is a protein antigen that is highly useful as an immunogen because it contains a diverse repertoire of epitopes against which antibodies are generated in a subject or host that encounters the HA antigen of influenza viruses during infection. HA is responsible for adhesion of the viral particle to, and its penetration into, a host cell, particularly, in the respiratory epithelium, in the early stages of infection. Cleavage of the virus HAO precursor into the HA1 and HA2 sub-fragments is a necessary step in order for the virus to infect a cell. Thus, cleavage is required in order to convert new virus particles in a host cell into virions capable of infecting new cells. Cleavage is known to occur during transport of the integral HAO precursor membrane protein from the endoplasmic reticulum of the infected cell to the plasma membrane. In the course of transport, HA undergoes a series of co- and post-translational modifications, including proteolytic cleavage of the precursor HA into the amino-terminal fragment HA1 (“head”) and the carboxy terminal HA2 (“tail” or“stalk”). One of the primary difficulties in growing influenza strains in primary tissue culture or established cell lines arises from the requirement for proteolytic cleavage activation of the influenza hemagglutinin in the host cell.

[0133] NA is a glycoprotein involved in the egress of replicated virus from an infected cell. This protein enzymatically cleaves sialic acid groups from other glycoproteins on the cell surface, thereby promoting the release of progeny viruses. NA also facilitates virus entry into respiratory cells by cleaving the sialic acid residues that act as receptor decoys from mucin glycoproteins expressed by the potential host cells.

[0134] The viral expression profile allows discrimination between different influenza

A subtypes based on viral HA/NA expression (e.g., H1N1, H1N2) (compare influenza B viruses, which are categorized as either the B/Yamagata or B/Victoria lineage viruses). These structural proteins are components of the mature virus particle, while nonstructural proteins, such as the heterotrimeric RNA polymerase and interferon-antagonist NS1 protein, are involved in replication of the viral genome and host immune evasion, respectively. (For a review of influenza viruses, see, e.g., Bouvier, NM et ah, Vaccine , Vol. 26 (Suppl. 4): D49- D53, 2008).

Virus Entry

[0135] Entry of influenza virus into a host cell is mediated by hemagglutinin (HA). HA is comprised of two structurally distinct regions, a stem and a globular head comprising a N- acetylneuraminic (sialic) acid receptor binding site. This binding site, highly conserved among different HA subtypes, recognizes and binds to the sialic acid on the host cell surface, and once bound to this host cell receptor, the virus is endocytosed. During trafficking of the virus, early endosomes mature into late endosomes with a lower pH. The acidic environment of the late endosome causes conformational change in HA that exposes a fusion peptide and facilitates the merger of the viral envelope and the late endosomal membrane. After fusion, the ribonucleoproteins (RNPs), composed of viral RNA segments coated with nucleoprotein (NP), including matrix proteins Ml and M2 both encoded on RNP unit 7, are released via a resulting endosomal membrane pore into the cytoplasm and uncoated. During uncoating, the interaction between the RNPs and Ml matrix protein is disrupted, and the unencumbered RNPs are in condition to be imported into the nucleus. Once in the nucleus, viral replication and transcription commences, and the resulting mRNA is exported to the cytoplasm. Packaging exported viral RNA (vRNA) into the viral core is believed to be facilitated by Matrix protein 1 (Ml) interactions with the vRNA, NPs, and the nuclear export protein (NEP).

The Humoral Immune Response Against Influenza

[0136] In infected subjects, the humoral immune response is hypothesized to be vital for controlling influenza infection and dissemination, and infection with one serotype provides long-lasting protection to that specific serotype (homotypic immunity). Subsequent infection by another influenza serotype results in short-lived protection (heterotypic immunity), but this transient immunity may increase the risk of flu due to reassortment of hemagglutinin viral RNA into circulating seasonal influenza viruses. The transient nature of heterotypic immunity is believed to be due to cross-reactive viral protein-specific antibodies that are protective above a certain concentration threshold.

[0137] An antigen capable of eliciting a natural antibody response to influenza infection in humans is the hemagglutinin (HA) protein. Neutralizing antibodies are directed against the viral HA protein and inhibit viral attachment, internalization, replication within cells, and egress from cells. Some embodiments may provide a neutralizing or protective antibody that binds and inhibits the function of antigens, such as an influenza virus antigen, or an influenza A virus antigen described here.

[0138] The influenza A virus antigen sequences, immunogenic compositions and vaccines described herein induce a broadly reactive immune response (antibodies) that target and are directed against the hemagglutinin Hl protein of influenza A virus Hl subtypes (e.g., H1N1, H1N2, H1N3, H1N4, or a combination thereof). Other embodiments provide for influenza A virus antigens, immunogens, virus-like particles (VLPs), subviral particles (SVPs), and the like which elicit an immune response against a broad range of different subtypes of influenza virus, including antigenically different subtypes, where the immune response is against more than one influenza virus (e.g., two, three, four, five, six). Further embodiments may provide compositions comprising these antigens, immunogens, VLPs, SVPs, or other components that elicit an anti-influenza immune response, such as those comprising the sequences or fragments selected from one or more of FIGs. 1A-1B (SEQ ID NOs: l-5), FIG. 7 (SEQ ID NOs:6-9), and/or TABLE 1 (SEQ ID NOs: 10-27). In certain embodiments, influenza A virus antigen sequences that elicit broadly reactive antibodies were affected by the glycosylation sites at residues 142 and 144, as well as the lysine residue at position 147 of the Sa antigenic site in HA antigens.

[0139] In one embodiment, non-naturally occurring, broadly reactive, pan-epitopic influenza A virus antigens are provided where these antigens generate an immune response against influenza viruses (e.g., one or more influenza A virus subtypes). The antigen in certain embodiments may be an influenza A virus hemagglutinin (HA) Hl protein or an antibody binding portion thereof. Another embodiment may provide an influenza A virus antigen subtype that is any one of H1N1, H1N2, H1N3, H1N4, H1N5, H1N6, H1N7, H1N8, H1N9, H1N10, H1N11, as well as other present and future influenza virus subtypes, or a combination thereof. In yet a further embodiment, the influenza A virus antigen of the disclosure comprises an amino acid sequence that has 80% or greater (e.g., 85%, 90%, 95%, 98%, 99%) identity to an amino acid sequence of an influenza A virus antigen or antigenic fragment selected from any one of SEQ ID Nos: 1 -27, or any combination thereof. Other embodiments may be directed to any influenza A virus antigen or antigenic fragment selected from the amino acid sequences of SEQ ID NOs: 1-27, or combinations thereof, where the antigen or antigenic fragment generates an immune response against one or more influenza A virus subtypes. A further embodiment provides for an influenza A virus antigen or antigenic fragment that is a broadly reactive immunogen, which elicits a broad immune response in a host subject immunized with such an antigen or antigenic fragment, thereby producing a cross-reactive immune response against any of the influenza A virus subtypes disclosed herein, any other present or future influenza A virus subtypes, or any combinations thereof. Broadly Reactive Influenza Virus Proteins and Fragments Thereof

[0140] Embodiments of the disclosure may provide for non-naturally occurring, broadly reactive, pan-epitopic influenza antigens that generate an immune response against influenza, for example and not limited to, one or more influenza A virus subtypes. In certain embodiments, the antigen may be an influenza A virus hemagglutinin (HA) protein or antibody-binding portion thereof. Further embodiments may provide for these antigens that include influenza A virus subtypes (e.g., H1N1, H1N2, H1N3, H1N4, H1N5, H1N6, H1N7, H1N8, H1N9, H1N10, H1N11, or a combination thereof). Certain embodiments of the disclosure are directed to non-naturally occurring, broadly reactive, pan-epitopic influenza virus proteins or polypeptides (e.g., antigens, immunogens), fragments of such influenza virus proteins or polypeptides, influenza virus-like particles (VLPs), Subviral Particles (SVPs), and the like. These VLPs and SVPs may comprise an influenza immunogen described herein containing diverse epitopes (e.g., antigenic determinants) that endow the antigen with the ability to generate a broadly reactive immune response against influenza and its symptoms, either prophylactically or therapeutically. The immune response may occur following administration and delivery of the influenza virus immunogen disclosed herein to a subject susceptible to influenza (e.g., humans). By way of example, representative HA antigenic sequences generated by the practice of methods (described in e.g., Crevar CJ, et al. Hum Vaccin Immunother 11 :572-583, 2015; Giles BM, et al. Clin Vaccine Immunol. 19: 128-139, 2012; Giles BM, et al. J Infect Dis. 205: 1562-1570, 2012; Giles BM and Ross TM. 201 1. Vaccine 29:3043-3054, 2016; Carter DM, et al. J. Virol. 90(9):4720-4734, 2016) are presented in FIGs. 1A-1B (e.g, SEQ ID Nos: 1-5), FIG. 7 (e.g, SEQ ID NOs:6-9) and TABLE 1 (e.g, SEQ ID Nos: 10-27). The influenza A virus antigen may comprise, in certain embodiments, an amino acid sequence that is at least 80% (e.g, 85%, 90% 95%, 97%, 99%) identical to an amino acid sequence of an influenza A virus antigen or antigenic fragment thereof selected from any one of SEQ ID NOs: l-27, or any combination thereof. These sequences or fragments thereof may be used in various embodiments disclosed here in order to generate a broadly reactive immune response to influenza, including but not limited to, influenza A virus.

[0141] Also provided are non-naturally occurring, broadly reactive, pan-epitopic influenza antigen polypeptides as described herein, such as pan-epitopic, broadly reactive influenza HA polypeptides. In certain embodiments, the amino acid sequence of the polypeptide is at least 95% to 99% (inclusive) identical to the amino acid sequence of a hemagglutinin polypeptide of influenza, or fragments thereof shown in FIGs. 1A-1B (SEQ ID NOs: l-5), FIG. 7 (SEQ ID NOs:6-9), and TABLE 1 (SEQ ID NOs: 10-27). In certain embodiments, the amino acid sequence of the influenza HA polypeptide that is at least 95% to 99% (inclusive) identical to the amino acid sequence of an HA polypeptide shown in FIGs. 1A-1B (SEQ ID NOs: 1-5), FIG. 7 (SEQ ID NOs:6-9), and TABLE 1 (SEQ ID NOs: 10-27) lacks the N-terminal methionine residue. In yet another embodiment, the amino acid sequence of the influenza polypeptide is at least 80% to 100% (inclusive) identical to amino acids 1 to 566 or to amino acids 1 to 565 of the influenza HA polypeptides shown in FIGs. 1 A-1B (SEQ ID NOs: 1-5) and FIG. 7 (SEQ ID NOs:6-9).

[0142] Provided are non-naturally occurring, broadly reactive, pan-epitopic influenza virus immunogenic polypeptides (immunogens) and influenza virus-like particles (VLPs), or subviral particles (SVPs), comprising a broadly reactive immunogen (e.g., influenza A hemagglutinin (HA) polypeptide) containing diverse epitopes (antigenic determinants) that endow the immunogen with the ability to generate a broadly active immune response so as to treat influenza infection and its symptoms, either prophylactically or therapeutically, following administration and delivery to a susceptible subject. By way of example, representative influenza virus hemagglutinin immunogenic antigen sequences portions of thereof as described herein that are useful immunogens, VLPs, SVPs, are presented in FIGs. 1A-1B (SEQ ID NOs: 1-5), FIG. 7 (SEQ ID NOs:6-9), and TABLE 1 (e.g, SEQ ID Nos: 10-27). In other embodiments, such portions of the influenza HA antigens described herein may be selected from those fragments, such as but not limited to the Cb, Sa, and Sb antigenic sites, of VIPER V4, V5, V6, V7, and V8 sequences presented in FIGs. 1 A-1B (SEQ ID NOs: 1-5) and V9, V10, VI 1, and V12 sequences presented in FIG. 7 (SEQ ID NOs:6-9) that are immunogenic. See also , TABLE 1. In some embodiments, the broadly reactive, pan-epitopic immunogenic influenza hemagglutinin polypeptides may be administered as SVPs or VLPs comprising the influenza A virus antigen or portions thereof, where the antigen comprises an amino acid sequence that is at least 80% (e.g., 85%, 90%, 95%, 97%, 99%) identical to an amino acid sequence of an influenza A virus antigen or antigenic fragment selected from any one of SEQ ID NOs: 1-27, or any combinations thereof. Other embodiments may provide for an immunogen, immunogenic composition, subviral particle, virus-like particle, vaccine, or the like comprising polynucleotide sequences encoding the influenza antigen sequences as described herein.

[0143] It will be understood that influenza virus antigen sequences and the immunogens described and provided herein are non-naturally occurring, broadly reactive, and pan-epitopic, whether or not these characteristics and features are explicitly stated. It will also be appreciated that the influenza virus antigen proteins, e.g., influenza hemagglutinin proteins, as described herein and as used as immunogens are non-naturally occurring or synthetic antigens, or antigenic portions thereof, that elicit an immune response (e.g., neutralizing antibodies, production of T-lymphocytes) in a subject. These influenza virus antigens, including immunogens, are“broadly reactive” and“pan-epitopic” because they elicit the production of broadly reactive or cross-reactive antibodies that are directed against optimized influenza virus hemagglutinin protein comprising epitopes from multiple influenza strains (e.g., influenza A, influenza Hl), and thereby broadly protective or neutralizing. One embodiment may provide an influenza virus antigen that is cross-reactive or cross-protective with at least one (e.g., two, three, four, five, six) influenza viruses or subtypes of influenza. In another embodiment, the influenza virus antigen is cross-reactive with one or more influenza viruses, where the influenza virus is selected from human, avian, swine, equine, canine, and the like.

[0144] In one embodiment, the antigen sequences, immunogenic compositions (e.g., vaccines, medicinal product), VLPs, SVPs, or combinations thereof described herein for influenza virus are advantageous as they generate an immune response, (e.g., an antibody response, production of neutralizing antibodies or T-lymphocytes) directed against influenza A Hl serotypes, without enhancing disease. Such a property provides a highly useful immunogenic composition (e.g., vaccine, medicinal product which may be used instead of, or interchangeably with any of these terms), that protects against all serotypes of the virus to avoid antibody dependent enhancement (ADE). ADE may occur when pre-existing antibodies to an influenza A virus Hl serotype do not neutralize, but instead enhance, a heterotypic infection by a different influenza A virus Hl subtype. As described herein, an influenza virus-like particle (VLP) immunogenic composition (e.g., vaccine, medicinal product) which targets the hemagglutinin (HA) glycoprotein of influenza A virus and which is broadly reactive against different influenza A virus Hl subtypes is provided (e.g., Examples 3, 7-13 and 17-18; FIGS. 5-6, 8-14, TABLE 1). As will be appreciated by one skilled in the art, a subviral particle (SVP) relates to or is a molecule (particle) comprising either genetic material or protein, and which is smaller than the intact influenza virus particle, while maintaining features and properties of the virus. Other embodiments may provide for an influenza SVP immunogenic composition (e.g., vaccine, medicinal product) which targets the hemagglutinin (HA) glycoprotein of influenza A virus and which is broadly reactive against different influenza A virus Hl subtypes, where the VLP or SVP comprise the influenza A virus antigen, or fragments thereof, as described herein (e.g., an amino acid sequence of an influenza A virus antigen or antigenic fragment thereof selected from any one of SEQ ID NOs: 1-27, or any combinations thereof, at least 80% identical to an amino acid sequence of an influenza A virus antigen or antigenic fragment thereof selected from any one of SEQ ID NOs: 1-27, or any combinations thereof).

[0145] The broadly reactive and immunogenic influenza A virus Hl antigen sequences that are capable of generating an immune response against influenza virus subtypes, as well as present and future influenza virus strains, may be generated by a method such as described in co-pending provisional patent application number 62/697,818, filed July 13, 2018, the contents of which are incorporated herein by reference, which involves a consideration of parameters associated with influenza virus, such as geography and time (e.g., a season) of infection, for example, amino acid sequences of influenza virus subtypes and/or strains present in a geographical area or location, such as the Americas or Asia, during a selected period of time (e.g., a linear time range), in which the influenza virus was isolated.

[0146] In some embodiments, the influenza SVPs or VLPs include a viral protein, such as influenza hemagglutinin protein. In embodiments, the SVPs or VLPs may include other structural proteins of influenza virus. The production of SVPs and VLPs has been described in the art and is within the skill and expertise of one of ordinary skill in the art. Briefly, and as described herein, influenza SVPs or VLPs can be produced by transfection of host cells with one or more plasmids containing polynucleotide sequences that encode an influenza protein, e.g., the hemagglutinin (HA) protein of influenza. After incubation of the transfected cells for an appropriate time to allow for protein expression (such as for approximately 72 hours), influenza SVPs or VLPs can be isolated from cell culture supernatants. Influenza SVPs or VLPs can be purified from cell supernatants using procedures practiced in the art; for example, VLPs can isolated by low speed centrifugation (to remove cell debris), vacuum filtration and ultracentrifugation through 20% glycerol. [0147] In some embodiments, the antigen sequence of a broadly reactive and immunogenic influenza antigen as described herein, such as an influenza HA protein antigen, contains a diverse repertoire of epitopic determinants that can reflect antigenic drift and sequence variability in the virus’s antigenic proteins, for example, over time periods (e.g., seasons), or in different geographic locations. Moreover, an influenza hemagglutinin HA protein antigen as described herein can comprise an amino acid sequence that contains antigenic determinants (epitopes) derived from sequence diverse influenza subtypes and strains, including drift variants, against which broadly reactive neutralizing antibodies can be raised, especially when the antigen is used as an immunogenic product, (an immunogen), e.g., an antiviral vaccine, that is introduced into a subject.

[0148] Yet in further embodiments, the influenza antigen amino acid sequences provide a composite, immunogenic antigen sequence, which includes epitopic determinants ultimately derivable from both past and more recent influenza viruses causing infection or disease, and/or from viruses in different geographical locales, and/or from different subtypes of influenza, i.e., a“pan-epitopic” antigen that elicits a broadly reactive immune response when used as an immunogen, a vaccine, SVP, or VLP. In some embodiments, the immunogenic influenza hemagglutinin protein antigen sequences encompass epitopes that generate antibodies that are directed against the protein of more than one, or all, of the serotypes/subtypes of influenza. In some embodiments, the immunogenic influenza A virus Hl protein antigen sequences encompass epitopes that result from antigenic changes in the sequences of influenza A virus Hl protein surface antigens that arise from point mutations during viral replication, giving rise to new influenza A virus Hl protein variants. As a result, the administration to a subject of an influenza immunogen as described herein can elicit a broadly reactive immune response in the subject that is directed against epitopes reflecting such antigenic changes.

[0149] Because the broadly reactive influenza antigens and the sequences thereof as described herein and used as an immunogen or immunogenic composition, such as a vaccine, elicit a broadly reactive immune response in an immunocompetent subject, they provide a superior vaccine that captures the antigenic epitopes of many different influenza A virus Hl isolates (subtypes or strains), against which broadly active immune responses (e.g., broadly active neutralizing antibodies) are generated. It is noted that the terms“broadly active” and “broadly reactive” are used synonymously herein. [0150] In some embodiments, the influenza virus antigen as described herein is a polypeptide or peptide antigen of influenza virus that currently causes disease or infection and its symptoms, such as the flu in its various forms. In another embodiment, the influenza virus antigen is a polypeptide or peptide antigen that will, in future, cause disease and symptoms of influenza virus infection. In some embodiments, the influenza antigen is a polynucleotide sequence. In some embodiments, the influenza virus antigen is a polynucleotide sequence that encodes a polypeptide or peptide antigen as described herein. By way of example, representative broadly reactive influenza immunogen sequences are shown in FIGs. 1A-1B (SEQ ID NOs: l-5), FIG. 7 (SEQ ID NOs:6-9), and TABLE 1 (SEQ ID NOs: 10-27).

[0151] In another embodiment, the influenza immunogen sequence described herein is expressed in a cell as a polypeptide, protein, or peptide. In some embodiments, the influenza immunogen is isolated, purified, or both isolated and purified. In some embodiments, the immunogen is formulated for administration to a subject in need thereof. In some embodiments, the immunogen is administered to a subject in need thereof in an effective amount to elicit an immune response in the subject. In some embodiments, the immune response elicits neutralizing antibodies. In some embodiments, the immune response is prophylactic or therapeutic.

[0152] In some embodiments, a non-naturally occurring influenza immunogen (e.g., antigen or immunogen sequence), e.g., a vaccine, is provided that elicits a broadly reactive immune response in a subject following introduction, administration, or delivery of the immunogen to the subject. The route of introduction, administration, or delivery is not limited and may include, for example, intravenous, subcutaneous, intramuscular, oral, or other routes. The vaccine may be therapeutic (e.g., administered to a subject following a symptom of disease caused by influenza) or prophylactic (e.g., administered to a subject prior to the subject having or exhibiting a symptom of an infection, or full-blown infection, caused by influenza).

[0153] In some embodiments, the final amino acid sequence of the antigen, e.g., the

HA protein of influenza, is reverse translated and optimized for expression in mammalian cells. As will be appreciated by a skilled practitioner in the art, optimization of the nucleic acid sequence includes optimization of the codons for expression of a sequence in mammalian cells and RNA optimization (such as RNA stability).

[0154] In some embodiments, a polynucleotide or an isolated nucleic acid molecule

\comprising a nucleotide sequence encoding a polypeptide or peptide antigen (e.g., an influenza A virus antigen, an influenza A virus Hl polypeptide, an influenza A virus HA polypeptide) is provided. In certain embodiments, the nucleotide sequence encoding the influenza A virus polypeptide is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to a polynucleotide encoding an influenza A virus HA polypeptide sequence, or fragment or portion of an influenza A virus HA polypeptide sequence shown in FIGs. 1A-1B (SEQ ID NOs: l-5), FIG. 7 (SEQ ID NOs:6-9), and TABLE 1 (SEQ ID NOs: 10-27). A further embodiment may be directed to a polynucleotide comprising a nucleotide sequence encoding an influenza A virus HA polypeptide or at least 80% (e.g., 85%, 90%, 95%, 97%, 99%) identical to the influenza A virus HA polypeptide or fragment shown in FIGs. 1 A-1B (SEQ ID NOs: 1-5), FIG. 7 (SEQ ID NOs:6-9), and TABLE 1 (SEQ ID NOs: 10-27). Nucleic acid sequences encoding these polypeptides can be used to generate virus-like particles (VLPs) containing the influenza protein antigens, which are used as immunogens/vaccines to generate neutralizing antibodies in immunized subjects.

[0155] In other embodiments, the polynucleotide comprises a nucleotide sequence encoding the influenza polypeptide (e.g., influenza hemagglutinin (HA) polypeptide, influenza A virus HA antigen), where the nucleotide sequence is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to a polynucleotide encoding an HA polypeptide sequence shown in FIGs. 1A-1B (SEQ ID NOs: 1-5), FIG. 7 (SEQ ID NOs:6-9), and TABLE 1 (SEQ ID NOs: 10-27) that further lacks the start codon encoding an N-terminal methionine of the HA polypeptide sequences.

[0156] Further embodiments of the disclosure comprising vectors containing a nucleotide sequence encoding a non-naturally occurring, broadly reactive polypeptide or peptide antigen (e.g., influenza polypeptide, influenza A virus hemagglutinin Hl polypeptide) are provided. In some embodiments, the vectors comprise a nucleotide sequence encoding the polypeptide or peptide antigen, (e.g., influenza polypeptide, influenza A virus Hl polypeptide antigen) disclosed hereinwhere the nucleotide sequence is at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to a polynucleotide encoding an HA polypeptide sequence, or a fragment thereof shown in FIGs. 1A-1B (SEQ ID NOs: 1-5), FIG. 7 (SEQ ID NOs:6-9), and TABLE 1 (SEQ ID NOs: 10-27). Other embodiments may provide a vector comprising a nucleic acid sequence encoding the provided influenza hemagglutinin (HA) polypeptide, where the polypeptide comprises a sequence that is at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to at least one of the influenza A vims HA sequence, or fragment thereof, shown in FIGs. 1A-1B (SEQ ID NOs: l- 5), FIG. 7 (SEQ ID NOs:6-9), and TABLE 1 (SEQ ID NOs: 10-27). In some embodiments, the vector further includes a promoter operably linked to the nucleotide sequence encoding the HA polypeptide. In another embodiment, the promoter is a cytomegalovirus (CMV) promoter. In some embodiments, the nucleotide sequence of the vector is at least 80% (e.g., 80%, 90%, 95%, 98%, 99%) identical to a polynucleotide encoding an HA polypeptide sequence, or a fragment thereof shown in FIGs. 1A-1B (SEQ ID NOs: 1-5), FIG. 7 (SEQ ID NOs:6-9), and TABLE 1 (SEQ ID NOs: 10-27). Yet in further embodiments, the nucleotide sequence of the vector comprises the polynucleotide encoding an HA polypeptide sequence, or fragments thereof shown in FIGs. 1A-1B (SEQ ID NOs: 1-5), FIG. 7 (SEQ ID NOs:6-9), and TABLE 1 (SEQ ID NOs: 10-27). In some embodiments, the vector is a prokaryotic or eukaryotic vector. In some embodiments, the vector is an expression vector, such as a eukaryotic (e.g., mammalian) expression vector. In another embodiment, the vector is a plasmid (prokaryotic or bacterial) vector. In another embodiment, the vector is a viral vector.

[0157] The vectors used to express an influenza virus antigen, e.g., an influenza A virus

Hl protein, as described herein may be any suitable expression vectors known and used in the art. The vectors can be, for example, mammalian expression vectors or viral vectors. In some embodiments, the vector is the pTR600 expression vector (e.g., U.S. Patent Application Publication No. 2002/0106798, herein incorporated by reference; Ross et al., 2000, Nat Immunol. 1(2): 102-103; and Green et al., 2001, Vaccine 20:242-248).

[0158] Provided are influenza virus-derived, non-naturally occurring polypeptide antigens, e.g., HA polypeptide antigens, produced by transfecting a host cell with an expression vector as known and used in the art under conditions sufficient to allow for expression of the polypeptide, (e.g., an influenza virus polypeptide, influenza HA polypeptide, influenza A virus HA Hl polypeptide) in the cell. Isolated cells containing the vectors disclosed herein are also provided.

[0159] In some embodiments, fusion proteins comprising the broadly reactive, pan- epitopic influenza antigen polypeptides described herein, e.g., without limitation, the influenza hemagglutinin polypeptides disclosed herein, are also provided. In some embodiments, the influenza hemagglutinin polypeptide can be fused to any heterologous amino acid sequence to form the fusion protein. By way of example, peptide components of influenza polypeptides may be generated independently and then fused together to produce an intact influenza polypeptide antigen, (e.g., comprising 565 or 566 amino acids or any shorter amino acid sequence that induces an immune response), for use as an immunogen. Other embodiments of the disclosure may provide an influenza hemagglutinin polypeptide comprising a fusion of the Cb, Sa, and Sb antigenic sites from the same or various influenza viruses or synthetic HA antigens as presented in FIGs. 1A-1B (SEQ ID NOs: l-5), FIG. 7 (SEQ ID NOs:6-9), and TABLE 1 (SEQ ID NOs: 10-22). For example, the Cb (SEQ ID NO: 12), Sa (SEQ ID NOs: l8 and 21), and Sb (SEQ ID NO:26) sites of V6 may be fused together to produce an influenza polypeptide antigen. Another embodiment may be directed to an influenza polypeptide antigen comprising the antigenic sites from various HA antigens. For example, combining portions from Pl (Sa (aa 170-181): SEQ ID NO:2l; Sb: SEQ ID NO: 26) and X6 (Sa (aa 141-147): SEQ ID ON: 13) with the Cb site of Pl (SEQ ID NO: 12) and of X6 (SEQ ID NO: 10) form V2 and V3 fusion proteins, respectively, comprising the Cb, Sa, and Sb antigenic sites.

[0160] Also provided are subviral particles (SVPs) or virus-like particles (VLPs), for example, influenza SVPs or VLPs, containing a pan-epitopic, broadly reactive protein antigen, e.g., influenza hemagglutinin (HA) protein, as described herein. In certain embodiments, the HA protein of the VLP is at least or equal to 90% (e.g., 94%, 95%, 96%, 97%, 98%, 99% 100%) identical to the influenza HA proteins shown in FIGs. 1 A-1B (SEQ ID NOs: 1-5), FIG. 7 (SEQ ID NOs:6-9), and TABLE 1 (SEQ ID NOs: 10-27). The influenza SVPs or VLPs can further include any additional viral proteins necessary to form the virus particle. In certain embodiments, the virus or influenza VLPs or SVPs further include another influenza virus protein.

[0161] Also provided is an influenza SVP or VLP containing an influenza polypeptide, e.g., a hemagglutinin polypeptide, as described herein, produced by transfecting a host cell with a vector containing a polynucleotide encoding the influenza polypeptide. Also provided in some embodiments is an influenza SVP or VLP containing an influenza polypeptide, or influenza HA polypeptide, as described herein, produced by transfecting a host cell with a vector encoding the influenza polypeptide, e.g., hemagglutinin Hl, under conditions sufficient to allow for expression of the influenza protein. Such SVPs or VLPs comprising the sequences presented in FIGs. 1A-1B (SEQ ID NOs: 1-5), FIG. 7 (SEQ ID NOs:6-9), and TABLE 1 (SEQ ID NOs: 10-27) and used as immunogens generate antibodies having high neutralization titers against subtypes of influenza and strains thereof, as illustrated in, e.g., FIGs. 8, 10, 11, 13, and 14. [0162] Collections of plasmids (vectors) are also contemplated. In certain embodiments, the collection of plasmids includes plasmid encoding a broadly reactive influenza hemagglutinin protein as described herein, as well as plasmids encoding other influenza proteins, such as a structural protein other than an envelope protein, or another envelope protein. In some embodiments, the nucleotide sequence encoding a hemagglutinin protein of the influenza hemagglutinin-encoding plasmid is at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a polynucleotide encoding an influenza hemagglutinin polypeptide amino acid sequence shown in, for example, FIGs. 1A- 1B, FIG. 7, and TABLE 1. In some embodiments, the nucleotide sequence encoding a codon- optimized influenza protein of the influenza hemagglutinin-encoding plasmid is at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a polynucleotide encoding an influenza hemagglutinin polypeptide amino acid sequence shown in FIGs. 1A-1B, FIG. 7, and TABLE 1.

[0163] In the context of the present disclosure,“broadly reactive” or“broadly active” means that the hemagglutinin protein (e.g., an influenza hemagglutinin protein sequence) is immunogenic and contains a diversity of epitopes (antigenic determinants; pan-epitopic) that elicit in a subject an immune response (e.g., neutralizing antibodies directed against the diversity of influenza protein, e.g., hemagglutinin protein or epitopes, frequently accompanied by a T-cell response) sufficient to treat influenza disease or infection, and/or to inhibit, neutralize, or prevent infection, caused by most or all influenza viruses within a specific subtype, or by other influenza subtypes. In embodiments of the disclosure, the broadly reactive, influenza-derived antigen protein, e.g., the influenza hemagglutinin protein, may elicit a protective immune response against most, all known, or future influenza virus subtypes, such as at least 80% (e.g., 85%, 90%, 95%, or 96%, 99%) of the known or future influenza virus subtypes or isolates thereof.

Compositions and Pharmaceutical Compositions for Administration

[0164] The influenza antigens, immunogens, SVPs, or VLPs can be used as immunogenic compositions (e.g., vaccines, medicinal products) to elicit an immune response against influenza viruses, subtypes and strains thereof. For example, the non-naturally occurring, broadly reactive, pan-epitopic influenza polypeptides of the immunogenic compositions (e.g., vaccines, medicinal products) (or SVPs or VLPs) contain antigenic (pan- epitopic) determinants that are broadly reactive and serve to elicit an immune response in a subject (e.g., the production of neutralizing antibodies and/or activated T-cells, T-lymphocytes) that can treat an influenza virus-infected subject (e.g., neutralize the infecting virus) and/or protect a subject against full-blown virus infection or the signs and symptoms thereof. In some embodiments, such immunogenic compositions (e.g., vaccines, medicinal products) as described herein are effective in treating a secondary infection by an influenza subtype that is different from the influenza subtype that caused the primary infection. In some embodiments, such immunogenic compositions as described herein provide highly useful products (e.g., immunogenic compositions, vaccines) that protect against serotypes of the virus to avoid antibody dependent enhancement (ADE).

[0165] Compositions comprising a broadly reactive, pan-epitopic influenza virus protein, (e.g., HA protein), or a fusion protein, antigen, immunogen, SVP, or VLP comprising such a broadly reactive influenza protein as described herein are provided. Other embodiments may provide an immunogen comprising DNA encoding an influenza virus protein, fragment thereof, or fusion protein described here, that elicits a neutralizing immune response against one or more influenza viruses (e.g., two, three, four, five, six). Further embodiments provide a composition comprising an influenza virus protein, (e.g., HA protein), or a fusion protein, antigen, immunogen, SVP, VLP, or component that elicits a neutralizing immune response against one or more influenza viruses as described herein, where the composition provides protection against one or more seasonal or pandemic influenza viruses, wherein the component that elicits an anti-influenza virus immune response may be selected from any of the sequences in FIGs. 1A-1B (SEQ ID NOs: l-5), FIG. 7 (SEQ ID NOs:6-9), TABLE 1 (SEQ ID NOs: lO- 27), and sequences of FIGs. 27A-27B (SEQ ID NOs:35-38, where the protection is in humans, swine, avian, equine, etc.

[0166] In some embodiments, the compositions (e.g., pharmaceutical, immunogenic) further comprise a pharmaceutically acceptable carrier (e.g., excipient, vehicle). In some embodiments, an adjuvant (i.e., a pharmacological or immunological agent that modifies or boosts an immune response, e.g. to produce more antibodies that are longer-lasting) may also be employed with the composition (e.g., pharmaceutical, immunogenic). For example, without limitation, the adjuvant can be an inorganic compound, such as alum, aluminum hydroxide, or aluminum phosphate; mineral or paraffin oil; squalene; detergents such as Quil A; plant saponins; Freund's complete or incomplete adjuvant, a biological adjuvant (e.g., cytokines such as IL-l, IL-2, or IL-12); bacterial products such as killed Bordetella pertussis , or toxoids; or immunostimulatory oligonucleotides (e.g., CpG oligonucleotides).

[0167] Compositions and preparations (e.g., physiologically or pharmaceutically acceptable compositions) containing the non-naturally occurring, broadly reactive, pan- epitopic influenza polypeptides (e.g., influenza hemagglutinin polypeptides, influenza A virus polypeptides) and influenza subviral particles (SVPs) or virus-like particles (VLPs) for parenteral administration include, without limitation, sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Non-limiting examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and canola 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, for example, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include, for example, fluid and nutrient replenishers, electrolyte replenishers (e.g., those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present in such compositions and preparations, such as, for example, antimicrobials, antioxidants, chelating agents, colorants, stabilizers, inert gases and the like.

[0168] 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-, tri-alkyl and aryl amines and substituted ethanolamines.

[0169] Provided herein are pharmaceutical compositions which include a therapeutically effective amount of a non-naturally occurring, broadly reactive, pan-epitopic, influenza A virus Hl protein antigen, immunogen, influenza virus SVPs or VLPs, alone, or in combination with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. In certain embodiments, carrier proteins may be utilized, such as but not limited to, Keyhole Limpet Hemocyanin (KHL) or Bovine Serum Albumin (BSA). The carrier and composition can be sterile, and the formulation suits the mode of administration as understood by a person of ordinary skill in the art. The compositions disclosed herein may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition of the disclosure comprising an influenza A virus antigen, immunogen, SVP, or VLP as described herein can be formulated as a liquid or aqueous solution, suspension, emulsion, dispersion, tablet, pill, capsule, powder, or sustained release formulation. A liquid or aqueous composition can be lyophilized and reconstituted with a solution or buffer prior to use. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can also include standard carriers (e.g., pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate, sterile saline solution, sesame oil, mineral oil) can be used. The medium can also contain conventional pharmaceutical adjunct materials such as, for example, pharmaceutically acceptable salts to adjust the osmotic pressure, buffers, preservatives, and the like. Other media that can be used in the compositions and administration methods as described are normal saline and oil.

Methods of Treatment, Administration, and Delivery

[0170] Methods of treating a disease or infection, or symptoms thereof, caused by influenza virus are provided. The methods comprise administering a therapeutically effective amount of a broadly reactive, pan-epitopic antigen, immunogen, SVP, VLP, or immunogenic composition (e.g., vaccine, medicinal product) as described herein, or a pharmaceutical composition comprising the antigen, immunogen, SVP, VLP, or a vaccine (e.g., an influenza virus SVP vaccine, an influenza virus VLP vaccine) as described herein to a subject (e.g., a mammal), such as for example, a human subject. One embodiment involves a method of treating a subject suffering from, or at risk of or susceptible to disease or infection, or a symptom thereof, caused by influenza virus. The method includes administering to the subject (e.g., a mammalian subject), an amount or a therapeutic amount of an immunogenic composition or a vaccine comprising a non-naturally occurring, broadly reactive, pan-epitopic, influenza virus antigen polypeptide, such as influenza hemagglutinin (HA) polypeptide, or influenza polypeptide SVPs or VLPs, sufficient to treat the disease, infection, or symptoms thereof, caused by influenza virus under conditions in which the disease, infection, and/or the symptoms thereof are treated. [0171] Other embodiments provide for immunogenic compositions, including vaccines, comprising an immunogenic protein or recombinant protein, or fragments thereof, and in some instances, with an adjuvant, where the immunogenic portion is selected from VI, V2, V3, V4, V5, V6, V7, V8, V9, V10, VI 1, V12, SW1, SW2, SW3, SW4, SEQ ID NOs: l- 38, or fragments thereof.

[0172] In some embodiments, the methods herein include administering to the subject

(including a human subject identified as in need of such treatment) an effective amount of a non-naturally occurring, broadly reactive, pan-epitopic, influenza virus antigen polypeptide, such as influenza HA polypeptide, or a vaccine, or a composition as described herein to produce such effect. The treatment methods are suitably administered to subjects, including humans, suffering from, having, susceptible to, or at risk of having an influenza disease, disorder, infection, or symptom thereof, namely, the flu, characterized by at least one of the characteristics of: high fever, runny nose, sore throat, muscle pain, headache, cough, and fatigue. Identifying a subject in need of such treatment can be based on the judgment of the subject or of a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method). Briefly, the determination of those subjects who are in need of treatment or who are“at risk” or“susceptible” can be made by any objective or subjective determination by a diagnostic test (e.g., genetic test, enzyme or protein marker assay), marker analysis, family history, and the like, including an opinion of the subject or a health care provider. The non-naturally occurring, broadly reactive, pan-epitopic influenza immunogens, such as influenza hemagglutinin polypeptide immunogens and vaccines as described herein, may also be used in the treatment of any other disorders in which infection or disease caused by influenza virus may be implicated. A subject undergoing treatment can be a non-human mammal, such as a veterinary subject, or a human subject (also referred to as a“patient”).

[0173] Another embodiment may be directed to a method of generating an immune response in a subject (e.g., mammalian, human), comprising administering to the subject, an effective amount of the influenza A virus antigen, immunogen, influenza VLP, influenza SVP, a pharmaceutical or immunogenic composition comprising the influenza A virus antigen, immunogen, influenza VLP, influenza SVP described herein. In yet another embodiment, the method of generating an immune response comprises the production of neutralizing antibodies, T-cells, T-lymphocytes In other embodiments, the method of generating an immune response in a subject may further comprise concomitantly administering an adjuvant (e.g., immune response enhancer, immune response stimulator, inducer of infection protection, Freund’s complete adjuvant, Freund’s incomplete adjuvant, Montanide™ ISA 720 and 51 (SEPPIC Inc., New Jersey), MF59 etc.) to the subject.

[0174] In addition, prophylactic methods of preventing or protecting against a disease or infection, or symptoms thereof, caused by influenza virus are provided. Such methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising an influenza immunogenic composition or vaccine (e.g., an influenza SVP or VLP vaccine) as described herein to a subject (e.g., a mammal such as a human), for example, prior to infection of the subject or prior to onset of the disease, such as an influenza-associated disease. Other embodiments may be directed to a method of vaccinating a subject against influenza A virus, comprising administering a vaccine comprising an influenza virus component (e.g., protein or fragment thereof, antigen, immunogen, VLP, SVP) that is cross- reactive with or cross-protective against at least one (e.g., two, three, four, five, six) influenza viruses or subtypes of influenza (seasonal or pandemic), where the influenza virus component may be an influenza A virus component selected from: SEQ ID NOs: 1-27, or fragment thereof, to a subject in need thereof, where administration may be by: intranasal, intramuscular, subcutaneous, transdermal, or sublingual administration. Further embodiments provide the use of the antigen, immunogen, VLP, SVP, vaccine, nucleic acid molecule, vector, composition, or component described herein that elicits a neutralizing immune response against one or more influenza viruses, to introduce an immune response against influenza in a subject. Yet another embodiment may be directed to a kit comprising an antigen, immunogen, VLP, SVP, vaccine, nucleic acid molecule, vector, composition, or component described herein that elicits a neutralizing immune response against one or more influenza viruses, such as but not limited to influenza A virus, any elements that assist in the administration of these components, and instructions for using the kit or any of its elements.

[0175] In another embodiment, a method of monitoring the progress of an influenza virus infection or disease caused by influenza virus, or monitoring treatment of the influenza infection or disease is provided. The method includes determining a level of a diagnostic marker or biomarker (e.g., an influenza virus protein, such as influenza hemagglutinin protein), or a diagnostic measurement (e.g., screening assay or detection assay) in a subject suffering from or susceptible to infection, disease or symptoms thereof associated with influenza virus, in which the subject has been administered an amount (e.g., a therapeutic amount) of a non- naturally occurring, broadly reactive, pan-epitopic influenza virus protein, e.g., influenza hemagglutinin protein, as described herein, or an immunogenic composition or vaccine as described herein, sufficient to treat the infection, disease, or symptoms thereof. The level or amount of the marker or biomarker (e.g., protein) determined in the method can be compared to known levels of the marker or biomarker in samples from healthy, normal controls; in a pre- infection or pre-disease sample of the subject; or in other afflicted/infected/diseased patients to establish the treated subject’s disease status. For monitoring, a second level or amount of the marker or biomarker in in a sample obtained from the subject is determined at a time point later than the determination of the first level or amount, and the two marker or biomarker levels or amounts can be compared to monitor the course of disease or infection, or the efficacy of the therapy/treatment. In certain embodiments, a pre-treatment level of the marker or biomarker in the subject (e.g., in a sample obtained from the subject) is determined prior to beginning treatment as described; this pre-treatment level of marker or biomarker can then be compared to the level of the marker or biomarker in the subject after the treatment commences and/or during the course of treatment to determine the efficacy of (monitor the efficacy of) the disease treatment.

[0176] The non-naturally occurring, broadly reactive, pan-epitopic, influenza virus antigen polypeptide, such as influenza hemagglutinin polypeptide as described, and VLPs comprising influenza polypeptides, or compositions thereof, can be administered to a subject by any of the routes normally used for introducing a recombinant protein, composition containing the recombinant protein, or recombinant virus into a subject. Routes and methods of administration include, without limitation, intradermal, intramuscular, intraperitoneal, intrathecal, parenteral, such as intravenous (IV) or subcutaneous (SC), vaginal, rectal, intranasal, inhalation, intraocular, intracranial, or oral. Parenteral administration, such as subcutaneous, intravenous or intramuscular administration, is generally achieved by injection (immunization). Injectables can be prepared in conventional forms and formulations, either as liquid solutions or suspensions, solid forms (e.g., lyophilized forms) suitable for solution or suspension in liquid prior to injection, or as emulsions. Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets. Administration can be systemic or local. [0177] The non-naturally occurring, broadly reactive, pan-epitopic, influenza virus polypeptides, such as influenza virus hemagglutinin polypeptides as described, and SVPs or VLPs comprising influenza polypeptides, or compositions thereof, can be administered in any suitable manner, such as with pharmaceutically acceptable carriers as described supra. Pharmaceutically acceptable carriers are determined in part by the particular immunogen or composition being administered, as well as by the particular method used to administer the composition. Accordingly, a pharmaceutical composition comprising the non-naturally occurring, broadly reactive, pan-epitopic, influenza virus antigen polypeptides, such as influenza hemagglutinin polypeptides, and VLPs comprising influenza polypeptides, or compositions thereof, can be prepared using a wide variety of suitable and physiologically and pharmaceutically acceptable formulations.

[0178] Administration of the broadly reactive, pan-epitopic, influenza virus antigen polypeptides, such as influenza hemagglutinin polypeptides, and VLPs comprising such influenza polypeptides, or compositions thereof, can be accomplished by single or multiple doses. The dose administered to a subject should be sufficient to induce a beneficial therapeutic response in a subject over time, such as to inhibit, block, reduce, ameliorate, protect against, or prevent disease or infection by influenza virus. The dose required will vary from subject to subject depending on the species, age, weight, and general condition of the subject, by the severity of the infection being treated, by the particular composition being used and by the mode of administration. An appropriate dose can be determined by a person skilled in the art, such as a clinician or medical practitioner, using only routine experimentation.

[0179] Further provided is a method of eliciting an immune response to influenza virus in a subject by administering to the subject a non-naturally occurring, broadly reactive, pan- epitopic, influenza protein antigen, e.g., an influenza hemagglutinin protein antigen as disclosed herein, fusion proteins containing the influenza protein, SVPs or VLPs containing the influenza protein, or compositions thereof as described herein. In some embodiments, the influenza A virus Hl protein, influenza fusion protein, SVP, or VLP can be administered using any suitable route of administration, such as, for example, by intramuscular injection. In some embodiments, the influenza A virus Hl protein, fusion protein, SVP, or VLP is administered as a composition comprising a pharmaceutically acceptable carrier. In some embodiments, the composition comprises an adjuvant selected from, for example, alum, Freund's complete or incomplete adjuvant, a biological adjuvant or immunostimulatory oligonucleotides (such as CpG oligonucleotides). In other embodiments, the composition may be administered in combination with another therapeutic agent or molecule.

[0180] Also provided is a method of immunizing a subject against infection or disease or the symptoms thereof caused by Hl influenza virus subtypes, in which the method involves administering to the subject SVPs or VLPs containing a non-naturally occurring, pan-epitopic, broadly reactive influenza A virus Hl protein as described herein, or administering an immunogenic composition thereof. In some embodiments of the method, the composition further comprises a pharmaceutically acceptable carrier and/or an adjuvant. For example, the adjuvant can be alum, Freund's complete or incomplete adjuvant, a biological adjuvant or immunostimulatory oligonucleotides (such as CpG oligonucleotides). In some embodiments, the influenza virus VLPs or SVPs (or compositions thereof) are administered intramuscularly.

[0181] In some embodiments of the methods of eliciting an immune response or immunizing a subject against virus infection or disease caused by or associated with influenza A virus, the subject is administered at least 1 pg of the SVPs or VLPs containing a non-naturally occurring, broadly reactive, pan-epitopic influenza A virus Hl protein. In some embodiments of the methods of eliciting an immune response or immunizing a subject against virus infection or disease caused by or associated with influenza A virus, at least 5 pg (e.g., 10 pg, 15 pg, 20 pg, 25 pg, 30 pg, 40 pg, 50 pg) of the SVPs or the VLPs containing the non-naturally occurring, broadly reactive, pan-epitopic influenza A virus Hl protein are administered. For example, in certain embodiments, 1 to 50 pg (e.g., 1 to 25 pg, 5 pg to 20 pg, 10 pg to 15 pg, 15 pg) of the SVPs or the VLPs containing the influenza virus protein may be administered. However, one of skill in the art is capable of determining therapeutically effective amounts of SVPs or VLPs (for example, an amount that provides a therapeutic effect or protection against influenza virus infection) suitable for administering to a subject in need of treatment or protection from influenza virus infection.

[0182] It is expected that the administration of SVPs or VLPs comprising a non- naturally occurring, broadly reactive, pan-epitopic influenza virus protein, (e.g., influenza HA protein), or an immunogen or immunogenic composition, as described herein will elicit high titers of neutralizing antibodies directed against the diverse repertoire of epitopic determinants on the influenza virus protein immunogen, as well as protective levels of influenza virus protein-inhibiting antibodies that are directed against a number of representative influenza subtypes and strains thereof, and will provide complete protection against lethal challenge with influenza vims and/or related influenza vims subtypes and strains thereof. The SVPs or VLPs containing a non-naturally occurring, broadly reactive, pan-epitopic influenza vims protein, (e.g., influenza HA), as described herein elicit a broad immune response (e.g., elicit neutralizing antibodies directed against a broad range of influenza subtypes, strains, isolates) compared with the immune response elicited by a wild-type (e.g., CA/09, Bris07) influenza vims vaccine. (FIG. 11).

[0183] An advantage of the immunogens and immunogenic compositions comprising non-naturally occurring, broadly reactive, pan-epitopic influenza vims antigens (e.g., hemagglutinin antigen) described herein is that a broadly reactive immune response is elicited against not only the influenza vims serotype from which the antigen was derived, but also against one or more, or all, other influenza vims serotypes or strains, e.g., H1N1, H1N2, H1N3, H1N4, H1N5, H1N6, H1N7, H1N8, H1N9, H1N10, H1N11, or a combination thereof. In some embodiments, the immunogens and immunogenic compositions described herein elicit immune responses against H1N1 and H1N2. Thus, the influenza vims immunogens are more cost effective to produce, and beneficially elicit a broadly reactive immune response, thus, obviating a need to make and administer a poly- or multivalent immunogenic composition or vaccine.

Adjuvants and Combination Therapies

[0184] The influenza vims immunogens or immunogenic compositions containing an influenza vims protein antigen, (e.g., influenza HA protein antigen), or containing influenza vims SVPs or VLPs as described herein, can be administered alone or in combination with other therapeutic agents to enhance antigenicity or immunogenicity, i.e., to increase an immune response, such as the elicitation of specific antibodies, in a subject. For example, the influenza vims SVPs or VLPs can be administered with an adjuvant, such as alum, Freund’s incomplete adjuvant, Freund's complete adjuvant, biological adjuvant, or immunostimulatory oligonucleotides (such as CpG oligonucleotides).

[0185] One or more cytokines, such as interleukin- 1 (IL-l), interleukin-6 (IL-6), interleukin- 12 (IL-12), the protein memory T-cell attractant“Regulated on Activation, Normal T Expressed and Secreted” (RANTES), granulocyte-macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-a), or interferon-gamma (IFN-g); one or more growth factors, such as GM-CSF or granulocyte-colony stimulation factor (G-CSF); one or more molecules such as the TNF ligand superfamily member 4 ligand (OX40L) or the type 2 transmembrane glycoprotein receptor belonging to the TNF superfamily (4-1BBL), or combinations of these molecules, can be used as biological adjuvants, if desired or warranted (see, e.g. , Salgaller et al., 1998, ./. Surg. Oncol. 68(2): 122-38; Lotze et al., 2000, Cancer J Sci. Am. 6(Suppl 1): S61-6; Cao et al., 1998, Stem Cells l6(Suppl l):251-60; Kuiper et al., 2000, Adv. Exp. Med. Biol. 465:381-90). These molecules can be administered systemically (or locally) to a subject.

[0186] Several ways of inducing cellular responses, both in vitro and in vivo , are known and practiced in the art. Lipids have been identified as agents capable of assisting in priming cytotoxic lymphocytes (CTL) in vivo against various antigens. For example, palmitic acid residues can be attached to the alpha and epsilon amino groups of a lysine residue and then linked (for example, via one or more linking residues, such as glycine, glycine-glycine, serine, serine-serine, or the like) to an immunogenic peptide (U.S. Patent No. 5,662,907). The lipidated peptide can then be injected directly in a micellar form, incorporated in a liposome, or emulsified in an adjuvant. As another example, E. coli lipoproteins, such as tripalmitoyl- S-glycerylcysteinlyseryl-serine can be used to prime tumor-specific CTL when covalently attached to an appropriate peptide (see, e.g., Deres et al., 1989, Nature 342:561). Moreover, the induction of neutralizing antibodies can also be primed with the same molecule conjugated to a peptide which displays an appropriate epitope, and two compositions can be combined to elicit both humoral and cell-mediated responses where such a combination is deemed desirable.

[0187] While treatment methods may involve the administration of SVPs or VLPs containing a non-naturally occurring, broadly reactive, pan-epitopic influenza virus immunogenic protein, (e.g., influenza HA protein), as described herein, one skilled in the art will appreciate that the non-naturally occurring, broadly reactive, pan-epitopic influenza virus protein itself (in the absence of a viral particle), as a component of a pharmaceutically acceptable composition, or as a fusion protein, can be administered to a subject in need thereof to elicit an immune response against influenza virus in the subject.

Kits

[0188] Also provided are kits containing a non-naturally occurring, broadly reactive, pan-epitopic influenza virus immunogen, polypeptide, nucleotide, SVP, VLP as described herein, or an immunogenic composition (e.g., a vaccine, medicinal product) or a pharmaceutically acceptable composition containing the influenza virus immunogen, polypeptide, nucleotide, SVP, VLP as described here, or composition containing any components thereof, and a pharmaceutically acceptable carrier, diluent, or excipient, for administering to a subject, for example. The immunogen may be in the form of an influenza A virus Hl protein (e.g., polypeptide, peptide, VLP, SVP) or a polynucleotide encoding an influenza A virus Hl polypeptide or influenza A virus Hl protein or fragment thereof, as described herein. Another embodiment may be directed to the kits containing one or more of the plasmids, or a collection of plasmids as described herein, are also provided. As will be appreciated by the skilled practitioner in the art, such a kit may contain one or more containers that house the immunogen, vaccine, or composition, diluents or excipients, adjuvants as necessary, and instructions for use.

[0189] The practice of embodiments of the disclosure employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984);“Animal Cell Culture” (Freshney, 1987);“Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987);“Current Protocols in Molecular Biology” (Ausubel, 1987);“PCR: The Polymerase Chain Reaction”, (Mullis. 1994);“Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Useful techniques for particular embodiments will be discussed in the sections that follow.

EXAMPLES

[0190] The following examples illustrate specific aspects of the instant description.

The examples should not be construed as limiting, as the example merely provides specific understanding and practice of the embodiments and its various aspects.

EXAMPLE 1 : ANTIGEN CONSTRUCTION AND SYNTHESIS

[0191] Influenza A hemagglutinin (HA) nucleotide sequences isolated from human

H1N1 infections were downloaded from the NCBI Influenza Virus Resource database. Nucleotide sequences were translated into protein sequences using the standard genetic code. Full-length sequences from H1N1 viral infections isolated from human sources between 1977 and 2007 were used to generate a first consensus sequence and a second consensus sequence was derived from H1N1 HA sequences from viruses isolated between 2009 and 2016. The consensus sequences were generated using AlignX (Vector NTI). The final amino acid sequences were compared to wild-type HA amino acid sequences (FIG. 3). Two sites, the Sa and Cb regions of the HA globular head were selected for mutational analysis. In seasonal Hl HA sequences (from 1977 to 2007), there is a lysine (Lys or K) at amino acid residue 90 in the Cb region. At this same position in pandemic Hl strains (from 2009 to 2016), as well as the NJ/1976 and SC/1918 swine-like strains), alanine (Ala or A) or valine (Val or V) are present. In the Sa site, there is a Lys (K) at amino acid residue 147 in pandemic Hl strains that is missing in seasonal Hl strains. In addition, there is a putative glycosylation motif at amino acid residues 142 to 144 in seasonal Hl strains that is not present in pandemic Hl strains. Therefore, HA constructs were optimized for expression in mammalian cells, including codon usage and RNA optimization (Genewiz, Washington, DC, USA). Three H1N1 HA constructs were synthesized and inserted into the pTR600 expression vector, as previously described (Ross et al, 2001, Nat Immunol). The V4 HA amino acid sequence was designed using the seasonal consensus sequence with the addition of a Lys (K) at amino acid residue 147. The V5 HA amino acid sequence was designed using the seasonal consensus sequence with a shift of the putative glycosylation motif from residues 142 to 144 to residues 145 to 147. The V6 HA amino acid sequence was designed using the pandemic consensus sequence with the addition of a threonine (Thr or T) at residue 142, thereby deleting the putative glycosylation motif.

EXAMPLE 2: CELLS AND VIRUSES

[0192] The cells and viruses of this disclosure were prepared and used in accordance with the descriptions here. HEK-293T cells and Madin-Darby Canine Kidney (MDCK) cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (10,000 U/ml). Influenza A H1N1 viruses were grown in eggs and purchased from VIRAPUR® (New York): A/Chile/l/l983, A/Singapore/6/l986, A/Texas/36/ 1991, A/Beijing/262/l995, A/New

Caledonia/20/l999, A/Solomon Islands/3/2006, A/Brisbane/59/2007, A/California/07/2009, A/Michigan/45/2015, or H1N1 influenza A viruses were obtained through the Influenza Reagent Resource, Influenza Division, WHO Collaborating Center for Surveillance, Epidemiology and Control of Influenza, Centers for Disease Control and Prevention, Atlanta, GA, USA..

EXAMPLE 3 : DESIGN AND CHARACTERIZATION OF MODIFIED COBRA Pl HA ANTIGEN

[0193] Epitopes in the Cb, Sa, or Sb antigenic sites of seasonal-like and pandemic-like wild-type or COBRA HA antigens were exchanged with homologous regions in the COBRA HA proteins to determine which regions and residues were responsible for the elicited antibody profile. Next-generation hemagglutinin (HA) head-based vaccines have been developed that elicit protective antibodies against H1N1 influenza viruses. An understanding of the specific amino acids around the receptor binding site (RBS) that were important in elicitation of these broadly reactive antibodies was studied. Specific glycan sites and amino acids located at the tip of the HA molecule enhanced the elicitation of these broadly reactive antibodies. While the effect of glycosylation on in vitro antigenicity and viral replication has been demonstrated, this study looks at the role of glycosylation on in vivo immunogenicity and the effect on glycosylation on the elicitation of broadly-reactive antibodies against H1N1 strains. A better understanding of the HA structures around the RBS was researched to find more effective HA immunogens.

[0194] HA antigens that elicited broadly protective antibodies against H1N1 influenza viruses were designed using a methodology known as Computationally Optimized Broadly Reactive Antigens (COBRA) (Carter DM et al. J Virol 90:4720-4734, 2016). Epitopes in the Cb and Sa sites were exchanged in the Pl and X6 COBRA HA proteins to determine which regions were responsible for the elicited antibody profile against a panel of H1N1 viruses. Two of these HA antigens, Pl and X6, elicited antibodies with HAI activity against a broad number of H1N1 influenza viruses isolated over the past 30 or more years (FIG. 8). Vaccines based upon wild-type HA antigens from NC/99, Bris/07, or CA/09 viruses elicited antibodies with a narrow range of HAI activity; recognizing 1-2 of the 15 viruses in the panel (FIG. 8). Specifically focusing on the differences in antibody breadth between Pl and X6, Pl elicited antibodies with HAI activity against pandemic H1N1 viruses (post 2009), but no HAI activity against the 2006 or 2007 seasonal H1N1 viruses. In contrast, X6 elicited antibodies with HAI activity against the 2006 and 2007 seasonal H1N1 viruses, but not the 2009 pandemic or post pandemic H1N1 strains (FIG. 8). The Pl HA was designed to cover the antigenic space from 1933-1957, 2009-2011 and swine sequences from 1931-1998. The COBRA X6 sequence (antigenic space from 1999-2012) was used to determine potentially important epitopes that could lead to the difference in antibody breadth and virus recognition or neutralization.

[0195] The COBRA X6 HA elicited antibodies with HAI activity that increased the number of seasonal H1N1 strains recognized as compared to antibodies elicited following Bris/07 virus vaccination or infection. However, these COBRA X6 HA induced antibodies that do not bind well or have HAI activity against Bris/07. The Pl COBRA HA elicited antibodies with HAI activity against pandemic-like H1N1 viruses, as well as some seasonal H1N1 strains, but not against Bris/07. Therefore, the amino acids in the Sa, Sb, and Cb regions of various seasonal and pandemic influenza virus (e.g., CA/09, Bris/07, COBRA Pl, COBRA X6) HA antigens were the basis to identify residues that differed among these four HA antigens as part of potential epitopes associated with antibody binding, neutralization, and protection against H1N1 influenza viruses.

[0196] Initial review identified 53 amino acids that that differed at 17 locations in these regions, with two being associated with putative glycosylation sites (FIG. 20). Since the introduction of H1N1 influenza viruses into the human population in 1918, the HA of H1N1 influenza viruses have acquired multiple N-linked glycosylation sites. The sequon, N-X-S/T (X being any amino acid except Pro), is associated with glycosylation of accessible Asn (N) residues (Kim JI, Park MS. Yonsei MedJ. 53 :886-893, 2012; Wei et al. Sci TranslMed. 2:24- 21, 2010). Glycoslation of the Hl HA globular head is associated with five dominant sites in various combinations (Asn at amino acids 142, 144, 172, 177, and 179 of HA) (Das et al. PLoS Pathog. 6:el00l2l l, 2010). From 1942 to 1985, H1N1 influenza viruses maintained several glycosylation combinations on HA at residues 144, 172, 177/179. In 1986, the glycan shifted from residue 144 to residue 142 with the primary glycosylated residues on HA located at 142 and 177. Introduction of the pandemic-like H1N1 viruses in 2009 brought back naked, or glycan-free, globular head HA antigens (Kim JI, Park MS. Yonsei Med J. 53 :886-893, 2012; Wei et al. Sci TranslMed. 2:24-21, 2010; Hong et al. J Virol. 87: 12471-12480, 2013). These glycans on HA not only shielded critical neutralizing epitopes, but they played a role in viral replication, virulence, and transmissibility (Zhang et al. PLoS One. 8:e6l397, 2013; Zost et al. PNAS. 114: 12578-12583, 2017; Pentiah et al. Glycobiology . 25: 124-132, 2015; Tate et al. Viruses. 6: 1294-1316, 2014; Laursen NS and Wilson IA Antiviral Res . 98:476-483, 2013; Sun et al. J Virol. 87:8756-8766, 2013; Medina et al. Sci Transl Med. 5: l87ral70, 2013). [0197] Mutations were introduced in these two COBRA HA antigens (Pl and X6) to exchange amino acids or antigenic sites between COBRA HA sequences, as well as between wild-type H1N1 HA proteins to better understand the epitopes involved in HAI specificity. This technique is referred to as Vaccines Intelligently Produced by Epitope Recombination (VIPER). VIPER technology incorporates specific changes into influenza HA sequences to induce increases in the elicited antibody breadth and effectiveness of these HA vaccines. Sequence analysis determined the potential epitopes of importance among various HA antigens, including, for example, COBRA Pl, COBRA X6, CA/09 (pandemic H1N1), and Bris/07 HA (seasonal) antigens (TABLE 1), as well as others. TABLE 1 shows portions or fragments of the Cb, Sa, and Sb antigenic sites. Residues that are underlined are associated with Pl HA vaccine, in bold type are associated with the X6 HA antigen (TABLE 1). The Sa and Cb antigenic sites were identified as different in Pl and CA/09, but similar in X-6 and Bris/07 HA proteins. The Sa residues 141-147 in Pl (PNHNTTK; SEQ ID NO: 15) and CA/09 (PNHDSNK; SEQ ID NO: 14), as well as the Sa residues 142-148 in Pl similarly differed from those residues in the Bris/07, X6, V2, V3, V9, V10, VI 1, and V12 HA molecules . Cb residues 87-92 in Pl (LLSARS; SEQ ID NO: 12) differed from the residues in X6 and Bris/07 (LISKES; SEQ ID NO: 10) or Cb residues 88-91 in Pl (LSAR; SEQ ID N028) differed from those residues in X6 and Bris/07 (ISKE; SEQ ID NO:29). Both the Cb and Sa site mutants based on COBRA X6 were introduced into the Pl COBRA HA backbone sequence simultaneously to result in VIPER 3 (V3) having LISKES (SEQ ID NO: 10) and PNHTVT- (SEQ ID NO: 13). Initially, H1N1 VIPER sequences utilized the COBRA Pl HA backbone sequence, and amino acid residues were exchanged for the amino acids found primarily in the Sa and/or the Cb regions, and also for some sequences, in the Sb region of COBRA X6 HA (TABLE 1).

[0198] For example, specific amino acids of the Pl COBRA HA antigen sequence were mutated to corresponding sites in wild-type Hl Influenza HA proteins by site-directed mutagenesis (Carter DM et al. J Virol 90:4720-4734, 2016; U.S. Patent Number 9,555,095). These modified COBRA Pl HA antigens were produced and designated by VIPER numbers. The Cb, Sa, and Sb antigenic sites of the Pl HA antigens have the sequences SEQ ID NO: 12, both SEQ ID NO: 15 (PNHNTTK at residues 141-147) and SEQ ID NO:2l (KKGGSYPKLSKS at residues 170-181), and TSTDQQSLYQNE (SEQ ID NO:26), respectively. [0199] For VIPER 1, the amino acids at residues 88-91 were changed from LSAR (SEQ

ID NO:28) to ISKE (SEQ ID NO:29) in the Cb region of the COBRA Pl HA antigen (TABLE 1). The ISKE (SEQ ID NO:29) amino acids are located at this position in the NC/99, Bris/07, and COBRA X6 HA molecules. Also, the amino acids at residues 87-92 in the Cb region of the COBRA Pl HA antigen were changed from LLSARS (SEQ ID NO: 12) to LISKES (SEQ ID NO: 10) of the VIPER-l (VI), V3, V9, and V12 HA antigen (TABLE 1). The LISKES (SEQ ID NO: 10) amino acids were located at this position in the NC/99, Bris/07, COBRA X6, VI, V3, V9, and V12 HA molecules. These sequence changes transformed the Cb antigenic site from in the Pl HA from pandemic-like to seasonal-like in the resulting VIPER antigens. The amino acids at residues 87-92 in the Cb region of the COBRA Pl HA antigen with the residues LLSARS (SEQ ID NO: 12) were unchanged for V2, V4, V5, V6, V7, V8, V10, and VI 1 HA molecules (TABLE 1).

[0200] The amino acid residues 144-147 of Pl were the same for VIPER 1. However, for VIPER 2, the amino acids at residues 144-147 were changed from NTTK (SEQ ID NO: 30) in the Sa region of the COBRA Pl HA antigen to TVT- (TABLE 1). For VIPER 7 (V7) and VIPER 8 (V8), the amino acids at residues 144-147 were changed from NTTK (SEQ ID NO:30) in the Sa region of the COBRA Pl HA antigen to NTNK (SEQ ID NO: 33) in V7 and V8 (TABLE 1). Additionally, the amino acids at residues 144-147 in the Sa region of the COBRA Pl HA antigen, NTTK (SEQ ID NO:30), were also changed to residues TVT- in the V3, V6, V9, V10, VI 1, and V12 HA molecules (TABLE 1).

[0201] In other embodiments, the amino acids at residues 141-147 PNHNTTK (SEQ

ID NO: 15) located in the Sa region of Pl were modified to produce the residues in the V2, V3, V9, V10, VI 1, and V12 HA (PNHTVT-; SEQ ID NO: 13) Sa region, which is the same as those in the Sa regions of Bris/07 and COBRA X6. Similarly, the 142-148 amino acids NHNTTKG (SEQ ID NO:3 l) located in the Sa region of Pl were modified to match the residues in the X6 and Bris/07 HA (NHTVT-G; SEQ ID NO:32) Sa region to produce those residues in the V2, V3, V9, V10, VI 1, and V12 HA. This exchange introduced a deletion at residue 147 that is also present in late seasonal H1N1 sequences, where a lysine (Lys; K) is present at this location in pandemic HA antigens (TABLE 1).

[0202] For VIPER 4, the sequence is identical to VIPER 2, except a Lys is included at position 147. For VIPER 5, the sequence is identical to VIPER 1 in the Sa and Sb regions, except there is no amino acid located at position 147 in V5, and there is a valine (V) at position 145 of V5 while VI has a Threonine (T). There is no corresponding amino acid in these seasonal HA proteins at position 147 (TABLE 1). For VIPER 6, the sequence is the same as VIPER 5, except at position 142, there is an Asparagine (Asn) in V5 that has been mutated to a threonine (Thr) in V6.

[0203] Other embodiments provide for sequences where the only difference between

V2 and V4 over the portions of the Cb, Sa, and Sb antigenic sites was the inclusion of a lysine (Lys; K) at residue 147 in V4. The VIPER 5 (V5) sequence over the portions of the Cb, Sa, and Sb antigenic sites was identical to V2 except that the amino acid located at residue 144 was an asparagine (Asn; N) in V5, which replaced the threonine (Thr; T) of V2. The only difference between VIPER 6 (V6) and V2 over the portions of the Cb, Sa, and Sb antigenic sites was the mutation of the asparagine at residue 142 in V2 to the threonine in V6. The VIPER 7 (V7) sequence in the Sa antigenic site were mutated from residues 144-147 from NTTK (SEQ ID NO:30) in the COBRA Pl HA antigen sequence to NTNK (SEQ ID NO:33) (TABLE 1). In addition, the amino acids at residue 179-180 were mutated from SK (serine (Ser; S)-lysine (Lys; K)) in the Pl HA antigen sequence to NQ (asparagine (Asn; N)-glutamine (Gln; Q)) in the V7 sequence as well as the amino acids located in A/Michigan/45/20l5. For VIPER 8 (V8), the amino acid at residue 146 was changed from threonine (Thr; T) in the Sa region of the COBRA Pl HA antigen to asparagine (Asn; N) (TABLE 1).

[0204] VIPER 9 to 12 (V9-V12) HA vaccines were designed based on the COBRA X6

HA sequence. The Cb antigenic sites (residues 87-92) of COBRA X6, VI, V3, V9, V12, and Bris/07 (LISKES; SEQ ID NO: 10) were identical and mutated from the Cb site of COBRA Pl (LLSARS; SEQ ID NO: 12). For VIPER 9, the Cb antigenic site from COBRA Pl (residues 87-92) was exchanged in favor of the Cb site of COBRA X6. The VIPER 10 (VI 0) sequence has the same Sa and Sb antigenic sites from COBRA X6 and the amino acid sequence from residues of the Cb site from COBRA PL The Sb antigenic site of COBRA Pl (residues 202- 213) was mutated from the sequence, TSTDQQSLYQNE (SEQ ID NO:26), to produce the sequence NIGDQRALYHTE (SEQ ID NO:27) in COBRA X6, VIPER 10 (V10), and V12. In order to produce VIPER 11 (VI 1), both the Cb and Sb antigenic sites of COBRA X6 (LISKES; SEQ ID NO: 10 and SEQ ID NO:27, respectively) were mutated in favor of the sequences in the respective antigenic sites of COBRA Pl (i.e., SEQ ID NO: 12 and SEQ ID NO: 26, respectively), while the Sa sequence of VI 1 was the same as that of X6 (SEQ ID NO: 13 and SEQ ID NO:20 at residues 141-147 and 170-181, respectively). Finally, to produce VIPER 12 (V12), the putative N-linked glycosylation site (N-X-S, where X is any amino acid except for Proline (P)) beginning at the asparagine residue 177 in the COBRA X6 Sa antigenic site was mutated from the amino acid sequence NLS (asparagine (Asn; N)-leucine (Leu; L) -serine (Ser; S)) to the amino acid sequence KLS (lysine (Lys; K)-leucine (Leu; L)-serine (Ser; S)) found in V12, CA/09, Pl, V1-V6, and V8 (TABLE 1). The HA genes were codon-optimized for expression in mammalian cells and then biochemically synthesized by GENEWIZ® (New Jersey, USA).

TABLE 1 :

[0205] The COBRA X6 HA has an identical Sa and Cb region as Bris/07, and therefore has the same glycan pattern with the putative glycan site at residue 142. The COBRA Pl HA has a glycosylation sequon in the Sa antigenic site at residue 144, but this differs from the CA/09 HA, which has no putative glycosylation sites (TABLE 1). To determine how the Cb and Sa antigenic sites contributed to the immunogenicity of these COBRA HA antigens, a series of mutations were introduced to identify the specific amino acids or glycosylation sites that are critical for broadly reactive antibody responses to H1N1 viruses. Exchanging the Cb site in COBRA Pl HA (LLSARS; SEQ ID NO: 12) with the Cb site located in the COBRA X6 HA (LISKES; SEQ ID NO: 10) to generate the VI HA did not alter the elicited antibody profile of HAI activity against the panel of H1N1 influenza viruses, which was statistically similar to the Pl HAI antibody profile (FIGs. 10B-10C Panels (D)-(E)). Addition of the Sa site from the COBRA X6 HA alone (V2 HA) or in combination with the Cb site (V3 HA), lowered the HAI titers against pandemic-like strains, but raised the titers to seasonal-like strains, including Bris/07. Therefore, it appeared that the Cb antigenic site was not substantially involved in the COBRA Pl or COBRA X6 HA induced HAI activity; however, the Sa region appeared to be responsible for inducing these phenotypes.

[0206] Introduction of the COBRA X6 Sa region not only deleted a glycosylation site in the COBRA Pl HA, but also deleted the Lysine (K) at residue 147. The l9l8-like H1N1 viruses had a Lys located at position 147. However, later viruses had an arginine (Arg; R) or isoleucine (He; I) at this position or alternatively, the amino acid was deleted altogether. The amino acid residue at 147 was deleted from most seasonal-like H1N1 viruses isolated from 1995-2008. However, with the introduction of the pandemic-like swine H1N1 influenza viruses in 2009, the lysine at position 147 in HA returned to circulating human H1N1 influenza strains. The presence of this lysine (K) 147 residue in the CA/09 HA actually stabilized the interaction between HA and sialic acid (SA) (Das SR, et al. PLoS Pathog 6:el00l2l 1, 2010; Xu R, et al. J Virol 86:982-990, 2012; Matsuzaki Y, et al. J Virol 88: 12364-12373, 2014).

EXAMPLE 4: VACCINE PREPARATION

[0207] Human embryonic kidney (HEK) 293T cells (1 x 10 ⁶ ) were transiently transfected with 3 pg DNA expressing each COBRA or wild-type HA gene cassette in addition to the influenza neuraminidase (A/mallard/Alberta/24/0l, H7N3), the HIV p55 Gag sequences, and one of the various H1N1 wild-type, COBRA HA, or VIPER HA expressing plasmids on mammalian expression vectors (Green TD, et al. J Virol. 77:2046-2055, 2003). Following incubation for 72 hours at 37 °C, supernatants from transiently transfected cells were collected, centrifuged to remove cellular debris, and filtered through a 0.22 pm pore membrane. Mammalian virus-like particles (VLPs) were purified and sedimented by ultracentrifugation on a 20% glycerol cushion at 27,000 x g (or 135,000 x g) for 4 hours at 4 °C. VLPs were resuspended in phosphate buffered saline (PBS) and the total protein concentration assessed by conventional bicinchoninic acid assay (BCA), and HA concentration was quantified by Western blot.

[0208] For Western blot, cell lysates were electrophoresed on a 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane. The blot was probed with pooled mouse antisera from infections with A/Brisbane/57/2007 and A/Califomia/07/2009 viruses. HA-antibody complexes were then detected using goat anti-mouse IgG labeled with horseradish peroxidase (HRP) (Southern Biotech, Birmingham, AL, EISA). HRP activity was detected using chemiluminescent substrate (Pierce Biotechnology, Rockford, IL, EISA) and exposed to X-ray film (Thermo Fisher, Pittsburgh, PA, EISA).

[0209] The hemagglutination activity of each preparation of VLP was determined by adding equal volumes of turkey red blood cells (RBCs) to a V-bottom 96-well plate and incubating with serially diluted volumes of VLPs for a 30 minute incubation at room temperature. The highest dilution of VLP with full agglutination of RBCs was considered the endpoint HA titer.

EXAMPLE 5: HA QUANTIFICATION OF PURIFIED INFLUENZA VIRUS VLPs

[0210] Purified VLPs and different known concentrations of standard recombinant HA

(HA1 H1N1 A/California/07/2009) were electrophoresed on a 10% sodium dodecyl sulfate- polyacrylamide gel (SDS-PAG) and transferred to a polyvinylidene difluoride (PVDF) membrane. The blot was probed with monoclonal antibody 15B7 (Immune Technology Corporation, New York, NY, USA). HA-antibody complexes were then detected using goat anti-mouse IgG labeled with horseradish peroxidase (HRP) (Southern Biotech, Birmingham, AL, USA). HRP activity was detected using Clarity™ Western ECL substrate (Bio Rad), and the digital images were captured by Chemi-Doc imaging system (Bio Rad). Linear regression standard curve analysis was generated using the known concentrations of recombinant standard HA, and the HA contents in VLPs were calculated by interpolation from the data of standard curve. Experiment were performed in duplicate, and multiple exposure times were analyzed for all iterations.

EXAMPLE 6: DETERMINATION OF HA CONTENT

[0211] A high-affinity, 96-well flat bottom ELISA plate was coated with 5-10 pg of total protein of VLP and serial dilutions of a recombinant Hl HA antigen (for measuring, e.g., VI -V8) (Protein Sciences, Meriden, CT) in ELISA carbonate buffer (50 mM carbonate buffer, pH 9.5) orH3 antigen (for measuring, e.g., V9-V12) (3006_H3_Vc, Protein Sciences, Meriden, CT) in ELISA carbonate buffer (50 mM carbonate buffer, pH 9.5) was added. The plate was incubated overnight at 4 °C on a rocker. After incubation, the plate was washed in PBS with 0.05% Tween-20 (PBST), then non-specific epitopes were blocked with 1% bovine serum albumin (BSA) in PBST solution for 1 hour at room temperature. Buffer was removed from the plate and then stalk-specific Group 2 antibody CR8020 or Group 1 antibody CR6261 (Tan GS, et al. J Virol 88: 13580-13592, 2014) was added to the plate and incubated for 1 h at 37 °C. The plate was washed, then probed with goat anti-human IgG horseradish-peroxidase- conjugated secondary antibody (2040-05, Southern Biotech, Birmingham, AL) for 1 hour at 37 °C. The plate was washed and then freshly prepared o-phenylenedi amine dihydrochloride (OPD) (P8287, Sigma Aldrich, St. Louis, MO, USA) substrate in citrate buffer (P4922, Sigma Aldrich, St. Louis, MO, USA) was added to the plate wells, followed by 1 N H ₂ S0 ₄ stopping reagent. The plates were read at an absorbance of 492 nm using a microplate reader (Powerwave XS, Biotek, Winooski, VT) and the calculated background was subtracted from the negative wells. Linear regression standard curve analysis was performed using the known concentrations of recombinant standard antigen to estimate HA content in VLP lots.

EXAMPLE 7: VACCINATED MICE CHALLENGED WITH H1N1 INFLUENZA VIRUSES

[0212] Mice were vaccinated with virus-like particles (VLP) expressing one of the 12 modified HA antigens (designated V1-V12), COBRA HA antigens, or wild-type HA antigens. The elicited antisera were assessed for hemagglutination-inhibition activity against a panel of historical seasonal -like and pandemic-like H1N1 influenza viruses. Primarily, the pattern of glycosylation sites and residues in the Sa antigenic region, around the receptor binding site (RBS), served as signatures for the elicitation of broadly-reactive antibodies by these HA immunogens. Mice were vaccinated with VLPs expressing HA antigens that lacked a glycosylation site at residue 144 and a deleted lysine at position 147 residue were most effective at protecting against morbidity and mortality following infection with pandemic-like and seasonal-like H1N1 influenza viruses.

[0213] BALB/c mice (Mus musculus , females, 6 to 8 weeks old) negative for antibodies to circulating influenza A (H1N1, H3N2) and influenza B viruses were purchased from Envigo (Indianapolis, IN, USA) and housed in stainless steel cages (Shor-line, Kansas City, KS) microisolator units containing Sani-Chips laboratory animal bedding (P.J. Murphy Forest Products; Montville, NJ). Mice were provided with Rodent Chow Diet (Harlan Teklad; Madison, WI) and fresh water ad libitum. They were allowed free access to food and water in accordance with USDA guidelines for laboratory animals. All procedures were reviewed and approved by the University of Georgia Institutional Animal Care and Use Committee (IACUC) #2016-02-011-Y3-A7, which were conducted in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals, The Animal Welfare Act, and the CDC/NIH’s Biosafety in Microbiological and Biomedical Laboratories guide. To determine the protective efficacy of each VIPER vaccine, BALB/c mice were randomly divided into 10 groups (n=l l/group). Each VIPER vaccine was prepared by vaccinating BALB/c mice three times at 4-week intervals via intramuscular injection with 1 pg of purified virus-like particles (VLPs) expressing VIPER 1-12 (V1-V12), COBRA Pl, X6, wild-type HA antigens (Brisbane/59/2007 (Bris/07), Califomia/07/2009 (CA/09) VLPs) (3 pg based upon HA content), or PBS as a mock vaccination, each formulated with an adjuvant, AF03, (oil-in- water emulsion) (provided by Sanofi Pasteur) for a final volume of 50 mΐ, and then boosted with the same amount of VLPs or PBS at week 4 (Day 28) and week 8 (Day 56) post- vaccination (FIG. 4). The final concentration after mixing 1 : 1 with VLPs was 2.5% squalene. Blood was harvested from all anesthetized mice at days 28, 56, and 70 or collected at weeks 6, 10, and 12 post-vaccination. Serum was centrifuged at 6000 rpm. Clarified serum was harvested or removed and frozen at -20 ± 5°C.

[0214] All mice were challenged with the wild-type CA/09 H1N1 influenza virus (1 x

10 ⁶ PFU) (10X 50% lethal dose [LD50] on week 13 post-vaccination (FIG. 9 (A)) in a volume of 50 mΐ. Mice were monitored daily for 14 days for weight loss, disease signs and deaths. Three mice from each group were euthanized on Day 2 post-infection, and lungs were harvested and snap frozen on dry ice, then stored at -80°C for viral titration in the future. Mice were humaely euthanized when they reached the humane endpoint by losing 20% of their original body weight or accumulated disease score of up to 3 (lethargy=l, hunched posture=l, rough fur=l, weight loss l5%~20% = 1, weight loss > 20% of original body weight = 3). The experimental endpoint was defined as > 20% weight loss. All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (Carter et al. J Virol. 91(24): e0l283-l7, 2017), Animal Welfare Act (Das, et al. PLoS Pathog. 6(1 l):el00l2l 1, 2010), and Biosafety in Microbiological and Biomedical Laboratories (Hong, et al. J. Virol. 87(22): 12471-12480, 2013).

[0215] Mice vaccinated with CA/09, Pl, or VIPER VI VLPs all survived challenge with little or no weight loss. Mock-vaccinated animals rapidly lost weight and reached experimental endpoints by day 6 post-infection (dpi). Mice vaccinated with the V2 or V3 VLP vaccines, as well as the COBRA X6 VLP vaccine all survived challenge (FIG. 9 (A)), but lost -8-11% body weight by day 6 post-infection and then slowly began to recover weight (TABLE 2)·

[0216] A different set of vaccinated mice were challenged on week 13 post-vaccination with 8.75 x 10 ⁶ plaque forming unit (PFU) A/Brisbane/59/2007xPR8 (6:2 or 7: 1) reassortment influenza virus (lOx LD50) () in a volume of 50 pl (FIG. 9 (B)). Mice vaccinated with the V2 VLP were not protected against Bris/07 challenge (i.e., 20% survival) and 60% of the V3 VLP mice survived, with the average weight loss of the survivors having lost -11% weight by day 6 post-infection (TABLE 2). [0217] The mice were monitored daily for 14 days for signs of weight loss, disease, and death. Three mice from each group Bris/07 were euthanized on Day 3 post-infection, and their lungs were harvested and snap frozen on dry ice, then stored at -80 °C for future viral titration. Once the mice reached the humane endpoint of losing 20% of their original body weight or an accumulated a disease score of 3 (lethargy=l, hunched posture=l, rough fur=l, weight loss 15%~20%=1, weight loss> 20% of original body weight=3), the mice were humanely euthanized. The experimental endpoint was defined as more than 20% a weight loss of the original body weight. All procedures were performed according to the Guide for the Care and Use of Laboratory Animals (Carter DM, et al. J Virol 9l(24):e0l283-l7, 2017), Animal Welfare Act (Das SR, et al. PLoS Pathog 6:el00l2l l, 2010), and Biosafety in Microbiological and Biomedical Laboratories (Hong M, et al. J Virol 87: 12471-12480, 2013). TABLE 2 shows hemagglutination-inhibition, weight loss, and survival of vaccinated mice. For the average hemagglutination-inhibition (HAI) titer per vaccine group that are positive (POS) as indicated by a HAI titer greater than 1 :40, low positive (LOW) as designated by a HAI titer of 1 :20, and negative (NEG) as indicated by a HAI titer of less than 1 : 10. The weight loss ranged from 0% to 20%, while the survival was from 0% to 100%. N.D. was defined as Not Done.

TABLE 2

EXAMPLE 8: HA REGIONS THAT ELICIT ANTIBODIES WITH H1N1 HAI ACTIVITY

[0218] To determine if the lysine at residue 147 (i.e., K147) played a role in the elicited immune responses by the COBRA HA antigens, a second series of mutations in the COBRA Pl HA antigen was introduced. The HA residue at 147 and the N-linked glycosylations cooperated to elicit broadly-reactive antibodies with HAI activity against H1N1 influenza viruses. Adding the K147 residue to the V2 HA generated an HA antigen with a Cb region from COBRA Pl and the Sa region from the COBRA X6 HA with the addition of K147 (named V4 HA). Returning the lysine residue at 147 with the N-linked glycan at residue 144 in HA elicited antibodies with HAI activity with similar profiles as those antibodies elicited by Pl or VI HA antigens (FIG. 11 A (A)). These antibodies had HAI activity against both seasonal and pandemic H1N1 influenza viruses, except SI/06 and Bris/07. Therefore, in all four cases where the lysine was present at residue 147 (i.e., CA/09, Pl, VI, and V4 HA), the HA antigen elicited a COBRA Pl HAI antibody profile, regardless if the glycan was located at residue 142 or 144.

[0219] In HA antigens that lacked the K147 residue, the location of the glycans were more important for the elicitation of antibodies with a breadth of HAI activity against the panel of H1N1 influenza viruses. The V5 HA lacks the K147 residue and the glycan located at residue 144. The V5 HA elicited antibodies with an HAI profile identical to those antibodies elicited by CA/09 HA (FIG. 11 A (B)). The V5 HA has a glycan at residue 144, but no K147 residue. In CA/09, the addition of the K147 residue compensated for the lack of glycans. But the addition of one glycan at position 144, plus the K147, (e.g., Pl COBRA HA) expands the CA/09 HAI titers to include recognition of seasonal-like H1N1 viruses. Lastly, HA antigens with no glycans at 142 or 144 and no K147 (e.g., V6 HA) elicited antibodies with HAI activity with a similar profile to X6 (FIG. 11B (C)). Both the V6 HA and the CA/09 612 HA lacked glycans at 142/144 residues, but only differed at the K147 site and again emphasizing that the K147 found in CA/09 was a signature for the elicitation of pandemic-like antibody phenotype. The V6 HA only differed from Bris/07 HA by the glycosylation site at 142, but the HAI antibody profile was quite different. The glycan at 142 without the K147 residue appeared to be a signature for the elicitation of antibodies with HAI activity specific for recent seasonal H1N1 influenza viruses, such as SI/06 or Bris/07.

[0220] In order to determine the breadth of the vaccine elicited antibodies, serum was collected from vaccinated mice at week 12, following the third vaccination. Mice vaccinated with Bris/07 VLPs had antisera with HAI activity against Bris/07, as well as SI/06 (FIG. 10A (A))) and mice vaccinated with CA/09 VLPs had antisera with HAI activity against CA/09 and Mich/l5 pandemic-like viruses (FIG. 10A (B)). Mice vaccinated with X6 had antisera with HAI activity against NC/99, SI/06, and Bris/07 (FIG. 10B (C)). In contrast, and as previously reported (Carter DM, et al. J Virol. 90:4720-4734, 2016; Carter DM, et al. J Virol. 91(24): e0l283-l7, 2017), mice vaccinated with COBRA Pl VLPs had antisera against both H1N1 pandemic viruses (CA/09; Mich/l5) and historical seasonal influenza viruses (FIG. 10B (D)). Mice vaccinated with the VIPER V 1 VLPs had antibodies with HAI activity against the CA/09, Mich/l5, Sing/86, TX/91, and Bei/95 (FIG. 10C (E)), as well as SC/18 and NJ/76 (data not shown). These VI VLP elicited antibodies did not have HAI activity against SE06 or Bris/07. This HAI pattern was similar to the HAI activity elicited by COBRA Pl . Mice vaccinated with V2 or V3 VLPs had antibodies with HAI activity against most all viruses in the panel to some degree (FIG. 10C (F) and FIG. 10D (G), respectively). The HAI activity against CA/09 elicited by V2 VLPs was very low (FIG. 10D (F)). The V3 VLP vaccines comprised immunogens that elicited antibodies with broad breadth of HAI activity and high HAI titers against the panel (FIG. 10D (G)). FIG. 10D (H) shows Mock vaccine control.

[0221] Introduction of the amino acids in the Sa site from seasonal-like influenza viruses into the V2 HA backbone increased the HAI titers to seasonal-like viruses and decreased the HAI activity in elicited sera to CA/09. In order to define in more detail, the amino acids in the Sa region that are critical for the elicitation of antibodies with HAI activity against seasonal -like and pandemic-like H1N1 viruses, a second set of VIPER HA antigens were designed and designated VIPER V4 (TABLE 1). Utilizing the V2 HA amino acid sequence, the Sa site was mutated to insert a lysine (Lys; K) at residue 147. This resulted in a hybrid Sa site with residues 142-146 representing the amino acids, NHTVT (SEQ ID NO:34), found in seasonal-like influenza HA antigens with the inserted lysine (Lys; K) at residue 147 located in pandemic-like HA proteins (TABLE 1). Similar to V2 and V3 HA antigens, the insertion may shift the putative glycosylation site from position 144 to position 142, which may further alter the site structure and antigenicity of the V4 HA molecule. Mice vaccinated with V4 HA VLPs (FIG. 11 A (A)) elicited antibodies with a similar HAI profile as antibodies elicited by VI HA VLP vaccines (FIG. 10C (E)). The antisera had HAI activity against Sing/86, TX/91, Bei/95, NC/99, and to a lesser extent CA/09 and Mich/l5, but no HAI activity against SE06 or Bris/07 (FIG. 11 A (A)).

[0222] The threonine (Thr; T) at position 144 in the Sa region of V2 HA was mutated into an asparagine (Asn; N) to form V5 HA. This resulted in a V5 Sa region that matched the seasonal-like Sa region, except for the asparagine at position 144 that was found in pandemic- like HA sequences. Mice vaccinated with the V5 HA VLP had antibodies with HAI activity against CA/09 and Mich/l5, both post-pandemic-like H1N1 viruses, with no detectable HAI titers against any of the seasonal-like H1N1 viruses (FIG. 11 A (B)). The asparagine at position 142 in V2 HA was mutated to a threonine to form V6 HA (TABLE 1). All seasonal-like and pandemic-like HA antigens have an asparagine at residue 142. Therefore, unlike any of the wild-type, COBRA, or other VIPER HA antigens, there is no putative glycosylation site at residue 142 or 144. Mice vaccinated with V6 HA VLP had antibodies with HAI activity against most seasonal and pandemic H1N1 influenza viruses (FIG. 11B (C)). The V6 HA antigen elicited antibodies with a pattern of HAI activity similar to V4 HA. This V6 HA had a Cb region sequence similar to that of Pl, no potential N-linked glycosylation sites in Sa, and the deletion of an amino acid at residue 147.

[0223] Overall, there were general signatures associated with each of these sequence combinations. The Cb mutations did not appear to have a significant impact on the elicitation of antibodies with specific HAI activity, therefore, the specific amino acids of the Sa region were focused on. The HA antigens were classified into five phenotypes: CA/09-like, Pl-like, V2/V3-like, X6-like, and Bris/07-like. HA antigens elicited antibodies with HAI activity against pandemic-like viruses {i.e. CA/09) if the HA had either 144 glycan or the K147 residue present. The CA/09 HA had no glycan at residues 142 or 144, but did contain the K147 residue and the V5 HA contained the 144 glycan, but no K147 residue. The Pl, VI, and V4 HA antigens that had the K147 residue had an expanded HAI antibody profile beyond the pandemic-like viruses to include seasonal H1N1 influenza viruses from 1986-2005. This HAI activity appeared to be independent of the presence of a glycan at residue 144, since the V4 HA antigen had a threonine at this residue. All five of these HA antigens (i.e., CA/09, Pl, VI, V4, and V5) protected mice against CA/09 infection with little weight loss or signs of morbidity observed, but did not protect mice against Bris/07 challenge.

[0224] HA antigens, such as, for example, V2, V3, or V6, which did not have either the 144 glycan or the K147 residue, elicited antibodies with HAI activity with a broad seasonal- like phenotype that recognized H1N1 viruses from 1986-2005 with low HAI titers against seasonal viruses from 2006-2008 and pandemic viruses from 2009-2015. Mice vaccinated with these HA antigens were protected against CA/09 challenge, but they did lose some weight. When these vaccinated mice that were challenged with Bris/07 virus, they had mild morbidity with -50% of the mice surviving challenge.

[0225] Although the Sa region, and specifically the glycans at 142/144 and K147 residue, were shown to be important in order to elicit broadly-protective antibodies, other regions or antigenic sites played a role in the HA structure and contributed to the elicitation of broadly-reactive antibodies. For example, the targeted Sa and Cb sequences in the Bris/07, X6, and V3 HA antigens are identical, but the X6 and V3 HA antigens provided protection against CA/09 challenge; whereas the Bris/07 HA vaccine could not protect mice against CA/09 challenge (FIG. 12). Therefore, other regions of HA may contribute to protection that were not included in this analysis. A second region of Sa was located at amino acids 170-181. Seven of the 12 amino acids differed between COBRA Pl and COBRA X6 HA and played a role in the overall HA structure, along with other regions of HA that contribute to the immunogenicity of these immunogens.

EXAMPLE 9: INFLUENZA VIRUS CHALLENGE OF VACCINATED MICE

[0226] Interestingly, all mice vaccinated with the V4, V5, or V6 VLP vaccines, and the mice vaccinated with the CA/09 VLP vaccine, survived challenge with CA/09 (FIG. 11C (D)). All mock-vaccinated mice died from infection by day 6 post-challenge. In contrast, only the mice vaccinated with a Bris/07 VLP vaccine or a V6 VLP vaccine had mice that survived challenge with Bris/07 (FIG.11C (E)). All the Bris/07 VLP vaccinated mice survived and 40% of the V6 VLP vaccinated mice survived. The other mice vaccinated with CA/09, V4, V5, or Pl VLP vaccines died or reached endpoints by day 3-4 post-challenge (FIG. 11C (E)).

[0227] A set of mice was sacrificed on day 3 post-challenge and the influenza virus titer in the lungs was assessed (FIG. 12). Mock vaccinated mice had high lung titers (2xl0 ⁷ pfu/ml). Mice vaccinated with CA/09, Pl, or VI VLP vaccines had no detectable virus in their lungs and V2, V3, or V6 VLP vaccinated mice had a 3-5 log drop in the average lung viral titer (average 5xl0 ³ - lxlO ⁵ pfu/g) compared to mock vaccinated mice (FIG. 12 (A)). Mice vaccinated with X6 VLP vaccines had detectable virus in one mouse, where the other mice had no detectable virus and Bris/07 VLP vaccinated mice had a 1 log drop in viral titer compared to mock vaccinated mice (FIG. 12 (A)). In contrast, mock vaccinated mice challenged with Bris/07 had ~2.2xl0 ⁷ pfu/g of virus in their lungs (FIG. 12 (B)). Mice vaccinated with Pl, VI, V4, or CA/09 VLPs had similar levels of virus in their lungs. However, mice vaccinated with V2, V3, V5, or V6 had a 1-2 log drop in lung viral titer compared to mock vaccinated mice (FIG. 12 (B)). One V3 vaccinated mouse, all of the Bris/07 vaccinated mice, and almost all the X6 VLP vaccinated mice had no detectable virus in the lungs (FIG. 12 (B)).

[0228] The HA immunogen structure is only one component for the effectiveness of the elicited antibodies. The antigenic HA target plays a significant role in the overall protective capacity of the elicited antibodies. These COBRA or VIPER HA-based vaccines did not effectively elicit antibodies against A/Chile/l/l983 (Chile/83) strain in the H1N1 panel. The Chile/83 virus has a glycosylation site at residue 144 on its HA, along with the K147 residue (Kim JI and Park MS. Yonsei Med J 53 :886-893, 2012). H1N1 swine isolated with an N- linked glycosylation site at residue 144 rendered these influenza viruses resistant to antibody- meditated neutralization, whereas viruses with an N142 residue instead of an N144 residue are sensitive to neutralization (Hause BM, et al. Clin Vaccine Immunol 19: 1457-1464, 2012). Therefore, even if the glycan is missing on the HA immunogens and the putative neutralizing epitope is exposed to elicit high titer antibodies to this specific epitope, if the epitope is shielded on the target HA antigen, the effectiveness of the vaccine to protect against influenza infection is decreased. This is a critical understanding in order to develop universal vaccines based upon the globular HA head. The development of HA immunogens that can elicit high titer antibodies to multiple HA epitopes on antigenically distinct viruses will advance the development of new influenza vaccine designs. A mixture of more than one broadly-reactive HA immunogen may be needed to elicit protective antibodies against all influenza strains within a subtype. Overall, the elicitation of broadly-reactive HA antigens can be improved by understanding how the residues around the receptor binding site are structured and exposed to the immune system.

EXAMPLE 10: HEMAGGLUTINATION INHIBITION (HAI) ASSAY

[0229] The hemagglutination inhibition (HAI) assay was used to assess functional antibodies to the HA that inhibit agglutination in guinea pigs and as well as in turkey erythrocytes. The protocols were adapted from the WHO laboratory influenza surveillance manual (Manual for the laboratory diagnosis and virological surveillance of influenza, Geneva: World Health Organization, 2011). To inactivate non-specific inhibitors, sera were treated with receptor-destroying enzyme (RDE) (Denka Seiken, Co., Japan) prior to testing.

[0230] A panel of six H1N1 viruses was used for the HAI assay, including

A/Chile/l/l983, A/Singapore/6/l986, A/Texas/36/ 1991, A/Beijing/262/l995, A/New

Caledonia/20/l999, A/Brisbane/59/2007, A/Solomon Islands/3/2006, A/California/07/2009, A/Michigan/45/20l5. The HAI assay was performed as previously described (Carter DM, et al. J Virol 90:4720-4734, 2016). Briefly, prior to being tested, sera were treated with receptor- destroying enzyme (RDE) ( Denka Seiken , Co., Japan) to inactivate nonspecific inhibitors. Three parts of RDE were added to one part of sera and incubated overnight at 37 °C. RDE was inactivated by incubation at 56 °C for 30 minutes. RDE-treated sera were diluted in a series of two-fold serial dilutions in V-bottom microtiter plates. An equal volume of each H1N1 virus, adjusted to approximately eight hemagglutination units (HAU)/50 pL, was added to each well. The plates were covered and incubated at room temperature for 20-30 minutes, and then 0.8% guinea pig erythrocytes ( Lampire Biologicals , Pipersville, PA, USA) or turkey erythrocytes ( Lampire Biologicals , Pipersville, PA, USA) in PBS were added. Red blood cells (RBCs) were stored at 4 °C and used within 72 hours of preparation. The plates were mixed by agitation and covered, and the RBCs were settled for 30 minutes to 1 hour at room temperature. The HAI titer was determined by the reciprocal dilution of the last well that contained non- agglutinated RBCs. Positive and negative serum controls were included for each plate. According to the WHO and European Committee for Medicinal Products to evaluate influenza vaccines (European Medicines Agency, Guideline on influenza vaccines: Non-clinical and clinical module [Draft], EMA/CHMP/VWP/457259/2014, London E14 4HB, UK), all mice were negative (HAI < 1 : 10) for pre-existing antibodies to the currently circulating human influenza viruses prior to vaccination. Seroprotection was defined as HAI titer >1 :40 and seroconversion as a 4-fold increase in titer compared to baseline; however, a more stringent threshold of >1 :80 was often examined. Mice were naive and seronegative at the time of vaccination, thus seroconversion and seroprotection rates were used interchangeable here.

[0231] HAI titers observed for sera obtained from mice vaccinated with HA antigens isolated from CA/09, NC/99, and seasonal H1N1, and subsequently exposed to pandemic H1N1 virus are shown in FIGs. 2A-2B (A-C). FIG. 5 illustrates HAI titers determined from collected antisera (day 84 post-vaccination) for each group of mice (n=l 1) against a panel of 9 H1N1 influenza viruses. Mice were naive and seronegative at the time of vaccination. Mice vaccinated with V4 against Bei/95, NC/99, CA/09, and Mich/l5 showed seroprotection, i.e., HAI Titers >1 :40. Those vaccinated with V5 against CA/09 and Mich/l5 showed seroprotection, while those mice vaccinated with V6 against Vei/95, NC/99, Bris/07, CA/09, and Mich/l5 showed seroprotection. Mice vaccinated with CA/09 against CA/09 virus showed seroprotection, while mice vaccinated with Bris/07 against NC/99 showed seroprotection. Mice vaccinated with V4, V5, or V6 against A/Califomia/07/09 (CA/09) H1N1 virus also showed less weight loss than mice administered with a mock vaccine. At day 5, mice vaccinated with V4, V5, V6, and Mock vaccine are depicted as points from 100% of original weight down to less than 80%, which correspond to lines in order from top to bottom, respectively (FIG. 6).

EXAMPLE 11 : FOCUS REDUCTION ASSAY (FRA)

[0232] The Focus Reduction Assay (FRA) used in this study was initially developed by the World Health Organization collaborating Centre in London, U.K. (Matrosovich M, et al. Virol J. 3:63, 2006; Sullivan K, et al. J Virol. 179:81-89, 2012) and modified by U.S. Center for Disease Control and Prevention (CDC) (Thomas Rowe, personal communication). MDCK-SIAT1 cells were plated at 2.5 - 3 x 10 ⁵ cells/ml (lOOuL/well in 96-well plate) a day before the assay was run. Cells were grown overnight in 96-well plates to reach 95%-l00% confluency at the time of the assay, forming a confluent monolayer in Dulbecco’s Modified Eagle Medium (DMEM) containing 5% heat-inactivated fetal bovine serum and antibiotics. The following day, the cell monolayers were rinsed with 0.01 M phosphate-buffered saline pH 7.2 (PBS, Gibco) followed by the addition of 2-fold serially diluted RDE-treated serum at 50 pl per well starting at a 1 :20 dilution in Virus Growth Medium containing 1 ug/mL of a serine protease inhibitor, N-Tosyl-L-phenylalanyl chloromethyl ketone (TPCK)-treated Trypsin, VGM-T, [DMEM containing 0.1% BSA, penicillin/streptomycin, and lpg/ml TPCK-treated trypsin (Sigma, St. Louis, MO, EISA)]. Afterwards, 50 pl virus for all FRAs shown here were standardized to 1.2 x 10 ⁴ FFU/ml (Focus Forming Units) (corresponds to 600 FFU/50pl) in VGM-T was added to each plate or VGM-T to cell control wells. The virus stocks were standardized by previous titration in the FRA. Following a 2 hour incubation period at 37 °C with 5% C0 ₂ , the cells in each well were then overlaid with 100 mΐ of equal volumes of 1.2% Avicel® RC/CL, microcrystalline cellulose and carboxymethylcellulose sodium, NF, BP (Matrosovich M, et al. Virol J. 3 :63, 2006) (Type: RC581 NF; FMC Health and Nutrition, Philadelphia, PA, USA) in 2X Modified Eagle Medium containing lpg/ml TPCK-treated trypsin, 0.1% BSA and antibiotics. Plates were incubated for 18-22 hours at 37 °C, 5% CO2. The overlays were then removed from each well and the monolayer was washed once with PBS to remove any residual Avicel®. The plates were fixed with ice-cold 4% formalin in PBS for 30 minutes at 4 °C, followed by a PBS wash and permeabilization using 0.5% Triton-X-lOO in PBS/glycine at room temperature for 20 minutes. Plates were washed three times with wash buffer (PBS, 0.1% Tween-20; PBST), incubated for 1 hour with a monoclonal antibody against influenza A nucleoprotein (Walls HH, et al. J Clin Microbiol. 23 :240-245, 1986) (Influenza Reagent Resource; IRR) in ELISA buffer (PBS, 10% horse serum, 0.1% Tween-80). After washing three times with PBST, the cells were incubated with goat anti-mouse peroxidase- labelled IgG (SeraCare, Inc., Milford, MA) in ELISA buffer for 1 hour at room temperature. Plates were washed three times with PBST and infectious foci (spots) were visualized using TrueBlue™ substrate (SeraCare, Inc., Milford, MA USA) containing 0.03% H2O2 incubated at room temperature for 10-15 minutes. The reaction was stopped by washing five times with distilled water. Plates were dried and foci enumerated using a CTL BioSpot® Analyzer with ImmunoCapture 6.4.87 software (CTL, Shaker Heights, OH). The FRA titer was reported as the reciprocal of the highest dilution of serum corresponding to 50% foci reduction compared to the virus control minus the cell control.

[0233] In order for a plate to pass quality control, both the average of the octuplet virus control wells (VC) as well as the average of the octuplet cell control wells (CC) must pass. The virus controls initially were between 150 to 650 foci (spots) and the cell controls were to have fewer than 21 foci. The virus control wells were subsequently expanded to between 200 and 1600 spots. Additionally, the reference, vaccine strain, virus was run in triplicate plates in each individual assay and at least two out of three plates had to pass the VC and CC criteria and homologous ferret antisera must have the same titer. Each assay plate (one virus per plate) contained a panel of reference antisera as well as human vaccine serum control to assess overall assay consistency.

EXAMPLE 12: ADDITIONAL COBRA Pl HA GLYCOSYLATION MOTIFS ENHANCED PROTECTIVE ANTIBODIES TO SEASONAL Hl INFLUENZA STRAINS

[0234] The CA/09 HA does not have a putative glycosylation motif at residue 144, but has an aspartic acid (Asp; D) amino acid at this residue. Seasonal influenza strains, such as Bris/07, also do not contain a glycosylation site at residue 144 (threonine; Thr; T), but the COBRA Pl HA has a putative glycosylation motif at this residue (TABLE 1). The addition of the asparagine (Asn; N) amino acid at residue 144 in Pl and VI enhanced HAI activity against CA/09, but not Bris/07 (FIG. 12). The lack of a putative N-linked glycosylation site also enhanced HAI activity to NC/99 and Bris/07. Since CA/09 has an asparagine (Asn; N) at residue 146, N-linked glycosylations at all three asparagine residues in the Sa site were hypothesized, so the Pl COBRA HA sequence was used and a third asparagine was inserted into this Sa region (Asn at residues 142, 144, 146) and the lysine (Lys; K) at residue 147 was retained to determine the HAI activity against seasonal and pandemic-like H1N1 viruses (FIGs. 13A-13D). Mice vaccinated with VLPs expressing a modified Pl COBRA HA with the addition of the asparagine at residue 146, as well as asparagine and glutamic acid (Asn-Glu) at residues 179-180 (V7) did not significantly alter this phenotype (FIG. 13B (C)), and all mice survived both Bris/07 and CA/09 challenges (FIGs. 13C (E) and (F)). Mice challenged with CA/09 had little or no weight loss (FIG. 13D (G)), but mice challenged with Bris/07 lost between -7-11% body weight by day 7 post-infection and then recovered. Mice vaccinated with VLPs expressing the V8 HA, which had the same Sa sequence as the V7 HA, except the HA had the addition of only the asparagine (Asn; N) at residue 146 (TABLE 1) elicited protective antibodies similar to V7, but with a higher magnitude of HAI activity against the panel of H1N1 viruses (FIG. 13 A (D)) and again all the mice were protected against infection (FIGs. 13B-13C, panels (E)-(G)).

EXAMPLE 13 : DETERMINING EPITOPES IN SA AND SB THAT MAY ELICIT ANTIBODIES WITH HAI ACTIVITY AGAINST MULTIPLE H1N1 STRAINS

[0235] The region of Sa from amino acid 142-147 has been critical for eliciting antibodies with a seasonal-like or pandemic-like phenotype (FIG. 12 and FIG. 13). In order to determine if other regions of Sa (amino acids 170-181) or the Sb region (202-213) play a role in determining the breadth of elicited antibodies, mutations were engineered in the COBRA X6 HA to mimic homologous regions of CA/09 or COBRA Pl HA (TABLE 1). The corresponding Sb region from Pl was exchanged with X6 to generate V9 HA, and the corresponding Cb region was exchanged with X6 to generate the VI 0 HA. For VI 1, both regions were exchanged. For V12, the Asn (N) at amino acid 177 in the Sa region was mutated to a Lys (K). Mice vaccinated with X6 VLP vaccine had antibodies with HAI activity against NC/99, SE06, and Bris/07 (FIG. 14B (D)), which was similar to the HAI activity from sera collected from VIPER V9, V10, VI 1, or V12 (FIGs. 14C-14D, Panels (E)-(F) and (G)-(H)). None of these epitope exchanges significantly altered the pattern of elicited antibody recognition compared to antibodies elicited by COBRA X6 VLPs. Mice vaccinated with any of these VIPER vaccines had similar weight loss (peak 15-20% of original weight) (TABLE 2), however, most mice, including all the V12 mice, recovered and survived the CA/09 526 challenge (FIG. 15 (A)). Viral lung titers were ~2 logs lower at 3 day post-challenge in COBRA X6 or VIPER V9-V12 VLP vaccinated mice compared to mock vaccinated mice (FIG. 15 (B)). Mice vaccinated with CA/09 or Pl VLPs had low to undetectable viral lung titers.

EXAMPLE 14: ELICITATION OF ANTIBODIES BY VIPER HA ANTIGENS IN FERRETS PRE-IMMUNE TO A HISTORICAL H1N1 INFLUENZA VIRUS

[0236] Using a previously developed pre-immune ferret model, ferrets were infected with the Sing/86 H1N1 influenza virus (FIG. 16). As previously observed (Carter et al. J Virol 9l(24):e0l283-l7), all ferrets seroconverted and had high HAI titers to Sing/86 and the TX/91 virus. At day 84, ferrets were vaccinated with one of six recombinant HA (rHA) vaccines representing the VIPER V3, V6, or V12, the COBRA Pl or X6, or the wild-type CA/09 HA antigens (FIGs. 17A-17D; panels (A)-(G)). Mock vaccinated pre-immune ferrets were used as controls. At day 98 (two week post-vaccination), ferrets vaccinated with Pl, X6, or V12 had HAI titers against all nine H1N1 viruses in the panel, except Bris/07 (FIGs. 17B-17D; panels (C), (D), and (G)). Pre-immune ferrets that were vaccinated with V3 or V6 had HAI titers elicited by the Sing/86 infection (Sing/86 and TX/91) and low HAI titers to the other H1N1 viruses in the panel (FIGs. 17C; panels (E) and (F)). In contrast, ferrets vaccinated with CA/09 HA had high HAI titers to Sing/86 and TX/91, as well as CA/09 and Mich/l5 (FIG. 17A (B)).

EXAMPLE 15: INFLUENZA VIRUS CHALLENGE OF VACCINATED FERRETS

[0237] Pre-immune ferrets vaccinated with the CA/09, Pl, X6, and VI 2 HA vaccines survived challenge with CA/09 with little or no weight loss (FIG. 18 (A)). Mock vaccinated ferrets lost on average 8% of their body weight by day 7 post-challenge and then began to recover. In contrast, pre-immune ferrets vaccinated with V3 or V6 HA vaccines lost ~5% body weight by day 3 post-infection and then began to recover (FIG. 18 (A)). Virus was detectable in all ferret nasal washes at day 1 post-infection (FIG. 18 (B)). However, all pre-immune ferrets vaccinated with V12 or X6 had less than 40 pfu/ml at day 1 post-infection that was statistically lower than pre-immune ferrets vaccinated with the other vaccines or mock vaccinated (FIG. 18 (B)), where the vaccines from top to bottom correspond to the sets of data days post-infection from left to right, respectively.

EXAMPLE 16: VIRAL NEUTRALIZATION TITERS

[0238] In order to determine if vaccine-elicited antibodies would neutralize various

H1N1 virus infections, an in vitro focal reduction assay (FRA) of serum was performed (FIGs. 19A-19C). Mock ferrets vaccinated with any of the wild-type, COBRA, or VIPER VLP vaccines had low to undetectable neutralizing antibody titers against all the H1N1 viruses tested, i.e., Chile/83, Sing/86, NC/99, Bris/07, and CA/09. Ferrets pre-immune to Sing/86 and then vaccinated with one of the VIPER V3, V6, or V12, the COBRA X6 or Pl, or wild-type CA/09 VLP vaccines had considerably higher neutralization titers compared to naive ferrets (data not shown). Sing/86 pre-immune ferrets that were vaccinated with Pl VLPs had antisera with high 50% and 80% neutralizing titers against all the H1N1 viruses except Bris/07. Only sera collected from ferrets pre-immune to Sing/86 and vaccinated with CA/09 VLPs had a log2 titer equal to Pl against CA/09 virus (FIG. 19C (E)). Sera from these pre-immune CA/09 VLP vaccinated ferrets did not neutralize the other seasonal strains any better than X6 COBRA or the VIPER HA antigens (FIGs. 19A-19B; panels (A)-(D)). Sing/86 pre-immune ferrets that were vaccinated with the X6 VLP had a log2 serum dilution neutralization titer of 9.2 (50% inhibition) against C A/09, which was 2-3 logs lower than Pl or CA/09 VLP vaccinated pre- immune ferrets (FIG. 19C (E)). Basically, all of the elicited antisera had a similar neutralization titer against Bris/07 (FIG. 19B (D)), despite the wide range of HAI titers observed against the Bris/07 virus (FIG. 17).

EXAMPLE 17: VIRAL LUNG TITERS

[0239] A plaque assay was performed according to known protocols. In brief, lungs were homogenized in lml DMEM and the supernatant was collected by centrifuging the homogenized samples at 2000 rpm for 5 minutes. Low passage MDCK cells were plated at a confluency of lxlO ⁶ in each well of a six -well plate (Greiner bio-one, NC, USA) one day before the assay. MDCK cells were infected with different dilutions of samples in 100 pL of DMEM supplemented with penicillin-streptomycin. After a 1 hour incubation at room temperature, the medium was removed, and cells were washed twice with fresh DMEM, and 2 mL of Modified Eagle Medium (MEM) medium plus 0.8 % agarose (Cambrex, East Rutherford, NJ, USA) was added. Cells were incubated for 72 hours at 37 °C with 5% C0 ₂ . Agarose was removed, and the cells were then fixed with 10% buffered formalin and stained with 1% crystal violet (Fisher Science Education) for 15 minutes. The crystal violet was removed by rinsing thoroughly in distilled water. The numbers of plaques were counted and virus titer presented as PFU/lung was calculated in lung tissue.

EXAMPLE 18: DETERMINATION OF VIRAL NASAL WASH TITERS

[0240] Madin-Darby canine kidney cells (MDCK) cells were seeded at (5 x 10 ⁵ ) in each well of a six-well plate. Samples were diluted (final dilution factors of 100 to 10-6) and overlaid onto the cells in 100 pl of Dulbecco modified Eagle medium (DMEM) supplemented with penicillin-streptomycin and incubated for 1 hour with intermittent shaking every 15 minutes. Samples were removed, cells were washed twice, and medium replaced with 2 mL of L15 medium plus 0.8% agarose (Cambrex; East Rutherford, NJ, USA) and incubated for 72 hours at 37°C with 5% C0 ₂ . Agarose was removed and discarded. The cells were fixed with 10% buffered formalin and then stained with 1% crystal violet for 15 minutes. The plates were then thoroughly washed in distilled water (dH ₂ 0) to remove excess crystal violet, being air- dried, followed by counting the number of plaques, and calculating the number of PFU per milliliter. EXAMPLE 19: STATISTICAL ANALYSES

[0241] All data were reported as absolute mean values ± S.EM. Weight loss comparisons among the different vaccinated groups were compared using a nonparametric two- way ANOVA test, and viral lung titers and HAI titers were compared using a nonparametric one-way ANOVA test. All statistical analyses were performed using GraphPad Prism 7 software (San Diego, California, USA) and a p<0.05 was considered statistically significant (*p <0.05; **p<0.0l; ***p<0.00l; ****p< 0.0001).

EXAMPLE 20: SW AND Pl HA ANTIGEN VLPS VACCINATED MICE STUDIES

[0242] Computationally Optimized Broadly Reactive Antigens (COBRAs) were generated based on both human and/or swine (SW) Hl HA sequences. BALB/c mice (n=l 1) were vaccinated with virus-like particles (VLP) expressing COBRA or wild-type Hl HA proteins. Mice were intranasally challenged with either A/Califomia/07/2009 H1N1 (CA/09) npdm or A/Swine/NorthCarolina/l52702/20l5 H1N2 Delta-2 virus (NC/15). Sera were collected pre-challenge and weights monitored until 14 days post-challenge.

[0243] Mice vaccinated with VLPs expressing SW1 (SEQ ID NO: 35), SW2 (SEQ ID

NO:36), SW4 (SEQ ID NO:38), and Pl COBRA HA antigens elicited antibodies with hemagglutinin-inhibition (HAI) activity against a panel of swine influenza viruses representing all four clades of SIV. Higher survival rates were observed with groups vaccinated with the homologous vaccine, SW1 or X3 VLP vaccine for NC/15 challenge and SW1, SW2, SW4, and Pl for CA/09 challenge. SW1 had higher survival rates for both challenge viruses.

[0244] SW1 was the first observed vaccine to increase the survival against two challenge viruses located in separate swine lineages. The remaining COBRA HA antigens elicited an antibody response against strains from multiple lineages and clades. Pl also elicited antibodies that cross reacted with human Hl strains. Targeting the HA to create a vaccine that is cross reactive to multiple lineages and clades of swine and human Hl influenza (FIG. 21) is a more economical approach than current methods.

[0245] The swine Hl influenza viruses are separated into three distinct lineages. The

Eurasian lineage: A/swine/Denmark/WVL9/l993 H1N1 1C.2 Other;

A/Swine/Spain/50047/2003 H1N1 1C.2.2 Other; A/swine/Zhejiang/l/2007 H1N1 1C.2.3 Other (Grey), the Classical lineage (Alpha clade: A/swine/Iowa/l973 H1N1 1A.1 Alpha; A/NewJersey/l 1/1976 H1N1 lA. l-like Alpha; A/Swine/Wisconsin/l25/l997 H1N1 1A.1 Alpha (Blue); Beta clade = A/Wine/Nebraska/AO 1444614 /2013 H1N1 1A.2 Beta; A/swine/Iowa/00737/2005 H1N1 1A.2 Beta; A/swine/Colorado/SGl322/ 2009 H1N1 1A.2 Beta (Magenta); Gamma clade = A/Swine/North_Carolina/93523/2001 H1N2 1A.3.3 Gamma- 2; A/swine/Korea/Asan04/2006 H1N2 1A.3.3.3 Gamma; A/swine/Minnesota/ AO 1489606/2015 H1N1 1A.3.3.3 Gamma; A/swine/North_Carolina/00485/2005 H1N1 1A.3.3.3 Gamma; A/swine/Ohio/511445/20074 H1N1 1A.3.3.3 Gamma (Pink); Pandemic clade = A/Swine/Indiana/P 12439/2000 H1N2 1A.3.3 npdm; A/Califomia/07/2009 H1N1

IA.3.3.2 npdm; A/swine/NorthCarolina/34543/2009 H1N1 1A.3.3.2 npdm;

A/swine/Missouri/A0l203163/2012 H1N1 1A.3.3.2 npdm (Red), and the Human Seasonal- like lineage: A/Swine/Oklahoma/A0l4950l/20l l H1N2 1B.2.2.2 Delta-l;

A/Swine/NorthCarolina/02744/2009 extraction; A/swine/NorthCarolina/ 5043-1/2009 H1N2

IB.2.1 Delta-2; A/Swine/NorthCarolina/AO 1377454/2014 H1N2 1B.2.1 Delta-2; A/Swine/NorthCarolina/l52702/20l5 1B.2.1 Delta-2 (Green). Designed COBRA vaccines are boxed in red. Black sequences are human Hl . Challenge viruses were chosen from two separate lineages: Classical (A/CA/07/2009 or CA/09) and Human Seasonal-like (A/SW/NC/152702/2015 or NC/15) indicated with arrows. Distance of the tree equal to amino acids substitutions per site.

[0246] SW 1 is the first observed vaccine to increase the survival against two challenge viruses located in separate swine lineages. The remaining COBRA HA antigens elicited an antibody response against strains from multiple lineages and clades. Pl also elicited antibodies that cross reacted with human Hl strains.

EXAMPLE 21 : STUDY FOR TESTING COBRA VACCINE EFFECTIVENESS IN MICE

[0247] COBRA vaccines were designed by initially downloading HA reference sequences from GISAID or an online flu genome database (FIG. 22A (A)). Consensus layering of the HA1 was performed until final consensus amino acid sequences were obtained. Ten groups were designed (FIG. 22A (B)). Seven were prepared using the COBRA method with differing input parameters. Two were the wild-type HAs of challenge viruses, and the last being a mock vaccine. The VLP was constructed with the HA of interest, an N3 subtype neuraminidase, and HIV Gag protein. The HIV Gag protein is shown as a great double ring structure, with N3 and Vaccine HA alternating around the double ring structure to form the HA/NA VLP. The VLP was mixed 1 : 1 with an oil-in-water adjuvant to prepare the vaccine (FIG. 22B (C)). BALB/C mice (n=l 1) were vaccinated (1 pg HA/mouse) intramuscularly with a prime, boost, boost schedule with three bleeds at week 4, week 6, and week 10 (FIG. 22B (D)). Sera were separated from collected blood for HAI assays. On week 12, mice were intranasally challenged with either NC/15 (10 ⁷ pfu/50 mΐ) or CA/09 (5xl0 ⁴ pfu/50 mΐ) (DO). Lungs were harvested for viral titers on day 2 (D2) post-challenge (n=3), and weight loss and survival (20% weight-loss cut-off) were monitored over the course of infection to day 14. See, FIG. 22B (D).

EXAMPLE 22: SWINE COBRA SW1 LIMITS MORTALITY FOR CHALLENGE VIRUSES

[0248] Survival curves of vaccinated mice (n=5) after challenge with

A/Swine/NorthCarolina/l 52702/2015 (Sw/NC/l5) (FIG. 23 (A)-(B)) or A/Califomia/07/2009 (CA/09) (FIG. 23 (C)-(D)). At 20% weight loss or overt clinical symptoms mice were humanely euthanized. Weights were monitored up to 14 days post challenge. SW1 of the SW COBRA Survival curve (A) and X3 of the Hu and Pl COBRA Survival curve (B) exhibited curves similar to that of Sw/NC/l5, which is presented as the uppermost horizontal line at 100% survival in both survival curves, i.e., SW 1 and X3 are presented as horizontal lines below that of Sw/NC/l5. SW3 (A) and X6 and CA/09 (B) curves exhibited curves similar to PBS, where the percent survival of PBS decreases at day 5, and further decreases at about day 7, then plateaus to a level above 50% survival. SW2 and SW4 (A) and Pl (B) groups had 100% lethality at about day 6 and day 4, respectively. SW1, SW2, and SW4 (C) and Pl (D) had curves similar to CA/09 (100% survival). X3 and X6 (D) were intermediate (above 50% survival), and SW3 (C) performed more similar to PBS (i.e., < 50% survival).

EXAMPLE 23 : SWINE COBRA SW1 LIMITS WEIGHT LOSS FOR CHALLENGE VIRUSES OVER TIME

[0249] Weight loss of vaccinated mice (n=5) after challenge with

A/Swine/NorthCarolina/l 52702/2015 (Sw/NC/l5) (FIG. 24A (A)-(B)) or

A/Califomia/07/2009 (CA/09) (FIG. 24B (C)-(D)). Vaccines are identified in the legend, including PBS control. At 20% weight loss (i.e., 80% weight), mice were humanely euthanized. Weights were monitored for up to 14 days post challenge. SW1 (A) and X6 (B) exhibited curves similar to Sw/NC/l5. SW3, X3, and CA/09 vaccinated groups exhibited curves similar to PBS control. SW2, SW4, and Pl groups had the steepest curves. SW2 and SW4 (C) and Pl (D) had curves similar to CA/09. SW1 was intermediate (C). SW3 (C) and X3 and X6 (D) performed similar to PBS control.

EXAMPLE 24: SWINE COBRA SW1 LIMITS WEIGHT LOSS FOR CHALLENGE VIRUSES

[0250] The percent original weight of vaccinated mice (n=5) after challenge with

A/Swine/NorthCarolina/l 52702/2015 (Sw/NC/l5) (FIG. 25 (A)-(B)) or A/Califomia/07/2009 (CA/09) (FIG. 25 (C)-(D)) was analyzed on Day 6. At 20% weight loss, mice were humanely euthanized. Mice vaccinated with SW1 (A) and X6 (B) and challenged with NC/15 exhibited percent weight data similar to NC/15. Mice vaccinated with SW3 (A) and X3 (B) and CA/09 (A & B) exhibited percent weight data similar to PBS (A& B). Mice vaccinated with SW2 and SW4 (A) and Pl (B) groups had lost 20% of their original weight. Mice vaccinated with SW2 and SW4 (C) and Pl (D) and challenged with CA/09 had percent weight data similar to CA/09 (C & D). Mice vaccinated with SW1 (C) was intermediate with respect to percent weight. Whereas, mice vaccinated with SW3 (C) and X3 and X6 (D) had percent weight data similar to PBS (C & D). The significance was determined using the Student’s T-test. Mice euthanized before Day 6 were analyzed with a percent weight of 80%. Amino acid sequences of SW 1 (SEQ ID NO: 35), SW2 (SEQ ID NO: 36), SW3 (SEQ ID NO: 37), and SW4 (SEQ ID NO: 38) are presented in FIGs. 27A-27B.

EXAMPLE 25: HEMAGGLUTINATION INHIBITION (HAI) ACTIVITY TITERS FROM SERA OF MICE VACCINATED WITH COBRA-BASED HA VACCINES OR WILD TYPE HA-BASED VACCINES

[0251] Sera collected from mice at weeks 10-11 were tested against a panel consisting of human viruses (X6; CA/09; Bris/07; Chile/83 : black), classical swine lineage VLPs (alpha=SW/IA/73, SW/WE97: blue; beta= SW/CO/09; SW/NE/13 : magenta; gamma 2= SW/NC/01; gamma l=SW/Korea/06; SW/OH/07; SW/MN/15: pink; pandemic= SW/IN/00; SW/NC/09; SW/MO/12: red), human seasonal-like swine lineage VLPs (delta 1= SW/OK/01; delta 2: SW/NC/l 52702/15; SW/NC/5043-1/09; SW/NC/15; SW/NC/02744/09: green), and Eurasian swine lineage (Eur: SW/Spain/03; SW/Zhejiang/07: grey) (FIGs. 26A-26E). All PBS mock vaccinated mice had HAI titers below the limit of detection against every panel virus. The limit of detection was <5 HAI Titer. The horizontal grey bar indicates an HAI titer between 1 :40 and 1 :80 titers, and the assumption was that at or above this area, the titers correlated with increased protection.

[0252] SW1, SW2, SW4, and Pl elicited antibodies with HAI activity against strains across all three swine lineages, i.e., classical, human seasonal-like, and Eurasian swine lineages. SW2, SW4, and Pl, however, also elicited antibodies with reactivity against CA/09. SW3 elicited antibodies with less overall reactivity in comparison to the number of strains that were recognized by antibodies were elicited by wild type (WT) HA antigens. X3 and X6 COBRA HA antigens also elicited limited cross reactivity, but greatly recognized an A/Sw/NC/02744/09 from the Human seasonal-like lineage delta clade.

[0253] No antibodies against the challenge virus NC/15 were elicited before challenge, however, SW1, X3, and X6 vaccinated mice experienced reduced morbidity. SW1 and X3 vaccinated mice had 100% survival.

[0254] Antibodies against CA/09 were elicited in SW2, SW4 and Pl vaccinated mice.

When challenged, SW2, SW4, and Pl mice had reduced morbidity and 100% survival. SW1 vaccinated mice, which elicited a minimal antibody response, experienced intermediate weight loss with 100% survival.

[0255] Cross protection against both challenge viruses was achieved with SW1 vaccination, which increased the survival rates. The COBRA HA antigens elicited antibodies not targeting the receptor binding site of the HA. Potential differences in the viral loads of the lungs need to be better understood. Overall, these COBRA-based HA vaccines elicited broadly protective antibodies that can benefit both human health and the swine industry.

Other Embodiments

[0256] As various changes can be made in the above-described subject matter without departing from the scope and spirit of the present invention, it is intended that all subject matter contained in the above description, or defined in the appended claims, be interpreted as descriptive and illustrative of the invention. From the foregoing description, it will be apparent that many modifications and variations may be made to the invention and are possible in light of the above teachings described herein to adopt it to various usages and conditions. Accordingly, the description herein is intended to embrace all such alternatives, modifications, and variances which fall within the scope of the appended claims. [0257] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

SPECIFIC EMBODIMENTS

[0258] Non-limiting specific embodiments are described below each of which is considered to be within the present disclosure.

[0259] Specific embodiment 1) A non-naturally occurring, broadly reactive, pan- epitopic influenza A virus antigen that generates an immune response against one or more influenza A virus subtypes.

[0260] Specific embodiment 2) The influenza A virus antigen of claim 1, wherein the antigen is influenza A virus hemagglutinin Hl protein or an antibody-binding portion thereof.

[0261] Specific embodiment 3) The influenza A virus antigen of claim 1 or claim

2, wherein the influenza A virus subtype is: H1N1, H1N2, H1N3, H1N4, H1N5, H1N6, H1N7, H1N8, H1N9, H1N10, H1N11, or a combination thereof.

[0262] Specific embodiment 4) The influenza A virus antigen of any one of specific embodiments 1 to 3, wherein the influenza A virus antigen comprises an amino acid sequence that is at least 80% (e.g., 85%, 90%, 95%, 97%, 99%, 100%) identical to an amino acid sequence of an influenza A virus antigen or antigenic fragment thereof selected from any one of SEQ ID NOs: 1-38, or any combination thereof.

[0263] Specific embodiment 5) A virus-like particle (VLP) comprising the influenza A virus antigen of any one of specific embodiments 1 to 4.

[0264] Specific embodiment 6) The VLP of claim 5, wherein the VLP comprises a polynucleotide encoding the influenza A virus antigen.

[0265] Specific embodiment 7) A subviral particle (SVP), wherein the SVP comprises the influenza A virus antigen of any one of specific embodiments 1 to 4.

[0266] Specific embodiment 8) The SVP of claim 7, wherein the SVP comprises a polynucleotide encoding the influenza A virus antigen. [0267] Specific embodiment 9) The influenza A vims antigen of specific embodiments 1 to 4, wherein the antigen is an immunogen that generates an immune response against one or more influenza A vims subtypes.

[0268] Specific embodiment 10) The influenza A vims antigen, immunogen, VLP, or SVP of any one of specific embodiments 1 to 9, wherein the immune response comprises the production of neutralizing antibodies.

[0269] Specific embodiment 11) The influenza A vims antigen, immunogen, VLP, or SVP of any one of specific embodiments 1 to 9, wherein the immune response comprises the production of T-lymphocytes.

[0270] Specific embodiment 12) A pharmaceutical composition comprising the influenza A vims antigen, immunogen, VLP, or SVP of any one of specific embodiments 1 to 11, and a pharmaceutically acceptable carrier.

[0271] Specific embodiment 13) The pharmaceutical composition of claim 12, further comprising an adjuvant.

[0272] Specific embodiment 14) An immunogenic composition comprising the influenza A vims antigen, immunogen, VLP, or SVP of any one of specific embodiments 1 to 11

[0273] Specific embodiment 15) The immunogenic composition of claim 14, wherein the immunogenic composition is a vaccine.

[0274] Specific embodiment 16) A pharmaceutical composition comprising the immunogenic composition of claim 14 or claim 15 and a pharmaceutically acceptable carrier.

[0275] Specific embodiment 17) The pharmaceutical composition of claim 16, further comprising an adjuvant.

[0276] Specific embodiment 18) A method of generating an immune response in a subject, comprising administering to the subject, an effective amount of the influenza A virus antigen, immunogen, VLP, or SVP of any one of specific embodiments 1 to 11.

[0277] Specific embodiment 19) A method of generating an immune response in a subject, comprising administering to the subject, an effective amount of the pharmaceutical composition of any one of specific embodiments 12-13, 16-17, or any combination thereof. [0278] Specific embodiment 20) A method of generating an immune response in a subject comprising administering to the subject an effective amount of the immunogenic composition of claim 14 or claim 15.

[0279] Specific embodiment 21) The method of any one of specific embodiments

18 to 20, wherein the immune response comprises the production of neutralizing antibodies.

[0280] Specific embodiment 22) The method of any one of specific embodiments

18 to 21, wherein the immune response further comprises the production of T -lymphocytes.

[0281] Specific embodiment 23) The method of any one of specific embodiments

18 to 22, wherein an adjuvant is concomitantly administered to the subject.

[0282] Specific embodiment 24) A polynucleotide encoding the influenza A virus antigen of any one of specific embodiments 1 to 4.

[0283] Specific embodiment 25) A composition comprising the polynucleotide of claim 24 and a pharmaceutically acceptable carrier.

[0284] Specific embodiment 26) Use of an effective amount of the influenza A virus antigen, immunogen, VLP, SVP, pharmaceutical composition, or immunogenic composition of any one of specific embodiments 1-17, wherein the administration is sufficient to stimulate production of antigen-specific antibodies, thereby inducing an immune response.

[0285] All patents, applications, and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent, application, and publication were specifically and individually indicated to be incorporated by reference.