Truncated influenza neuraminidase and methods of using same - Patents.com

Abstract

Provided are modified influenza virus subtype 2 neuraminidase molecules lacking all or substantially all of the stalk region that form active soluble tetrameric neuraminidase when expressed in a host cell, and vaccine compositions comprising nucleic acids encoding the tetrameric neuraminidase or modified monomeric influenza virus subtype 2 neuraminidase molecules that form tetrameric NA when expressed in a cell. Also provided are methods of vaccinating or immunizing a subject against influenza virus using the vaccine compositions.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to and relies on the filing date of U.S. Provisional Patent Application No. 63 / 231,790, filed August 11, 2021, which is incorporated herein by reference. [Background technology]

[0002] Influenza is caused primarily by a virus that attacks the upper respiratory tract (the nose, pharynx, and bronchi, and rarely the lungs). Infection usually lasts for about a week. Influenza is characterized by sudden episodes of high fever, muscle pain, headache, and severe fatigue, dry cough, sore throat, and rhinitis. Most affected individuals recover within one to two weeks without requiring medical treatment. However, influenza poses a serious risk in the very young, the elderly, and those with medical conditions such as lung disease, diabetes, cancer, kidney, or heart problems. In these individuals, infection can lead to severe complications of the underlying disease, pneumonia, and death, although healthy adults and older children can be affected as well. Annual seasonal influenza epidemics are thought to result in between 3 and 5 million cases of severe illness and between 250,000 and 500,000 deaths each year worldwide.

[0003] Influenza viruses are members of the Orthomyxoviridae family. There are three major subtypes of influenza viruses, designated influenza A, influenza B, and influenza C. Influenza virions contain a segmented negative-stranded RNA genome that encodes the following proteins: hemagglutinin (HA), neuraminidase (NA), matrix (M1), proton ion-channel protein (M2), nucleoprotein (NP), polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), polymerase acidic protein (PA), and nonstructural protein 2 (NS2). HA, NA, M1, and M2 are membrane-associated proteins, whereas NP, PB1, PB2, PA, and NS2 are nucleocapsid-associated proteins. The HA and NA proteins are primarily envelope glycoproteins that are responsible for virus attachment and penetration of viral particles into and release from cells, respectively.

[0004] Both HA and NA proteins are the source of major immunodominant epitopes for virus neutralization and protective immunity, and therefore they are important components for preventive influenza vaccines. The genetic makeup of influenza viruses allows frequent and minute genetic changes, known as antigenic drift. Thus, the amino acid sequences of major influenza antigens, including HA and NA, are highly variable across a particular group, subtype, and / or strain. For this reason, current seasonal influenza vaccines are recommended annually and require annual monitoring to accommodate mutations in HA and NA proteins (antigenic drift) and match fast-evolving virus strains.

[0005] Influenza NA is a homotetrameric type II transmembrane glycoprotein, with each monomer having a globular head domain, a stalk region, a hydrophobic transmembrane domain, and a short N-terminal cytoplasmic domain. Tetramerization of the head domain is important for the formation of the enzymatic active site and for the sialidase activity required for the release of new virus particles from infected cells. Sialidase activity is also thought to be important for the virus to cross the mucus barrier within the host. The head domain is also the most immunologically relevant part of the NA. Antibodies against the influenza NA head region block the enzymatic activity of NA and interfere with viral pathogenesis, especially cell-to-cell spread and transmission.

[0006] Recombinant NA or purified NA virus obtained by proteolysis or detergent solubilization of the viral membrane has been studied for structural, enzymatic, and immunological analysis. If a soluble form of NA is desired for use as an immunogen, the NA molecule must be expressed without the anchoring transmembrane domain, which frequently results in the loss of the stabilizing force that helps to keep the NA molecule in its tetrameric form. Without the transmembrane domain and the ability to embed the HA protein in the membrane, the stability of the tetrameric NA head is compromised, resulting in partial degradation and loss of immunogenicity. Furthermore, the secondary and tertiary structures of the NA stalk and associated transmembrane domain are unknown, complicating rational protein engineering approaches based on native structures.

[0007] Therefore, the expression of soluble tetrameric NA in cells is extremely difficult, and in particular the expression of soluble tetrameric NA in high yield and / or in suitable host cells that is compatible with the large-scale production of therapeutic recombinant proteins is extremely difficult. These challenges arise in part from the failure of the assembly of tetrameric NA derived from recombinant NA constructs, resulting mainly in the expression of inactive soluble monomers and soluble dimers, or inclusion bodies (large protein aggregates that pose major challenges for the recovery of bioactive proteins from large-scale production), with little to no expression of active tetrameric NA (Non-Patent Document 1; Non-Patent Document 2; Non-Patent Document 3; Non-Patent Document 4; Non-Patent Document 5). In one case, a recombinant truncated NA was produced that, when expressed in insect cells, resulted in a mixed population of tetrameric, dimeric, and monomeric NAs; however, this specific construct, made from the NA of the A / Victoria / 3 / 1975 influenza strain, lacked amino acids 1-45 of the NA protein (i.e., only the first few amino acids of the cytoplasmic tail, transmembrane domain, and stalk domain) (Non-Patent Document 6). Further studies with the A / Victoria / 3 / 1975 influenza strain used by DeRoo et al. showed that when virtually all of the stalk domain was removed (i.e., recombinant constructs lacking amino acids 1-78 or 1-79 of the NA protein) and expressed in insect cells, only monomeric NA was produced (Non-Patent Document 3). Viral NA purified from the viral membrane via proteolysis or detergent solubilization is unstable and rapidly loses enzymatic activity (Non-Patent Document 7).

[0008] To overcome these challenges, the art teaches the use of full-length heterogeneous tetramerization domains fused to the NA head region to produce soluble recombinant NAs that form tetrameric NAs (Non-Patent Document 7; Non-Patent Document 8; Non-Patent Document 9; Non-Patent Document 10; Non-Patent Document 11). It has been shown that the structural role of the transmembrane and stalk domains in stabilizing the head region of NAs can be approximated by using full-length heterogeneous tetramerization domains that form four-helix bundles. This does not mean that the native stalk domain forms a four-helix bundle. If this were the case, the structure would be easily predictable using bioinformatics algorithms, but in reality it is not, and would be supported by using x-ray structural analysis and / or cryo-electron microscopy. Although full-length heterogeneous tetramerization domains provide a convenient recombinant tool for making soluble tetrameric NAs, the use of full-length heterogeneous tetramerization domains in recombinant proteins has the potential to induce immune responses against the foreign tetramerization domain. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Mather et al., 1992, Virus Res., 26:127-39 [Non-Patent Document 2] Castrucci et al., J. Virology, 1993, 67(2):759~64 [Non-Patent Document 3] Martinet, Eur.J.Biochem., 1997, 247:332~38 [Non-Patent Document 4] Yongkiettrakul et al., 2011, J. Virological Methods, 156:44~51 [Non-Patent Document 5] Romanik et al., 2012, FEBS Journal, 279(Suppl. 1):52-576, 339 [Non-Patent Document 6] DeRoo et al., Vaccine, 1996, 14(6):561-69 [Non-Patent Document 7] Schmidt et al., PLos ONE, 2011, 6(2):e16284 [Non-Patent Document 8] Da Silva et al., J Biol Chem, 2013, 288(1):644~53 [Non-Patent Document 9] Dai et al., 2016, J. Virology, 90(20):9457~70 [Non-Patent Document 10] Bosch et al., 2010, J. Virology, 84(19):10366-74 [Non-Patent Document 11] Prevato et al., 2015, PLos ONE, 10(8):e0135474 Summary of the Invention [Problem to be solved by the invention]

[0010] To effectively control seasonal influenza infections and help avoid pandemics, new recombinant influenza NA proteins (and nucleic acids encoding same) are needed, particularly recombinant influenza NA proteins expressed intracellularly in a manner that results in immunogenic, tetrameric NA without the use of full-length heterologous oligomerization sequences. [Means for solving the problem]

[0011] This application discloses modified monomeric influenza virus subtype 2 neuraminidase constructs lacking all or substantially all of the cytoplasmic tail, transmembrane region, and stalk region that form active, soluble tetrameric neuraminidase when expressed in a host cell. The recombinant design strategy disclosed in this application allows for large-scale production of recombinant tetrameric NA, thus enabling rapid and flexible responses to emerging influenza variants.

[0012] The first aspect is directed to a modified monomeric influenza virus subtype 2 neuraminidase (NA) that comprises a head region of an influenza virus subtype 2 NA, and all or substantially all of the cytoplasmic tail, transmembrane region, and stalk region of the influenza virus subtype 2 NA have been removed from the modified monomeric influenza virus subtype 2 NA, the modified monomeric influenza virus subtype 2 NA does not comprise a heterologous oligomerization domain, and expression of the modified monomeric influenza virus subtype 2 NA in a cell results in secretion of a tetrameric NA. In certain embodiments, the modified monomeric influenza virus subtype 2 neuraminidase comprises a signal peptide, and all or substantially all of the cytoplasmic tail, transmembrane region, and stalk region of the influenza virus subtype 2 NA have been replaced by the signal peptide. The first aspect is also directed to an artificial nucleic acid encoding a modified monomeric influenza virus subtype 2 NA. Various features and embodiments of this first aspect are described in further detail throughout the disclosure of this application.

[0013] A second aspect is directed to a tetrameric neuraminidase (NA) comprising four copies of a modified monomeric influenza virus subtype 2 NA, the modified monomeric influenza virus subtype 2 NA comprising the head region of influenza virus subtype 2 NA, but not including 1) all or substantially all of the cytoplasmic tail, transmembrane region, and stalk region of influenza virus subtype 2 NA, and 2) the heterologous oligomerization domain. Various features and embodiments of this second aspect are described in further detail throughout the disclosure of this application.

[0014] A third aspect is directed to a vaccine composition comprising a tetrameric NA (second aspect) or an artificial nucleic acid molecule encoding a modified monomeric influenza virus subtype 2 NA (first aspect). The vaccine composition may also include an adjuvant. The vaccine composition may further comprise an influenza virus hemagglutinin. In certain embodiments, the influenza virus hemagglutinin is derived from a different influenza strain than, or a mismatched influenza strain with, the modified monomeric influenza virus subtype 2 neuraminidase. Various features and embodiments of this third aspect are described in further detail throughout the disclosure of this application.

[0015] A fourth aspect is directed to an in vitro method of making tetrameric NA (second aspect), comprising culturing a host cell in a cell culture medium, the host cell containing an artificial nucleic acid encoding a modified monomeric influenza virus subtype 2 NA (first aspect), and expressing the modified monomeric influenza virus subtype 2 NA in the host cell, the host cell supernatant containing the tetrameric NA (second aspect) after expression of the modified monomeric influenza virus subtype 2 NA. The method may also further comprise purifying the secreted tetrameric NA to provide a purified influenza virus subtype 2 tetrameric NA. Various features and embodiments of this fourth aspect are described in more detail throughout the disclosure of the present application.

[0016] A fifth aspect is directed to a method of immunizing a subject against influenza virus comprising administering to the subject an immunologically effective amount of a vaccine composition (third aspect). Various features and embodiments of this fifth aspect are described in further detail throughout the disclosure of this application.

[0017] A sixth aspect is directed to a method of vaccinating a subject against influenza virus comprising administering to the subject an amount of a vaccine composition (third aspect) that is effective to vaccinate the subject against influenza virus. Various features and embodiments of this sixth aspect are described in further detail throughout the disclosure of this application.

[0018] A seventh aspect is directed to other methods of using the vaccine composition (third aspect), including methods of inducing an immune response against influenza virus NA, methods of preventing influenza virus disease, and methods of reducing one or more symptoms of influenza virus infection, such as weight loss or elevated body temperature (fever). Various features and embodiments of this seventh aspect are described in further detail throughout the disclosure of this application.

[0019] The foregoing general summary and the following detailed description are exemplary and explanatory but are not restrictive of the scope of the claims.

[0020] The accompanying drawings, which are incorporated in and constitute part of this specification, illustrate certain embodiments and, together with the description, explain certain principles of the neuraminidase molecules, compositions, and methods disclosed herein. [Brief description of the drawings]

[0021] [Figure 1]1 shows schematics for wild-type ("WT") subtype 2 influenza neuraminidase ("N2"), having a short N-terminal cytoplasmic tail, transmembrane region, stalk region, and head region, as well as a previously described recombinant N2 ("tetNA") in which substantially all of the cytoplasmic tail, transmembrane region, and stalk region have been replaced with a signal peptide (also known as a secretion signal), a histidine tag, and a full-length heterologous tetrabrachion tetramerization domain. FIG. 1 also includes a schematic of an engineered N2 ("dTM36") in which the cytoplasmic tail and transmembrane region have been replaced with a signal peptide (also known as a secretion signal), and an optional histidine tag; dTM36 contains the entire stalk and head region of N2. The final schematic in FIG. 1 is an exemplary engineered N2 ("dTM75") in which substantially all of the cytoplasmic tail, transmembrane region, and stalk region have been replaced with a signal peptide (also known as a secretion signal), and an optional histidine tag. The 6HIS tag in FIG. 1 corresponds to SEQ ID NO:135. [Diagram 2] Figure 1 shows the binding results to TAMIFLU® and the enzymatic activity of NAs from 37 different subtype 2 influenza strains expressed in cells after transfection. Tetrameric NAs expressed from modified A / Singapore / INFIMH-16-0019 / 2016 NAs and containing the full-length heterologous tetrabrachion tetramerization domain were used as a positive control. [Figure 3A] FIG. 1 shows neuraminidase sequence maps of 100 subtype 2 influenza test strains in the dTM75 high-throughput screening assay, with strain clusters showing a higher degree of NA sequence similarity compared to other strain clusters. [Figure 3B] FIG. 1 shows strain clusters ordered by the year in which the strains were isolated. [Figure 3C]Figure 1 shows binding results to TAMIFLU® (C) and NA enzymatic activity (D) from a high-throughput screen of dTM75 mutants derived from 100 subtype 2 influenza strains. The top 37 dTM75 TAMIFLU® binders are shown. [Figure 3D] Figure 1 shows binding results to TAMIFLU® (C) and NA enzymatic activity (D) from a high-throughput screen of dTM75 mutants derived from 100 subtype 2 influenza strains. The top 37 dTM75 TAMIFLU® binders are shown. [Figure 4] Figure 4 shows binding results to TAMIFLU® for a series of contiguous stalk deletion mutants from three strains: A / PERTH / 16 / 2009 (PERT09), A / BELGIUM / 4217 / 2015 (BELG15), and A / KANSAS / 14 / 2017 (KANS17). The N2 contiguous stalk deletion mutants contain a deletion of the neuraminidase stalk region starting at amino acid 60 and extending to amino acid 90 for PERT09, BELG15, and KANS17. Amino acids 59-94 (dTM60-dTM95) of PERT09 NA (SEQ ID NO:65), BELG15 NA (SEQ ID NO:5), and KANS17 NA (SEQ ID NO:45) are shown in Figure 4. The diameter of the circle indicates the amount of binding to TAMIFLU® by the engineered stalk deletion mutant NA molecules. FIG. 4 discloses SEQ ID NOs: 136-138, respectively, in order of appearance. [Diagram 5]Figure 1 shows size exclusion chromatography / multi-angle light scattering (SEC-MALS) analysis of purified NA proteins after large-scale production of dTM75 constructs from PERT09, BELG15, KANS17, A / Peru / 4617 / 2017 (PERU17), and A / Texas / 71 / 2017 (TEX17). The top panel shows the percentage of tetrameric NA in the samples, while the bottom panel shows the molecular weight (MW) of dTM75 NA in the samples, where monomeric dTM75 has a MW of about 60 kD and tetrameric dTM75 has a MW of about 210 kD, as discussed in Example 4. KANS17 dTM75 elutes in two peaks, pool 1 (monomer) and pool 2 (tetramer), while the other dTM75 constructs elute in a single peak containing tetrameric NA. [Figure 6] FIG. 1 shows the % tetrameric NA, thermal stability, binding to TAMIFLU®, and enzymatic activity of dTM75 mutants and tet-NA constructs derived from PERT09, BELG15, KANS17, PERU17, and TEX17 after large-scale production and purification of the dTM75 mutants and tet-NA constructs. [Figure 7] FIG. 1 shows the purity, thermostability, enzymatic activity (dTM to tet-NA MUNANA ratio), and binding to TAMIFLU® (dTM to tet-NA ratio) for PERT09 tet-NA constructs and PERT09 stalk truncation mutants dTM75, dTM74, dTM73, dTM72, and dTM71 after large-scale production and purification of tet-NA constructs and dTM75 mutants. [Figure 8A] 1 shows the dosing schedule for mice. Mice were immunized intramuscularly (IM) with a priming dose of recombinant N2 protein (+adjuvant) on day 0, followed by a boosting dose of recombinant N2 protein (+adjuvant) on day 21, and serum was collected on day 35 as described in Example 6. [Figure 8B]8A shows the immunogenicity of PERT09, BELG15, and KANS17 proteins, dTM75 and tet-NA, administered to mice as shown in FIG. 8A and described in Example 6. Mice were immunized with monomeric recombinant NA products from pool 1 of KANS17 dTM75, or tetrameric recombinant NA protein products from pool 2 of PERT09 dTM75, BELG17 dTM75, tet-NA, or KANS17 dTM75. NAI responses were measured against H6N2 reassortant viruses expressing homologous full-length NA by ELLA and expressed as IC50. NA-specific IgG responses were measured against homologous tet-NA (as coating antigen) by ELISA and expressed as EC50 (half-maximal effective concentration). Individual animal titers were normalized (Log2) and graphed as box-and-whisker plots, with values ​​representing group mean titers. The dashed line indicates the lower detection limit of the assay. [Figure 9A] 1 shows the dosing schedule for mice. Mice were immunized intramuscularly (IM) with a priming dose of recombinant N2 protein (+adjuvant) on day 0, followed by a boosting dose of recombinant N2 protein (+adjuvant) on day 21, and serum was collected on day 35 as described in Example 6. [Figure 9B] Figure 9A shows the immunogenicity of PERT09 truncated N2 proteins dTM75, dTM74, dTM73, dTM72, and dTM71 administered to mice as shown in Figure 9A and described in Example 6. NA-specific IgG responses were measured against homologous tet-NA by ELISA and expressed as EC50 (half maximal effective concentration). NAI responses were measured against H6N2 reassortant viruses expressing homologous full-length NA by ELLA and expressed as IC50. Individual animal titers were normalized (Log2) and graphed as box-and-whisker plots with values ​​representing group mean titers. The dashed line indicates the lower detection limit of the assay, while the shaded area represents titers within 4-fold of the tet-NA control protein. [Figure 10A]Figure 1 shows the dosing schedule for pre-immunizing ferrets. Ferrets were immunized intranasally on day 0 with a priming dose of influenza H1N2 reassortant virus expressing a wild-type NA of interest, followed by a boosting dose of non-adjuvanted, homologous recombinant NA (dTM75 or tet-NA) administered IM. Serum was collected on day 42 as described in Example 7. [Figure 10B] Figure 1 shows the immunogenicity of PERT09, BELG15, and KANS17 proteins, dTM75 and tet-NA, administered to pre-immunized ferrets as described in Example 7. NAI responses were measured by ELLA against H6N2 reassortant viruses expressing homologous full-length NA and expressed as IC50. NA-specific IgG responses were measured by ELISA against homologous tet-NA and expressed as EC50 (half-maximal effective concentration). Individual animal titers were normalized (Log2) and graphed as box-and-whisker plots, with values ​​representing group mean titers. The dashed line indicates the lower detection limit of the assay. The "mock" control was a group that was only pre-immunized with H1N2 virus on day 0 and not boosted with recombinant NA on day 21. [Figure 11-1] FIG. 1 shows the amino acid sequences of SEQ ID NOs: 1 to 133. [Figure 11-2] Continued from Figure 11-1. [Figure 11-3] Continued from Figure 11-2. [Figure 11-4] Continued from Figure 11-3. [Figure 11-5] Continued from Figure 11-4. [Figure 11-6] Continued from Figure 11-5. [Figure 11-7] Continued from Figure 11-6. [Figure 11-8] Continued from Figure 11-7. [Figure 11-9] Continued from Figure 11-8. [Figure 11-10] Continued from Figure 11-9. [Figure 11-11] Continued from Figure 11-10. [Figure 11-12] Continued from Figure 11-11. [Figure 11-13] Continued from Figure 11-12. [Figure 11-14] Continued from Figure 11-13. [Figure 11-15] Continued from Figure 11-14. [Figure 11-16] Continued from Figure 11-15. [Figure 11-17] Continued from Figure 11-16. [Figure 11-18] Continued from Figure 11-17. [Figure 11-19] Continued from Figure 11-18. [Figure 11-20] Continued from Figure 11-19. [Figure 11-21] Continued from Figure 11-20. [Figure 11-22] Continued from Figure 11-21. [Figure 11-23] Continued from Figure 11-22. [Figure 11-24] Continued from Figure 11-23. [Figure 11-25] Continued from Figure 11-24. [Figure 11-26] Continued from Figure 11-25. [Figure 11-27] Continued from Figure 11-26. [Figure 11-28] Continued from Figure 11-27. [Figure 11-29] Continued from Figure 11-28. [Figure 11-30] Continued from Figure 11-29. [Figure 11-31] Continued from Figure 11-30. [Figure 11-32] Continued from Figure 11-3. [Figure 11-33] Continued from Figure 11-32. [Figure 11-34] Continued from Figure 11-33. [Figure 11-35] Continued from Figure 11-34. [Figure 11-36] Continued from Figure 11-35. [Figure 11-37] Continued from Figure 11-36. [Figure 11-38] Continued from Figure 11-37. [Figure 11-39] Continued from Figure 11-38. [Figure 11-40] Continued from Figure 11-39. [Figure 11-41] Continued from Figure 11-40. [Figure 11-42] Continued from Figure 11-41. [Figure 11-43] Continued from Figure 11-42. [Figure 11-44] Continued from Figure 11-43. [Figure 12A] FIG. 1 shows study endpoints (serological antibody responses and protection against infection and / or disease severity) in naive ferrets following immunization with two doses of truncated PERT09 dTM75 followed by challenge with PERT09 H3N2 influenza virus as described in Example 8. [Figure 12B] Figure 1 shows the dosing schedule for the naive ferret study. Naive ferrets were immunized intramuscularly (IM) with a priming dose of recombinant N2 protein (dTM75 or tet-NA) with and without adjuvant on day 0, followed by a boosting dose of recombinant N2 protein (dTM75 or tet-NA) with and without adjuvant on day 21, as described in Table 4 and Example 8. Naive ferrets were then challenged intranasally with PERT09 H3N2 on day 43, as described in Example 8. Serum was collected on days 20, 42, and 57. [Figure 13] Figure 1 shows the immunogenicity of PERT09 dTM75 and control proteins as indicated by NAI antibody titers in ferret sera after prime (day 20), boost (day 42), and challenge (day 57) as described in Example 8. NAI responses were measured by ELLA against H6N2 reassortant viruses expressing the homologous full-length NA and expressed as half-maximal inhibitory concentrations (IC50) as described for mice in Example 6. Individual animal titers are shown with numbers representing the average titer for each group. [Figure 14]Figure 1 shows the immunogenicity of PERT09 dTM75 and control proteins as indicated by NA-binding antibody titers (ELISA) in ferret sera after prime (day 20), boost (day 42) and challenge (day 57) as described in Example 8. NA-specific IgG responses were measured by ELISA against the homologous tet-NA (as coating antigen) and expressed as EC50 (half maximal effective concentration) as described for mice in Example 6. Individual animal titers are shown with numbers representing the average titer of each group. [Figure 15] Figure 13 shows plots depicting disease severity as indicated by viral shedding (AUC), and peak temperature change (degrees Celsius), and peak weight change (percent change) following challenge with H3N2 influenza virus in naive ferrets pre-immunized with two doses of PERT09 dTM75, as described in Example 8. Results from a control group are also shown. [Figure 16] FIG. 1 shows the reduction in total viral shedding measured as AUC (means, range, and mean difference / p-values ​​for pairwise comparisons) following challenge with H3N2 influenza virus in naive ferrets pre-immunized with two doses of PERT09 dTM75 as described in Example 8. Results from a control group are also shown. [Figure 17] FIG. 1 shows the reduction in peak body temperature change, measured as the change in body temperature (mean, range, and mean difference / p-value for pairwise comparisons) above pre-challenge baseline, following challenge with H3N2 influenza virus in naive ferrets pre-immunized with two doses of PERT09 dTM75, as described in Example 8. Results from a control group are also shown. [Figure 18]FIG. 1 shows the reduction in peak body weight loss, measured as body weight change above pre-challenge baseline (mean, range, and distribution of % body weight change by treatment group), following challenge with H3N2 influenza virus in naive ferrets pre-immunized with two doses of PERT09 dTM75, as described in Example 8. Results from the control group are also shown. [Figure 19] Figure 1 shows the immunogenicity of PERT09 dTM75, SING16-rHA, and the combination of PERT09 dTM75 and SING16-rHA as indicated by NAI antibody titers in ferret sera after prime (day 20), boost (day 42), and challenge (day 57) as described in Example 9. NAI responses were measured by ELLA against H6N2 reassortant PERT09 viruses expressing the homologous full-length NA and expressed as half-maximal inhibitory concentrations (IC50) as described for mice in Example 6. Each symbol represents the NAI titer of each individual ferret within the group (n=9). Bar graphs and values ​​within bars represent geometric mean values ​​with 95% CI. "ns" indicates no significant difference. [Figure 20] Figure 1 shows the immunogenicity of PERT09 dTM75, SING16-rHA, and the combination of PERT09 dTM75 and SING16-rHA as indicated by NA-binding antibody titers (ELISA) in ferret serum after prime (day 20), boost (day 42), and challenge (day 57) as described in Example 9. NA-specific IgG responses were measured by ELISA against the homologous PERT09 tet-NA (as coating antigen) and expressed as EC50 (half maximal effective concentration) as described for mice in Example 6. Each symbol represents the ELISA titer of each individual ferret within the group (n=9). Bar graphs and values ​​within bars represent geometric mean values ​​with 95% CI. "ns" indicates no significant difference. [Figure 21]Figure 1 shows the immunogenicity of PERT09 dTM75, SING16-rHA, and the combination of PERT09 dTM75 and SING16-rHA as indicated by HAI titers to SING16 in ferret serum after prime (day 20), boost (day 42), and challenge (day 57) as described in Example 9. Each symbol represents the HAI titer of each individual ferret within a group (n=9). Bar graphs and numbers within bars represent geometric mean values ​​with 95% CI. "ns" indicates no significant difference. [Figure 22] Figure 1 shows the immunogenicity of PERT09 dTM75, SING16-rHA, and the combination of PERT09 dTM75 and SING16-rHA as indicated by HAI titers to PERT09 in ferret serum after prime (day 20), boost (day 42), and challenge (day 57) as described in Example 9. Each symbol represents the HAI titer of each individual ferret within a group (n=9). Bar graphs and numbers within bars represent geometric mean values ​​with 95% CI. "ns" indicates no significant difference. [Figure 23-1]Figures 23A-23C show weight loss following challenge with H3N2 influenza virus in naive ferrets pre-immunized with two doses of PERT09 dTM75, two doses of SING16 rHA, or two doses of a combination of PERT09 dTM75 and SING16 rHA, as described in Example 9. Results from the control groups are also shown. The lines confirm the mean percent change in weight over time (14 days) for the given group, as indicated in the caption. Data for groups immunized with 5μg+AF03 (Figure 23A), or 45μg (Figure 23B) are split into two graphs, and the PBS and PERT09 pre-infected control groups presented in each graph are from the same study. For comparison of peak weight loss (FIG. 23C), the mean value for each group is depicted next to the bar graph, and each symbol represents the weight loss value for each individual ferret (n=9) within a group. Bar graphs and numbers within bars represent the mean, and error bars represent SEM. Statistically significant differences between groups are noted with * and were estimated using pairwise comparisons by ANOVA followed by Tukey's test with p values ​​between 0.0001 and 0.005. [Figure 23-2] Continued from Figure 23-1. [Figure 24-1] 24A-24C are plots depicting the intensity and duration of viral shedding following challenge with H3N2 influenza virus in naive ferrets pre-immunized with two doses of PERT09 dTM75, two doses of SING16 rHA, or two doses of a combination of PERT09 dTM75 and SING16 rHA, as described in Example 9. Results from the control groups are also shown. The lines confirm the mean titer for a given group, as indicated in the caption. Data for groups immunized with 5 μg+AF03 (FIG. 24A), or 45 μg (FIG. 24B) are split into two graphs, and the PBS and PERT09 pre-infected control groups presented in each graph are from the same study. FIG. 24C shows total viral shedding (AUC), and viral shedding at day 45. [Figure 24-2] Continued from Figure 24-1. [Figure 25A] FIG. 1 shows a time course identifying the number of ferrets with detectable virus from days 0 to 8 after challenge with H3N2 influenza virus. [Figure 25B] Figure 1 shows the increase in peak body temperature change as change in body temperature above pre-challenge baseline measured in the morning and afternoon following challenge with H3N2 influenza virus in naive ferrets pre-immunized with two doses of PERT09 dTM75, two doses of SING16 rHA, or two doses of a combination of PERT09 dTM75 and SING16 rHA, as described in Example 9. Results from the control group are also shown. For peak body temperature, the mean value of each group is presented in a bar graph, each symbol represents the peak value of each individual ferret (n=9) within the group, the numbers within the bars represent the mean value, and error bars are presented as SEM. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] A.Definition In order that this disclosure may be more readily understood, certain terms are first defined below. Further definitions for the following terms and other terms are set forth throughout this specification. In the event that the definitions of terms set forth below conflict with definitions in applications or patents incorporated by reference, the definitions set forth in this application shall be used to understand the meaning of the terms.

[0023] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one or more methods, and / or steps of the kind described herein and / or that will be apparent to those skilled in the art upon reading this disclosure, and so forth.

[0024] The use of ordinal terms such as "first," "second," "third," etc. in the claims to modify claim elements does not, in itself, imply a priority, precedence, or order of one claim element over another, or the temporal order in which acts of a method are performed, but is merely used as a marker (for the purposes of ordinal terminology) to distinguish one claim element having a particular name from other elements having the same name.

[0025] Antigen: As used herein, the term "antigen" refers to an agent that, when exposed to or administered to an organism, (i) elicits an immune response; and / or (ii) binds a T cell receptor (e.g., when presented by an MHC molecule) or binds an antibody (e.g., produced by a B cell). In some embodiments, an antigen elicits a humoral response in an organism (e.g., including the production of antigen-specific antibodies); alternatively, or in addition, in some embodiments, an antigen elicits a cellular response in an organism (e.g., involving T cells whose receptors specifically interact with the antigen). It will be understood by those skilled in the art that a particular antigen may elicit an immune response in one or more members of a target organism (e.g., mouse, rabbit, primate, human), but not in all members of the target organism. In some embodiments, the antigen induces an immune response in at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of members of the target species. In some embodiments, the antigen binds to an antibody and / or a T cell receptor and may or may not induce a specific physiological response in the organism. In some embodiments, for example, the antigen may bind to an antibody and / or a T cell receptor in vitro, although such interactions may or may not occur in vivo. In some embodiments, the antigen reacts with the products of specific humoral or cellular immunity, including specific humoral or cellular immunity induced by a heterologous immunogen. The antigen includes truncated NA and tetrameric NA formed therefrom, as described herein.

[0026] "Approximately": As used herein, when applied to one or more values ​​of interest, the term "approximately" or "about" refers to a value that is close to a stated reference value. In some embodiments, the term "approximately" or "about" refers to a range of values ​​that is within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (above or below the reference value) relative to the stated reference value, unless otherwise stated or clear from the context (unless such number exceeds 100% of possible values).

[0027] Artificial nucleic acid molecule: As used herein, an "artificial nucleic acid molecule" is understood to be a nucleic acid molecule that typically does not occur in nature, such as DNA or RNA. In other words, an artificial nucleic acid molecule is understood to be a non-natural nucleic acid molecule. Such a nucleic acid molecule may be non-natural due to its individual sequence (not occurring in nature) and / or due to other modifications, such as structural modifications of nucleotides that do not occur in nature. An artificial nucleic acid molecule may be a DNA molecule, an RNA molecule, or a hybrid molecule that includes a DNA portion and an RNA portion. Typically, an artificial nucleic acid molecule is designed and / or created by genetic engineering techniques to correspond to a desired artificial sequence of nucleotides (heterologous sequence). Furthermore, the term "artificial nucleic acid molecule" is not limited to mean a "single molecule" and is typically understood to include a collection of identical molecules. Thus, an "artificial nucleic acid molecule" may relate to a plurality of identical molecules contained in an aliquot.

[0028] Carrier: As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a composition is administered. In some exemplary embodiments, a carrier can comprise a sterile liquid, such as, for example, water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as, for example, peanut oil, soybean oil, mineral oil, sesame oil, and the like. In some embodiments, a carrier is or comprises one or more solid ingredients.

[0029] Epitope: As used herein, the term "epitope" includes any moiety that is specifically recognized, in whole or in part, by an immunoglobulin (e.g., antibody or receptor) binding entity. In some embodiments, an epitope is composed of multiple chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface exposed when the antigen adopts a suitable three-dimensional conformation. In some embodiments, such chemical atoms or groups are physically proximate to one another in space when the antigen adopts such a conformation. In some embodiments, at least some of such chemical atoms and groups are physically separated from one another when the antigen adopts an alternative conformation (e.g., linearized).

[0030] Excipient: As used herein, the term "excipient" refers to a non-therapeutic agent that is incorporated into a pharmaceutical composition to provide or contribute, for example, to a desired consistency or stabilizing effect. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene, glycol, water, ethanol, and the like.

[0031] Glycosylation: As used herein, "glycosylation" refers to the addition of sugar units to a protein. As used herein, "N-glycan" refers to a sugar chain attached to a protein at the amide nitrogen of an N (asparagine) residue of the protein. Thus, an N-glycan is formed by the process of N-glycosylation. The glycan can be a polysaccharide.

[0032] Immune response: As used herein, the term "immune response" refers to a response of a cell of the immune system, such as a B cell, T cell, dendritic cell, macrophage, or polymorphonuclear cell, to a stimulus, such as an antigen, immunogen, or vaccine. An immune response can include any somatic cell involved in a host defense response, including, for example, epithelial cells that secrete interferons or cytokines. An immune response includes, but is not limited to, an innate immune response and / or an adaptive immune response. As used herein, a protective immune response refers to an immune response that protects a subject from infection (prevents infection or prevents the development of a disease associated with infection). Methods of measuring immune responses are well known in the art and include, for example, measuring lymphocyte (such as B or T cell) proliferation and / or activity, cytokine or chemokine secretion, inflammation, antibody production, and the like. An antibody response or humoral response is an immune response in which antibodies are produced. A "cellular immune response" is an immune response mediated by T cells and / or other white blood cells.

[0033] Immunogen: As used herein, the term "immunogen" or "immunogenic" refers to a compound, composition, or substance that, under appropriate conditions, is capable of stimulating an immune response, such as the production of antibodies or a T-cell response in an animal, including compositions that are injected or absorbed into an animal. As used herein, "immunizing" means rendering a subject protected against an infectious disease.

[0034] Immunologically effective amount: As used herein, the term "immunologically effective amount" means an amount sufficient to immunize a subject.

[0035] In some embodiments: As used herein, the term "in some embodiments" refers to multiple embodiments of any aspect of the disclosure, unless the context clearly dictates otherwise.

[0036] Monomeric influenza virus neuraminidase: Wild-type influenza virus neuraminidase (NA) is a tetramer of four identical monomers. Each NA monomer in wild-type influenza NA consists of four distinct structural domains: an enzymatic head region, a stalk region, a transmembrane region, and a cytoplasmic tail. As used herein, the term "monomeric influenza virus neuraminidase" refers to an NA monomer that combines with three other NA monomers to form a tetrameric NA. In some embodiments, the modified monomeric influenza virus neuraminidase described herein lacks all or substantially all of the cytoplasmic tail, the transmembrane domain, and the stalk domain.

[0037] N1: As used herein, "N1" refers to influenza virus subtype 1 neuraminidase (NA). Influenza A viruses are divided into groups 1 and 2. Groups 1 and 2 are further divided into subtypes, which refer to classification of viruses based on the sequences of two proteins on the surface of the virus, hemagglutinin (HA) and NA. Currently, 18 HA subtypes (H1-H18) and 11 NA subtypes (N1-N11) are recognized. Thus, N1 is significantly different from the other NA subtypes, N2-N11.

[0038] N2: As used herein, "N2" refers to influenza virus subtype 2 neuraminidase (NA). Influenza A viruses are divided into groups 1 and 2. Groups 1 and 2 are further divided into subtypes, which refer to classification of viruses based on the sequences of two proteins on the surface of the virus, hemagglutinin (HA) and NA. Currently, 18 HA subtypes (H1-H18) and 11 NA subtypes (N1-N11) are recognized. Thus, N1 is significantly different from the other NA subtypes, N2-N11.

[0039] NB: As used herein, "NB" refers to influenza type B neuraminidase (NA). Influenza type B strains are classified into two lineages: B / Yamagata and B / Victoria.

[0040] Pandemic strain: A "pandemic" influenza strain is an influenza strain that has caused or has the capacity to cause a pandemic infection of a population of interest, such as a human population. In some embodiments, a pandemic strain has caused a pandemic infection. In some embodiments, such a pandemic infection involves epidemic infection across multiple regions; in some embodiments, a pandemic infection involves infection across regions that are separated from one another such that infectious diseases do not normally pass between them (e.g., regions separated by mountains, bodies of water, as parts of significantly different continents, etc.).

[0041] Prevention: As used herein, the term "prevention" refers to the prevention, avoidance of disease symptoms, delay in onset, and / or reduction in the frequency and / or severity of one or more symptoms of a particular disease, disorder, or condition (e.g., infection with, for example, an influenza virus). In some embodiments, prevention is evaluated on a population basis such that an agent is considered to "prevent" a particular disease, disorder, or condition if a statistically significant reduction in the onset, frequency, and / or intensity of one or more symptoms of the disease, disorder, or condition is observed within a population susceptible to the disease, disorder, or condition.

[0042] Seasonal strain: A "seasonal" influenza strain is an influenza strain that has caused or is capable of causing seasonal infection (e.g., annual epidemic infection) in a population of interest, such as a human population. In some embodiments, a seasonal strain has caused seasonal infection.

[0043] Sequence identity: Similarity between amino acid or nucleic acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is often measured in terms of the percentage of identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. "Sequence identity" between two nucleic acid sequences refers to the percentage of nucleotides that are identical between the sequences. "Sequence identity" between two amino acid sequences refers to the percentage of amino acids that are identical between the sequences. Homologs or variants of a given gene or protein will possess a relatively high degree of sequence identity when aligned using standard methods.

[0044] The terms "% identical", "% identity" or similar terms are intended to refer in particular to the percentage of nucleotides or amino acids that are identical in optimal alignment between the two sequences being compared. Said percentage is purely statistical, and the differences between the two sequences may, but are not necessarily, randomly distributed over the entire length of the sequences being compared. Comparison of two sequences is usually carried out by comparing said sequences over segments or "comparison regions" in order to identify local regions of corresponding sequences after optimal alignment. Optimal alignment for comparison may be performed manually, with the aid of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math., 2, 482, with the aid of the local homology algorithm of Needleman and Wunsch, 1970, J. Mol. Biol., 48, 443, with the aid of the search for similarity algorithm of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA, 88, 2444, or with the aid of computer programs that use said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0045] The percentage of identity is obtained by determining the number of corresponding identical positions in the compared sequences, dividing this number by the number of positions being compared (e.g., the number of positions in the reference sequence) and multiplying this result by 100.

[0046] In some embodiments, the degree of identity is given for a region that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference sequence.For example, if the reference nucleic acid sequence is 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides in consecutive nucleotides in some embodiments.In some embodiments, the degree of identity is given for the entire length of the reference sequence.

[0047] A nucleic acid sequence or amino acid sequence that has a particular degree of identity to a given nucleic acid sequence or amino acid sequence, respectively, may have at least one functional and / or structural characteristic of the given sequence, e.g., in some cases, is functionally and / or structurally equivalent to the given sequence. In some embodiments, a nucleic acid sequence or amino acid sequence that has a particular degree of identity to a given nucleic acid sequence or amino acid sequence is functionally and / or structurally equivalent to the given sequence.

[0048] Stalk region: As used herein, the "stalk region" of influenza subtype 2 neuraminidase refers to the region of subtype 2 neuraminidase from about amino acid 36 to about amino acid 82. As used herein, "substantially all of the stalk region" refers to amino acid 36 to amino acid 69 of the stalk region of influenza virus subtype 2 NA. Thus, a modified N2 lacking the cytoplasmic tail, transmembrane region, and substantially all of the stalk region may lack amino acids 1-70, 1-71, 1-72, 1-73, 1-74, 1-75, 1-76, 1-77, 1-78, 1-79, 1-80, or 1-81 of influenza virus subtype 2 NA. In other words, a modified N2 as described herein may include 13 or less of the most C-terminal amino acids of the stalk region of influenza virus subtype 2 NA, where the most C-terminal amino acids of the stalk region typically refer to amino acids 70-82 of N2. In certain embodiments, the entire cytoplasmic tail, transmembrane region, and stalk region (eg, amino acids 1-82) are removed from modified N2.

[0049] Standard Therapeutic Strain: Each year, based on intensive surveillance efforts, the World Health Organization (WHO) selects influenza strains for inclusion in seasonal influenza vaccine products. As used herein, the term "standard therapeutic strain" or "SOC strain" refers to an influenza strain selected by the World Health Organization (WHO) for inclusion in seasonal influenza vaccine products. Standard therapeutic strains can include past standard therapeutic strains, current standard therapeutic strains, or future standard therapeutic strains.

[0050] Subject: As used herein, the term "subject" refers to any member of the animal kingdom. In some embodiments, "subject" refers to a human. In some embodiments, "subject" refers to a non-human animal. In some embodiments, subjects include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, the non-human subject is a mammal (e.g., a rodent, mouse, rat, rabbit, ferret, monkey, dog, cat, sheep, cow, primate, and / or pig). In some embodiments, the subject may be a transgenic animal, a genetically engineered animal, and / or a clone. In some embodiments, the subject is an adult, a juvenile, or an infant. In some embodiments, the terms "individual" or "patient" are used and are intended to be interchangeable with "subject."

[0051] Tetramerization domain: As used herein, the term "tetramerization domain" refers to an amino acid sequence that encodes a domain that causes tetrameric assembly of a polypeptide or protein. A tetramerization domain that is not native to a particular protein may be referred to as an artificial tetramerization domain or a heterologous tetramerization domain. Exemplary tetramerization domains include, but are not limited to, sequences derived from tetrabrachion, GCN4 leucine zipper, or vasodilator-stimulated phosphoprotein (VASP).

[0052] Vaccination: As used herein, the term "vaccination" or "vaccinating" refers to administration of a composition that generates an immune response against a pathogen, such as, for example, influenza virus. A vaccination is administered before, at, and / or after exposure to the pathogen and / or onset of one or more symptoms, and in some embodiments, before, at, and / or shortly after exposure to the pathogen. Vaccines can induce both prophylactic (preventative) and therapeutic responses. Methods of administration vary according to the vaccine, but can include inoculation, oral inhalation, or other forms of administration. The inoculum is delivered by any of a number of routes, including parenteral routes, such as intravenous, subcutaneous, intraperitoneal, intradermal, or intramuscular routes. The vaccine is administered with an adjuvant that boosts the immune response. In some embodiments, vaccination involves multiple administrations of the vaccination composition, appropriately spaced apart.

[0053] Wild type (WT): As understood in the art, the term "wild type" generally refers to the normal form of protein or nucleic acid found in nature.For example, wild type NA polypeptide is found in natural isolates of influenza virus.Various different wild type NA sequences are found in the NCBI influenza virus sequence database.

[0054] B. Cutting type N2 The present application discloses a recombinant influenza subtype 2 neuraminidase (N2) lacking all or substantially all of the cytoplasmic tail, transmembrane region, and stalk region that forms a soluble tetrameric neuraminidase (NA) when expressed in cells without the use of a heterologous tetramerization domain.

[0055] 1. Nomenclature for influenza viruses All nomenclatures used to classify influenza viruses are nomenclatures commonly used by those skilled in the art. Thus, influenza virus types or groups refer to three major types of influenza: influenza A, influenza B, or influenza C, which infect humans. Influenza A and influenza B cause significant morbidity and mortality each year. Those skilled in the art understand that the designation of a virus as a specific type refers to sequence differences within the respective M1 (matrix) protein or P (nucleoprotein). Influenza A viruses are further divided into group 1 and group 2. These groups are further divided into subtypes, which refer to classification of the virus based on the sequences of two proteins on the surface of the virus, HA and NA. Currently, 18 HA subtypes (H1-H18) and 11 NA subtypes (N1-N11) are recognized. Group 1 contains N1, N4, N5, and N8, and H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, and H18. Group 2 contains N2, N3, N6, N7, and N9, and H3, H4, H7, H10, H14, and H15. N10 and N11 have been identified in influenza-like genomes isolated from bats (Wu et al., Trends in Microbiology, 2014, 22(4):183-91). Potentially, there are 198 different influenza A subtype combinations, but only about 131 subtypes have been detected in nature. The most recent subtypes of influenza A viruses that commonly circulate in human populations and cause seasonal pandemics include A(H1N1) and A(H3N2). Influenza A subtypes are further divided into different genetic "clades" and "subclades." Finally, the term "strain" refers to viruses within a subtype that differ from each other in that they have small genetic variations in their genomes.

[0056] For convenience, certain abbreviations may be used to refer to the protein constructs and their parts described herein.For example, NA may refer to influenza neuraminidase protein or its parts.NA refers to neuraminidase from influenza subtype 2 strain.The term tet-NA refers to recombinant NA that contains a heterologous tetramerization domain, which forms tetrameric NA when expressed in cells.HA refers to hemagglutinin or its parts.

[0057] 2.Neuraminidase (NA) Along with hemagglutinin (HA), neuraminidase (NA) is one of the two major influenza surface proteins. The function of both NA and HA involves interaction with sialic acid, a terminal molecule that is attached to sugar moieties on glycoproteins or glycolipids expressed on the surface of cells. Binding of HA to sialic acid on the cell surface induces endocytosis of the virus by the cell, allowing the virus to enter and infect the cell. Sialic acid is also added to HA and NA as part of the glycosylation process that occurs within infected cells. NA removes sialic acid from intracellular glycoproteins and glycolipids, as well as from newly synthesized HA and NA on nascent virions. Removal of sialic acid by NA promotes the efficient release of virus particles from the surface of infected cells by preventing their aggregation. NA also prevents the virus from binding via HA to already infected and dying cells, promoting further spread of viral infection.

[0058] a. Structure of wild-type neuraminidase NA is a type II transmembrane glycoprotein that assembles on the viral surface as a tetramer of four identical monomers. The molecular weight of the wild-type monomer is about 55-72 kDa, depending on the influenza subtype; the molecular weight of the tetramer is about 240-260 kDa, depending on the influenza subtype. Each NA monomer consists of four distinct structural domains: the enzymatic head region, the stalk region, the transmembrane region, and the cytoplasmic tail. The largest domain is the head region, which is tethered to the viral membrane by the stalk region connected to the transmembrane region and finally to the N-terminal cytoplasmic tail. Techniques known to those skilled in the art are used to define the different domains of influenza virus neuraminidase.

[0059] The stalk region can vary significantly in size and amino acid structure among different influenza A virus subtypes, including N1 and N2 (Blok et al., Biochemistry, 1982, 21:4001-4007). Differences in stalk length are thought to modulate the distance of the enzymatic head region and affect the ability of NA to access sialic acid on cell surface receptors, with shorter stalk regions correlating with reduced sialidase activity (Da Silva et al., J Biol Chem, 2013, 288(1):644-53; McAuley et al., Frontiers in Microbiology, 2019, 10(39)). Despite the variability among the stalk regions of different subtypes, the NA stalk regions also share some structural features, including at least one cysteine ​​residue and a potential glycosylation site. The cysteine ​​residue(s) may participate in the formation of disulfide bonds between NA monomers and aid in the formation of stabilized NA tetramers, whereas the glycosylation sites may contribute to tetramer stabilization (McAuley et al., Frontiers in Microbiology, 2019, 10(39)). For example, a conserved cysteine ​​residue at or near amino acid 78 of N2 is thought to play a role in the tetramer assembly mechanism (Shtyrya et al., Acta Naturae., 2009, 1(2):26-32).

[0060] The enzymatic head region is composed of four monomers. Each monomer in the head forms a conserved six-bladed propeller structure. Each blade has four antiparallel β-sheets stabilized by disulfide bonds and connected by loops of variable length (McAuley et al., Frontiers in Microbiology, 2019, 10(39)). Tetramerization of the monomers is important for the formation of the active site and the synthesis of enzymatically active NAs (Dai et al., J. Virology, 2016, 90(20):9457-70).

[0061] B. Subtype 2 (N2) influenza virus neuraminidase Although the amino acid sequence and length of NA can vary significantly between different influenza A virus NA subtypes, such as N1 and N2, and in particular between the NA stalk regions of different influenza A virus NA subtypes, the length of the amino acid sequence of N2 from different influenza strains is typically about 469 amino acids, with a few strains typically having insertions or deletions of about one or two (or more) amino acids in the head region. When referring to specific amino acid residues in wild-type N2, the specific amino acid residue numbers are based on N2 numbering as understood in the art. The N-terminal cytoplasmic tail typically corresponds to amino acids 1-6 of the wild-type N2 sequence, while the transmembrane region typically corresponds to amino acids 7-35 of the wild-type N2 sequence. For example, within the wild-type NA sequence of the A / PERTH / 16 / 2009 strain (SEQ ID NO: 65), the cytoplasmic tail corresponds to amino acids 1-6 of SEQ ID NO: 65, whereas the transmembrane region corresponds to amino acids 7-35 of SEQ ID NO: 65. The length of the N2 stalk region is typically about 46 amino acids in length, beginning at about amino acid 36 and ending at about amino acid 82 of the wild-type N2 sequence. For example, within the wild-type NA sequence of the A / PERTH / 16 / 2009 strain (SEQ ID NO: 65), the stalk region corresponds to amino acid 36 to about amino acid 82 of SEQ ID NO: 65. However, the exact boundary between the end of the N2 stalk region and the beginning of the N2 head region has not been resolved by x-ray structural analysis.

[0062] 3. Removal of all or substantially all of the stalk region A recombinant N2 was designed that lacks all or substantially all of the cytoplasmic tail, transmembrane region, and stalk region. The recombinant NA does not contain a heterologous tetramerization domain, which the art teaches is important for making soluble recombinant NA proteins (Schmidt et al., PLos ONE, 2011, 6(2):e16284; Da Silva et al., J Biol Chem, 2013, 288(1):644-53; Dai et al., 2016, J. Virology, 90(20):9457-70; Bosch et al., 2010, J. Virology, 84(19):10366-74). However, it was surprisingly discovered that a truncated N2 lacking all or substantially all of the stalk region forms a soluble tetrameric NA when expressed in cells.

[0063] Initial experiments showed that recombinant N2 lacking amino acids 1-74 formed tetrameric NA when expressed in cells. This property was observed in truncated N2 sequences from different strains. When the deletion analysis was extended to include deletion mutants in which smaller and larger portions of the stalk region were removed, it was discovered that recombinant N2 lacking amino acid 1 through at least amino acids 70-82 formed tetrameric NA when expressed in cells. Indeed, it was discovered that in certain cases, N-terminal deletions encompassed the entire stalk region and could extend over several amino acids into the head region and still form soluble tetrameric NA.

[0064] One aspect of the present disclosure is directed to an artificial nucleic acid molecule encoding a modified monomeric influenza virus subtype 2 neuraminidase (N2). In certain embodiments, the modified monomeric N2 lacks all or substantially all of the cytoplasmic tail, transmembrane region, and stalk region, and comprises a head region of N2, where the modified monomeric influenza virus subtype 2 NA does not comprise a heterologous oligomerization domain, and expression of the modified monomeric N2 in a cell results in secretion of a tetrameric NA. In certain embodiments, the modified monomeric N2 comprises a signal peptide and a head region of N2, where all or substantially all of the cytoplasmic tail, transmembrane region, and stalk region of N2 are replaced by a signal peptide, and the modified monomeric N2 does not comprise a heterologous oligomerization domain. Expression of the modified monomeric N2 in a cell results in secretion of a tetrameric NA.

[0065] In some embodiments, the artificial nucleic acid molecule encodes a modified monomer N2, where amino acid 1 through at least amino acids 70-82 of N2 (eg, SEQ ID NOs: 1-115) are replaced by a signal peptide.

[0066] In some embodiments, the artificial nucleic acid molecule encodes a modified monomer N2, in which amino acids 1-70 of N2 (e.g., SEQ ID NOs: 1-115) are replaced by a signal peptide. Such an embodiment is also referred to as dTM71.

[0067] In some embodiments, the artificial nucleic acid molecule encodes a modified monomer N2, where amino acids 1-71 of N2 (e.g., SEQ ID NOs: 1-115) are replaced in the modified monomer N2 by a signal peptide. Such embodiments are also referred to as dTM72.

[0068] In some embodiments, the artificial nucleic acid molecule encodes a modified monomer N2, where amino acids 1-72 of N2 (e.g., SEQ ID NOs: 1-115) are replaced in the modified monomer N2 by a signal peptide. Such an embodiment is also referred to as dTM73.

[0069] In some embodiments, the artificial nucleic acid molecule encodes a modified monomer N2, where amino acids 1-73 of N2 (e.g., SEQ ID NOs: 1-115) are replaced in the modified monomer N2 by a signal peptide. Such embodiments are also referred to as dTM74.

[0070] In some embodiments, the artificial nucleic acid molecule encodes a modified monomer N2, where amino acids 1-74 of N2 (e.g., SEQ ID NOs: 1-115) are replaced in the modified monomer N2 by a signal peptide. Such embodiments are also referred to as dTM75.

[0071] In some embodiments, the artificial nucleic acid molecule encodes a modified monomer N2, where amino acids 1-75 of N2 (e.g., SEQ ID NOs: 1-115) are replaced in the modified monomer N2 by a signal peptide. Such an embodiment is also referred to as dTM76.

[0072] In some embodiments, the artificial nucleic acid molecule encodes a modified monomer N2, where amino acids 1-76 of N2 (e.g., SEQ ID NOs: 1-115) are replaced in the modified monomer N2 by a signal peptide. Such an embodiment is also referred to as dTM77.

[0073] In some embodiments, the artificial nucleic acid molecule encodes a modified monomer N2, where amino acids 1-77 of N2 (e.g., SEQ ID NOs: 1-115) are replaced in the modified monomer N2 by a signal peptide. Such an embodiment is also referred to as dTM78.

[0074] In some embodiments, the artificial nucleic acid molecule encodes a modified monomer N2, where amino acids 1-78 of N2 (e.g., SEQ ID NOs: 1-115) are replaced in the modified monomer N2 by a signal peptide. Such an embodiment is also referred to as dTM79.

[0075] In some embodiments, the artificial nucleic acid molecule encodes a modified monomer N2, where amino acids 1-79 of N2 (e.g., SEQ ID NOs: 1-115) are replaced in the modified monomer N2 by a signal peptide. Such embodiments are also referred to as dTM80.

[0076] In some embodiments, the artificial nucleic acid molecule encodes a modified monomer N2, where amino acids 1-80 of N2 (e.g., SEQ ID NOs: 1-115) are replaced in the modified monomer N2 by a signal peptide. Such an embodiment is also referred to as dTM81.

[0077] In some embodiments, the artificial nucleic acid molecule encodes a modified monomer N2, where amino acids 1-81 of N2 (e.g., SEQ ID NOs: 1-115) are replaced in the modified monomer N2 by a signal peptide. Such an embodiment is also referred to as dTM82.

[0078] In some embodiments, the artificial nucleic acid molecule encodes a modified monomer N2, where amino acids 1-82 of N2 (e.g., SEQ ID NOs: 1-115) are replaced in the modified monomer N2 by a signal peptide. Such an embodiment is also referred to as dTM83.

[0079] One embodiment of the present disclosure is directed to a tetrameric NA comprising four copies of modified monomeric N2.As discussed above, typically, the signal peptide is usually cleaved during post-translational processing, so that the secreted polypeptide does not contain the signal peptide.Thus, the modified monomeric N2, which is a part of the tetrameric NA, typically comprises only the head region of influenza virus subtype 2 NA, and does not comprise 1) all or substantially all of the cytoplasmic tail region, transmembrane region, and stalk region of influenza virus subtype 2 NA, and 2) heterologous oligomerization domain.

[0080] In some embodiments, the modified monomer N2 lacks all of the stalk region of N2, or lacks amino acid 1 through at least amino acids 70-82 of N2.

[0081] In some embodiments, the modified monomer N2 lacks amino acids 1-70 of N2 (e.g., SEQ ID NOs: 1-115). In addition to such embodiments, the tetramer NA formed by this modified monomer N2 is also referred to as dTM71.

[0082] In some embodiments, the modified monomer N2 lacks amino acids 1-71 of N2 (e.g., SEQ ID NOs: 1-115). In addition to such embodiments, the tetramer NA formed by this modified monomer N2 is also referred to as dTM72.

[0083] In some embodiments, the modified monomer N2 lacks amino acids 1-72 of N2 (e.g., SEQ ID NOs: 1-115). In addition to such embodiments, the tetramer NA formed by this modified monomer N2 is also referred to as dTM73.

[0084] In some embodiments, the modified monomer N2 lacks amino acids 1-73 of N2 (e.g., SEQ ID NOs: 1-115). In addition to such embodiments, the tetramer NA formed by this modified monomer N2 is also referred to as dTM74.

[0085] In some embodiments, the modified monomer N2 lacks amino acids 1-74 of N2 (e.g., SEQ ID NOs: 1-115). In addition to such embodiments, the tetramer NA formed by this modified monomer N2 is also referred to as dTM75.

[0086] In some embodiments, the modified monomer N2 lacks amino acids 1-75 of N2 (e.g., SEQ ID NOs: 1-115). In addition to such embodiments, the tetramer NA formed by this modified monomer N2 is also referred to as dTM76.

[0087] In some embodiments, the modified monomer N2 lacks amino acids 1-76 of N2 (e.g., SEQ ID NOs: 1-115). In addition to such embodiments, the tetramer NA formed by this modified monomer N2 is also referred to as dTM77.

[0088] In some embodiments, the modified monomer N2 lacks amino acids 1-77 of N2 (e.g., SEQ ID NOs: 1-115). In addition to such embodiments, the tetramer NA formed by this modified monomer N2 is also referred to as dTM78.

[0089] In some embodiments, the modified monomer N2 lacks amino acids 1-78 of N2 (e.g., SEQ ID NOs: 1-115). In addition to such embodiments, the tetramer NA formed by this modified monomer N2 is also referred to as dTM79.

[0090] In some embodiments, the modified monomer N2 lacks amino acids 1-79 of N2 (e.g., SEQ ID NOs: 1-115). In addition to such embodiments, the tetramer NA formed by this modified monomer N2 is also referred to as dTM80.

[0091] In some embodiments, the modified monomer N2 lacks amino acids 1-80 of N2 (e.g., SEQ ID NOs: 1-115). In addition to such embodiments, the tetramer NA formed by this modified monomer N2 is also referred to as dTM81.

[0092] In some embodiments, the modified monomer N2 lacks amino acids 1-81 of N2 (e.g., SEQ ID NOs: 1-115). In addition to such embodiments, the tetramer NA formed by this modified monomer N2 is also referred to as dTM82.

[0093] In some embodiments, the modified monomer N2 lacks amino acids 1-82 of N2 (e.g., SEQ ID NOs: 1-115). In addition to such embodiments, the tetramer NA formed by this modified monomer N2 is also referred to as dTM83.

[0094] Typically, in modified monomeric N2, the head region of N2 comprises the full length head region of wild type N2, including, for example, amino acid 83 to the terminal amino acid of wild type N2, which for most N2 strains is amino acid 469, although a few strains typically have insertions or deletions of about one or two (or more) amino acids in the head region. In some embodiments, so long as the modified monomeric N2 is capable of forming a tetrameric NA, the N-terminal NA truncation may extend beyond the stalk region into the head region when expressed in a cell. For example, as shown in the Examples, modified monomeric N2 in which all of the stalk region and several amino acids at the beginning of the head region have been deleted may form a tetrameric NA when expressed in a cell. In some embodiments, the modified monomer N2 lacks the cytoplasmic tail region, the transmembrane region, all of the stalk region of N2 (e.g., SEQ ID NOs: 1-115), and the first 1-5 amino acids of the head region, or an artificial nucleic acid molecule encodes such a modified monomer N2. In some embodiments, the modified monomer N2 lacks amino acid 1 to the first amino acid or the second amino acid of the head region, or an artificial nucleic acid molecule encodes such a modified monomer N2. For example, the modified monomer N2 can lack amino acids 1-83 of influenza virus subtype 2 NA (e.g., SEQ ID NOs: 1-115), or an artificial nucleic acid molecule encodes such a modified monomer N2 (also referred to as dTM84). For example, the modified monomer N2 can lack amino acids 1-84 of influenza virus subtype 2 NA (e.g., SEQ ID NOs: 1-115), or an artificial nucleic acid molecule encodes such a modified monomer N2 (also referred to as dTM85).

[0095] As mentioned above, the modified monomer N2 may lack amino acids 1-74 of N2 (e.g., SEQ ID NOs: 1-115). In addition to such embodiments, the tetrameric NA formed by this modified monomer N2 is also referred to as dTM75. Representative dTM75 constructs include, but are not limited to, any one of SEQ ID NOs: 117-131.

[0096] 4. Signal peptide The wild-type NA protein is a membrane-bound protein that contains a transmembrane domain. To create a soluble NA protein, the transmembrane domain can be deleted and a signal peptide can be added. The signal peptide targets the recombinant NA protein to the secretory pathway so that the recombinant NA protein is secreted from the host cell in which the recombinant NA is expressed. When the modified monomeric N2 nucleic acid is translated into a polypeptide in the host cell, the polypeptide contains a signal peptide. However, during post-translational processing, the signal peptide is cleaved so that the secreted polypeptide no longer contains the signal peptide. Thus, the nucleic acid construct described herein can code for a signal peptide to target the modified monomeric N2 to the secretory pathway, but the soluble tetrameric NA obtained from the host cell expressing the nucleic acid construct is composed of four modified N2 monomers that no longer contain a signal peptide.

[0097] Any signal peptide or nucleic acid encoding the same is used in the recombinant NA construct. The signal peptide shall be a signal peptide sequence recognized by the host cell used to express the recombinant N2. The signal peptide may be a heterologous signal peptide from a source other than influenza virus, or may be a signal peptide from influenza virus, such as the signal peptide for hemagglutinin. In some embodiments, the signal peptide is a mammalian signal peptide, including signal peptides that direct the secretion of the modified NA, such as signal peptides from CD5, immunoglobulin kappa light chain, serum albumin, azurocidin, trypsinogen, interleukin 2, and prolactin, or derivatives thereof. In some embodiments, the signal peptide is a human signal peptide (Kober et al., Biotechnology & Bioengineering, 2012, 110(4):1164-63; Dalton et al., Protein Sci., 2014, 23(5):517-25). In some embodiments, the signal peptide is a CD5 signal peptide. In some embodiments, the CD5 signal peptide comprises the amino acid sequence MPMGSLQPLATLYLLGMLVASVLS (SEQ ID NO: 132), or MPMGSLQPLATLYLLGMLVASCLG (SEQ ID NO: 133).

[0098] 5. Linker sequence In some embodiments, the modified monomer N2 may include an appropriate linker sequence that links the signal peptide to the truncated NA. In this way, the linker sequence may help separate the head region from the rest of the modified N2. For example, in some embodiments, where the entire stalk region is deleted, the linker sequence may link the signal peptide to the head region. In other embodiments, where substantially all of the stalk region is deleted, the linker sequence may link the signal peptide to the stalk region. When the modified NA contains a protein tag sequence, the linker sequence is inserted at the N-terminus and / or C-terminus of the protein tag sequence. The linker sequence is not part of the naturally occurring NA amino acid sequence. In some embodiments, the linker sequence includes glycine and / or serine residues, and optionally includes one or more alanine residues. In some embodiments, the linker sequence is about 2-10 amino acids in length, including 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, which helps provide flexibility between different regions or portions of the recombinant NA. In some embodiments, the linker sequence comprises or consists of the amino acid sequence GSG, AGS, or AGSG (SEQ ID NO: 134). In some embodiments, the linker sequence comprises or consists of the amino acid sequence GS or SG.

[0099] Any other linker sequence known in the art may be used in the recombinant NA construct. Other exemplary peptide linkers are those described in U.S. Patent Nos. 4,751,180, 4,935,233, and 5,073,627, each of which is incorporated herein by reference in its entirety. Using conventional techniques, the DNA sequence encoding the desired linker sequence may be inserted, for example, between the DNA sequence encoding the signal peptide and the DNA sequence encoding the head region in an embodiment in which the entire stalk region is deleted, and in the same reading frame as the DNA sequence encoding the head region, or between the terminal amino acid of the stalk region and the head region in an embodiment in which substantially the entire stalk region is deleted.

[0100] 6. Protein Tag Sequencing The modified monomer N2 may contain an appropriate protein tag sequence that is used to purify or otherwise identify the modified N2. For example, in some embodiments, the modified N2 contains a histidine tag sequence. Any protein tag sequence known in the art may be used in the recombinant NA construct.

[0101] 7. Protease cleavage site The modified monomeric NA may also include an appropriate protease cleavage site. The protease cleavage site is used to modify the recombinant NA by facilitating the removal of sequences (e.g., protein tag sequences, linker sequences, etc.) from the final soluble NA protein after the recombinant NA is expressed in a host cell. Any protease cleavage site known in the art may be used within the recombinant NA construct.

[0102] 8. Tetramer NA It was found that when nucleic acids encoding modified monomeric N2 described herein are expressed in cells, soluble tetrameric NA can be detected in the cell supernatant and purified in high yields. Although not all N2 strains produced detectable amounts of soluble tetrameric NA, the majority of the tested N2 strains produced detectable amounts of soluble tetrameric NA, indicating that this truncated stalk design strategy is broadly applicable to NA proteins from various N2 influenza strains. Certain N2 strains, and certain stalk deletion mutants of specific strains, produce high yields of soluble tetrameric NA when expressed in cells. As shown in the examples and discussed in more detail below, high-throughput screening can be used to easily identify strains that produce soluble tetrameric NA, as well as to quantify the amount of soluble tetrameric NA produced. The same high-throughput screen is also used to examine engineered NA stalk truncation mutants, varying the length of the stalk region, to identify mutants that produce soluble tetrameric NA or the highest amount of soluble tetrameric NA, as demonstrated in the examples.

[0103] The majority of soluble tetrameric NAs formed from modified monomeric N2 constructs described herein retain neuraminidase enzyme activity. In some embodiments, the soluble tetrameric NAs described herein have neuraminidase activity. Neuraminidase activity is measured using techniques known in the art, including, for example, MUNANA assay or NA-STAR® assay (ThermoFisher Scientific, Waltham, MA). In the MUNANA assay, 2'-(4-methylumbelliferyl)-alpha-DN-acetylneuraminic acid (MUNANA) was used as a substrate. Any enzymatically active neuraminidase contained in the sample cleaves the MUNANA substrate and releases the fluorescent compound, 4-methylumbelliferone (4-MU). Thus, the amount of neuraminidase activity in the test sample correlates with the amount of 4-MU released. In some embodiments, the soluble tetrameric NA described herein has an activity in the range of 1-25 moles per minute per μg as measured by the MUNANA assay.

[0104] In order to determine the neuraminidase activity of the soluble tetrameric NA of the present disclosure, the MUNANA assay shall be carried out using the following conditions: soluble tetrameric NA shall be mixed with buffer [33.3 mM 2-(N-morpholino)ethanesulfonic acid (MES, pH 6.5), 4 mM CaCl2, 50 mM BSA] and substrate (100 μM MUNANA) and incubated at 37 ° C with shaking for 1 hour; the reaction shall be stopped by adding alkaline pH solution (0.2 M Na2CO3); the fluorescence intensity shall be measured using excitation and emission wavelengths of 355 and 460 nm, respectively; and the enzyme activity shall be calculated against the 4MU reference. If necessary, an equivalent assay shall be used to measure the enzyme activity of neuraminidase.

[0105] As described in the Examples, a novel TAMIFLU® binding assay was developed to detect tetrameric NA. The TAMIFLU® screening assay is also described in more detail in U.S. Application No. 63 / 231,795, entitled "METHODS AND RELATED ASPECTS OF DETECTING AND PURIFYING INFLUENZA NEURAMINIDASE," filed August 11, 2021, and incorporated herein by reference in its entirety. Briefly, oseltamivir phosphate (TAMIFLU®) is a competitive inhibitor of the enzymatic activity of NA. In this application, the terms oseltamivir phosphate and TAMIFLU® are used interchangeably. The substrate of oseltamivir is specific for the enzymatic site of the tetrameric NA head. In contrast, the monomeric NA extracellular domain mutant, which is enzymatically inactive, does not bind to oseltamivir phosphate. Thus, the binding level of recombinant NA in the TAMIFLU® binding assay is proportional to the concentration of recombinant NA assembled as a tetramer and can be measured in μg / mL. For example, the soluble tetrameric NA described herein can have a TAMIFLU® binding value of at least 5 μg / mL, such as at least 20 μg / mL, at least 50 μg / mL, or at least 100 μg / mL. The soluble tetrameric NA described herein can have a TAMIFLU® binding value of 40-1000 μg / mL, 50-800 μg / mL, 100-800 μg / mL, 100-500 μg / mL, or 100-250 μg / mL. Alternatively, the binding level of recombinant NA in the TAMIFLU® binding assay is proportional to the concentration of recombinant NA, which can be measured in units relative to the binding of a positive control that is a tetrameric NA molecule, such as a modified influenza N2 that assembles as a tetramer and contains a heterologous tetramerization domain, including the positive control used in this application, such as the modified A / Singapore / INFIMH160019 / 2016 NA, which contains a full-length tetrabrachion tetramerization domain.For example, the soluble tetrameric NA described herein may have a TAMIFLU® binding value of about 0.5 to 10 units, such as about 2 to 10 or about 5 to 10 relative units, compared to the binding of modified A / Singapore / INFIMH160019 / 2016 NA (SEQ ID NO: 116), which contains a full-length tetrabrachion tetramerization domain, to TAMIFLU®.

[0106] For the purpose of determining the binding activity of the soluble tetrameric NA of the present disclosure to TAMIFLU®, the TAMIFLU® binding assay shall be performed using the following conditions: capture oseltamivir phosphate-biotin conjugate (5-10 μg / ml in 1× KB (Kinetic Buffer) buffer (0.1% BSA + 0.02% Tween in PBS)) onto the surface of a streptavidin-coated biosensor (e.g., High Precision Streptavidin (SAX) Dip and Read Biosensor; model number: 18-51182); immerse the oseltamivir phosphate-bound biosensor into wells containing 2-fold serial dilutions of recombinant NA samples (0.16-40 ug / ml in 1× KB); and measure the binding activity of the NAs using an Octet instrument (ForteBio, Molecular The binding kinetics of recombinant NA to oseltamivir phosphate was measured using BioLayer Interferometry (BLI) on a 100-well plate (Microbial Devices, LLC). When necessary, a comparable assay was used to measure binding to TAMIFLU®.

[0107] 10.N2 strain and NA sequence The modified monomeric influenza virus NA disclosed herein is derived from subtype 2 (N2) influenza virus, i.e., modified monomeric N2. As discussed elsewhere, current seasonal influenza vaccines must be administered annually and require annual renewal to accommodate mutations (antigenic drift) in HA and NA proteins and to match rapid evolution and emerging virus strains. HA induces virus-neutralizing antibodies and evolves more rapidly than NA. NA, which is more genetically stable, may be used as a supplement in different seasonal vaccines, thereby increasing efficacy. In other words, recombinant NA protein (or protein delivered by nucleic acid vaccine) may not require seasonal renewal as frequently as HA. The strategy disclosed in this application for generating truncated recombinant NA, which forms tetrameric NA when expressed in cells, is used to generate modified N2 from such emerging N2 influenza strains, including future N2 standard of care (SOC) strains, or, optionally, from past SOC or non-SOC strains.

[0108] The strategy disclosed in this application for producing a truncated recombinant NA that forms a tetrameric NA when expressed in a cell is used to generate a modified N2 from an engineered N2 protein, such as an engineered N2 protein obtained using the molecular modeling methods disclosed in U.S. Application Publication No. 2018 / 0298063, U.S. Application Publication No. 2019 / 0161519, and U.S. Application Publication No. 2021 / 0046176, which are incorporated herein by reference in their entireties.

[0109] The strategy disclosed in this application for generating truncated recombinant NAs that form tetrameric NAs when expressed in cells is also used to generate modified N2 from existing wild-type N2 influenza strains, including standard of care (SOC) N2 strains or non-SOC N2 strains. In some embodiments, N2 is derived from an influenza A virus strain. In some embodiments, N2 is derived from an A / (H3N2) strain. In some embodiments, the influenza A virus strain is the following strains: A / Perth / 16 / 2009, A / Kansas / 14 / 2017, A / Belgium / 4217 / 2015, A / Singapore / INFIMH-16-0019 / 2016, A / Switzerland / 8060 / 2017, A / Nevada / 32 / 2013, A / Yamagata / 62 / 1993, A / Nigeria / 120 / 2017 ... 4, A / Bangkok / 1 / 1979, A / Albany / 42 / 1975, A / Washington / 60 / 2014, A / HongKong / CUHK13510 / 2001, A / Marrakech / 79 / 2014, A / HongKong / 1774 / 99, A / Illinois / 34 / 2012, A / Kannur / MCVR5404 / 2010, or A / Peru / 3216 / 2016. The sequence number of the NA, the amino acids corresponding to the predicted head region, the GISAID reference number, and the isolate ID for each of these strains are provided in the table below.

[0110] [Table 1]

[0111] In some embodiments, the influenza A virus strain is one of the following strains: A / Ohio / 13 / 2017, A / Nanjing / 1663 / 2010, A / Fukuoka / DS7282 / 2017, A / Ohio / 62 / 2012, A / Minnesota / 11 / 2010, A / Utah / 11 / 2011, A / Hokkaido / 10H079 / 2011, A / Ishikawa / DS7157 / 2016, A / Gambia / G0071436 / 2012, A / Kagawa / DS722 / 2016, A / South_Australia / 85 / 2018, A / Victoria / 361 / 2011, A / Tokyo / DS7334 / 2017, A / Hochimi nh / 4596 / 2010, A / Kagawa / DS7144 / 2016, A / Peru / 4617 / 2017, A / Switzerland / 9715293 / 2013, A / Tokyo / UTSK1 / 2007, A / We sternAustralia / 13 / 2001, A / Tasmania / 1018 / 2015, A / Sweden / 3 / 2017, A / Kansas / 13 / 2009, A / Tokyo / DS763 / 2016, A / New castle / 67 / 2016, A / SouthAustralia / 34 / 2019, A / Stockholm / 32 / 2014, A / Stockholm / 14 / 2012, A / Stockholm / 15 / 2014, A A / Guangxigangbei / 190 / 2019, A / Xinjiangtianshan / 1411 / 2012, A / NewYork / 654 / 1994, A / Netherlands / 620 / 1989, A / NewYork / 758 / 1993, A / Catalonia / 9503S / 2017, A / Paris / 2379 / 2014, or A / Heilongjiangxiangyang / 1134 / 2011. The sequence number of the NA, the amino acid corresponding to the predicted head region, the GISAID reference number, and the isolate ID for each of these strains are provided in the table below.

[0112] [Table 2-1] [Table 2-2]

[0113] Influenza A strains have also been identified in the following: A / TEXAS / 50 / 2012 (SEQ ID NO: 87), A / PORTOALEGRE / LACENRS2376 / 2014 (SEQ ID NO: 69), A / MEMPHIS / 18 / 1978 (SEQ ID NO: 49), A / BELGIUM / 4217 / 2015 (SEQ ID NO: 5), A / TAIWAN / 1 / 1969 (SEQ ID NO: 81), A / SIENA / 3 / 1991 (SEQ ID NO: 71), A / BRISBANE / 273 / 2016 (SEQ ID NO: 7), A / ONTARIO / RV3236 / 2016 (SEQ ID NO: 63), A / TEHRAN / 996 / 2012 (SEQ ID NO: 85), A / ANKARA / 2396 / 2015 (SEQ ID NO: 3), A / KANSAS / 14 / 2017 (SEQ ID NO: 45), A / TEXAS / 71 / 2017 (SEQ ID NO: 88), A / HONGKONG / 4801 / 2014 (SEQ ID NO: 30), A / HONGKONG / 3089 / 2017 (SEQ ID NO: 29), A / FUKUOKA / DS729 / 2016 (SEQ ID NO: 12), A / TOKUSHIMA / DS5288 / 2015 (SEQ ID NO: 89), A / HELSINKI / 823 / 2013 (SEQ ID NO: 22), A / ALASKA / 25 1 / 2015 (SEQ ID NO: 1), A / HATAY / 4990 / 2016 (SEQ ID NO: 20), A / HANOI / ELI15597 / 2015 (SEQ ID NO: 19), A / MISSISSIPPI / 1 / 1985 (SEQ ID NO: 51), A / INDIANA / 18 / 2017 (SEQ ID NO: 36), A / POLAND / 19B / 2017 (SEQ ID NO: 68), A / TENNESSEE / 18 / 2017 (SEQ ID NO: 86), A / HONGKONG / 1774 / 1999 (SEQ ID NO: 28), A / DAKAR / 14 / 2014 (SEQ ID NO: 10), A / SAUDIARABIA / 21 / 1 999 (SEQ ID NO: 70), A / GIFU / DS7388 / 2017 (SEQ ID NO: 15), A / NEWYORK / 581 / 1997 (SEQ ID NO: 57), A / HELSINKI / 941 / 2013 (SEQ ID NO: 23), A / ISHIKAWA / DS7294 / 2017 (SEQ ID NO: 39), A / GUANGDONGDUANZHOU / 1227 / 2017 (SEQ ID NO: 16), A / AUCKLAND / 5 / 1996 (SEQ ID NO: 4), A / ISHIKAWA / DS7215 / 2016 (SEQ ID NO: 38), A / WESTVIRGINIA / 17 / 2012 (SEQ ID NO: 98),A / SINGAPORE / INFIMH160019 / 2016 (SEQ ID NO: 72), A / KAGAWA / DS7115 / 2016 (SEQ ID NO: 42), A / BILTHOVEN / 21801 / 1971 (SEQ ID NO: 6), A / FUKUOKA / DS72 / 2016 (SEQ ID NO: 11), A / MORAMANGA / 1907 / 2017 (SEQ ID NO: 52), A / HUNAN / 01 / 2014 (SEQ ID NO: 33), A / WISCONSIN / 16 / 2015 (SEQ ID NO: 100), A / CANBERRA / 13 / 2015 (SEQ ID NO: 8), A / HONGKONG / 107 / 1971 (SEQ ID NO: 27), A / LYON / 1242 / 2000 (SEQ ID NO: 48), A / NAGANO / 2153 / 2017 (SEQ ID NO: 53), A / NANJING / 49 / 1977 (SEQ ID NO: 54), A / ALBANY / 6 / 1970 (SEQ ID NO: 2), A / KAGAWA / DS769 / 201 6 (SEQ ID NO: 41), A / TAIWAN / 4183 / 2004 (SEQ ID NO: 83), A / KOREA / KUMCGR570 / 2011 (SEQ ID NO: 47), A / SYDNEY / 24 / 2015 (SEQ ID NO: 80), A / KOREA / KUMCGR99 / 2011 (SEQ ID NO: 46), A / TOKYO / DS7277 / 2017 (SEQ ID NO: 91), A / HONGKONG / CUHK18194 / 1998 (SEQ ID NO: 31), A / GUN It may also be one of MA / DS7107 / 2016 (SEQ ID NO: 18), A / HONGKONG / 8 / 1968 (SEQ ID NO: 26), A / TOKYO / DS7448 / 2017 (SEQ ID NO: 93), A / PERTH / 61 / 2015 (SEQ ID NO: 65), A / TW / 875 / 2004 (SEQ ID NO: 95), A / TAIWAN / 2332 / 2001 (SEQ ID NO: 82), A / INDIA / C058671 / 2005 (SEQ ID NO: 34).

[0114] The modified monomer N2 disclosed herein may comprise a portion of wild-type N2 (e.g., a portion of the head region or C-terminal region of the stalk region of wild-type N2). It is also possible to introduce one or more mutations into the head region and / or C-terminal region of the stalk region of wild-type N2, so long as the modified monomer N2 retains the ability to form a tetrameric NA when expressed in a cell. In certain embodiments, the head region and / or C-terminal region of the stalk region of the modified monomer N2 contains one, two, three, four, or five mutations compared to the head region and / or C-terminal region of the stalk region of wild-type N2. In certain embodiments, the modified monomer N2 comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the head region of wild-type N2. In certain embodiments, the modified monomer N2 comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the head region of any of the amino acid sequences of SEQ ID NOs: 1-115.

[0115] C. Composition In certain aspects, the present disclosure provides a vaccine composition comprising a tetrameric neuraminidase comprising four copies of the modified monomeric N2 described herein, or an artificial nucleic acid molecule encoding the modified monomeric N2 described herein. Nucleic acid vaccines based on plasmid DNA, vectors, or RNA (e.g., mRNA) are known in the art and have been effectively used against infectious diseases, such as viral infections. Vectors include, but are not limited to, recombinant expression vectors in vivo, such as polynucleotide vectors or plasmids (EP-A2-1001025; Chaudhuri P, Res.Vet.Sci.2001,70:255-6), adenovirus vectors, alphavirus vectors, flavivirus vectors, poxvirus vectors such as fowlpox virus vectors (U.S. Pat. Nos. 5,174,993; 5,505,941; and 5,766,599), or viral vectors, including but not limited to canarypox virus vectors (U.S. Pat. No. 5,756,103). RNA (e.g., mRNA) vaccines are formulated with lipid nanoparticles. The artificial nucleic acids encoding the modified monomer N2 described herein are stabilized by one or more chemical modifications to the nucleic acid molecule, including one or more backbone, sugar, and / or base modifications known in the art, including, for example, the modifications disclosed in PCT Publication No. WO2017 / 140905, which is incorporated by reference in its entirety.

[0116] In certain embodiments, the present disclosure provides a vaccine composition comprising the mRNA molecule encoding the modified monomeric N2 described herein.In some embodiments, the mRNA molecule is encapsulated in lipid nanoparticles (LNPs).The LNPs for delivering mRNA vaccines are known in the art, including the LNPs disclosed in, for example, US Patent Application Nos. 63 / 110,965 and 63 / 212,523, which are incorporated herein by reference in their entirety.

[0117] The vaccine composition may also further comprise an adjuvant.

[0118] As used herein, the term "adjuvant" refers to a substance or vehicle that nonspecifically enhances the immune response to an antigen. Adjuvants can include suspensions of minerals (e.g., alum, which are aluminum salts including aluminum hydroxide / oxyhydroxide (AlOOH), aluminum phosphate (AlPO4), aluminum hydroxyphosphate sulfate (AAHS), and / or aluminum potassium sulfate) to which antigens are adsorbed; or water-in-oil emulsions (e.g., Freund's incomplete adjuvant) in which antigen solutions are emulsified in mineral oils, optionally with killed acid-fast bacteria incorporated to further enhance antigenicity (Freund's complete adjuvant). Immunostimulatory oligonucleotides (such as those containing CpG motifs) are also used as adjuvants (see, for example, 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; and 6,429,199).Adjuvants also include biological molecules such as lipids and costimulatory molecules. Exemplary biological adjuvants include AS04 (Didierlaurent, A. M. et al., J. Immunol., 2009, 183:6186-6197), IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, and 41 BBL.

[0119] In certain embodiments, the adjuvant is a squalene-based adjuvant, comprising an oil-in-water adjuvant emulsion, comprising at least squalene, an aqueous solvent, a hydrophilic non-ionic surfactant polyoxyethylene alkyl ether, and a hydrophobic non-ionic surfactant. In certain embodiments, the emulsion is thermoreversible, and optionally, 90% of the population by volume of oil droplets is less than 200 nm in size.

[0120] In certain embodiments, the polyoxyethylene alkyl ether has the formula: CH3-(CH2) x -(O-CH2-CH2) n -OH, where n is 10 to 60 and x is 11 to 17. In a particular embodiment, the surfactant polyoxyethylene alkyl ether is polyoxyethylene (12) cetostearyl ether.

[0121] In certain embodiments, 90% of the population by volume of oil droplets are less than 160 nm in size. In certain embodiments, 90% of the population by volume of oil droplets are less than 150 nm in size. In certain embodiments, 50% of the population by volume of oil droplets are less than 100 nm in size. In certain embodiments, 50% of the population by volume of oil droplets are less than 90 nm in size.

[0122] In certain embodiments, the adjuvant further comprises at least one alditol, including, but not limited to, glycerol, erythritol, xylitol, sorbitol, and mannitol.

[0123] In certain embodiments, the hydrophilic nonionic surfactant has a hydrophilic / lipophilic balance (HLB) of 10 or more. In certain embodiments, the hydrophobic nonionic surfactant has an HLB of less than 9. In certain embodiments, the hydrophilic nonionic surfactant has an HLB of 10 or more and the hydrophobic nonionic surfactant has an HLB of less than 9.

[0124] In certain embodiments, the hydrophobic non-ionic surfactant is a sorbitan ester surfactant, such as a sorbitan monooleate surfactant, or a mannide ester surfactant. In certain embodiments, the amount of squalene is between 5-45%. In certain embodiments, the amount of the surfactant polyoxyethylene alkyl ether is between 0.9-9%. In certain embodiments, the amount of the hydrophobic non-ionic surfactant is between 0.7-7%. In certain embodiments, the adjuvant comprises i) 32.5% squalene, ii) 6.18% polyoxyethylene (12) cetostearyl ether, iii) 4.82% sorbitan monooleate, and iv) 6% mannitol.

[0125] In certain embodiments, the adjuvant further comprises a cryoprotectant, such as an alkyl polyglycoside and / or a sugar, in particular dodecyl maltoside and / or sucrose.

[0126] In certain embodiments, the adjuvant comprises AF03, as described in Klucker et al., J. Pharm. Sci. 2012, 101(12):4490-500, which is incorporated by reference in its entirety.

[0127] In addition to the artificial nucleic acid molecule encoding the tetrameric neuraminidase or modified monomeric N2 and an appropriate adjuvant, the vaccine composition may also further comprise one or more pharma-ceutically acceptable excipients. In general, the nature of the excipient will depend on the particular mode of administration to be used. For example, parenteral formulations usually contain injectable fluids that contain pharma-ceutically and physiologically acceptable fluids, such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, or the like, as a vehicle. For solid compositions (e.g., in the form of powders, pills, tablets, or capsules), conventional non-toxic solid carriers may include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to a biologically neutral carrier, the vaccine composition to be administered may contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pharma- ceutically acceptable salts for adjusting osmotic pressure, preservatives, stabilizers, buffers, sugars, amino acids, and pH buffering agents, such as, for example, sodium acetate or sorbitan monolaurate.

[0128] Typically, the vaccine composition is a sterile solution formulated for parenteral administration, such as intravenous, subcutaneous, intraperitoneal, intradermal, or intramuscular administration. The vaccine composition is also formulated for intranasal or inhalation administration. The vaccine composition is also formulated for any other intended route of administration.

[0129] In some embodiments, the vaccine composition is formulated for intradermal injection, intranasal administration, or intramuscular injection. In some embodiments, the injection is prepared in conventional form, as a solution or suspension, a solid form suitable for solution or suspension in liquid before injection, or as an emulsion. In some embodiments, the injectable solution and suspension are prepared from sterile powders or granules. General considerations in the formulation and manufacture of pharmaceutical agents for administration by these routes can be found, for example, in "Remington's Pharmaceutical Sciences", 19th Edition, Mack Publishing Co., Easton, PA, 1995, which is incorporated herein by reference. Currently, oral spray or aerosol routes or nasal spray or aerosol routes (e.g., by inhalation) are most commonly used to deliver therapeutic agents directly to the lungs and respiratory system. In some embodiments, the vaccine composition is administered using a device that delivers a metered dose of the vaccine composition. The device suitable for use in the delivery of intradermal pharmaceutical compositions described herein includes short needle devices such as those described in U.S. Patent No. 4,886,499, U.S. Patent No. 5,190,521, U.S. Patent No. 5,328,483, U.S. Patent No. 5,527,288, U.S. Patent No. 4,270,537, U.S. Patent No. 5,015,235, U.S. Patent No. 5,141,496, U.S. Patent No. 5,417,662 (all of which are incorporated herein by reference).Intradermal compositions can also be administered by devices that limit the effective penetration length of the needle into the skin, such as those described in WO1999 / 34850, which is incorporated herein by reference, and their functional equivalents.Jet injection devices are also suitable, which deliver liquid vaccines to the dermis through a liquid jet injector or through a needle, which creates a jet that penetrates the stratum corneum and reaches the dermis.Regarding jet injection devices, see, for example, U.S. Pat. Nos. 5,480,381, 5,599,302, 5,334,144, 5,993,412, 5,649,912, 5,569,189, 5,704,911, 5,383,851, 5,893,397, 5,466,220, and 5,339,163. US Patent No. 5,312,335, US Patent No. 5,503,627, US Patent No. 5,064,413, US Patent No. 5,520,639, US Patent No. 4,596,556, US Patent No. 4,790,824, US Patent No. 4,941,880, US Patent No. 4,940,460, WO1997 / 37705, and WO1997 / 13537 (all of which are incorporated herein by reference).Also suitable is ballistic powder / particle delivery device, which uses high pressure gas to accelerate the vaccine in powder form through the outer layer of the skin to the dermis.In addition, in the classical Mantoux method of intradermal administration, a conventional syringe may also be used.

[0130] Preparations for parenteral administration typically include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as electrolyte replenishers based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as, for example, antimicrobial agents, antioxidants, chelating agents, and inert gases.

[0131] The vaccine composition may further comprise an influenza virus hemagglutinin protein. In certain embodiments, the influenza virus hemagglutinin and the modified monomeric influenza virus subtype 2 neuraminidase are derived from different influenza virus strains. In certain embodiments, the influenza virus hemagglutinin and the modified monomeric influenza virus subtype 2 neuraminidase are derived from different influenza A virus strains. In certain embodiments, the influenza virus hemagglutinin and the modified monomeric influenza virus subtype 2 neuraminidase are derived from different A / (H3N2) virus strains.

[0132] D. Nucleic Acids, Cloning, and Expression Systems The present disclosure further provides artificial nucleic acid molecules that code for the disclosed modified monomer N2. The nucleic acid may comprise DNA, may comprise RNA, may be fully synthetic or recombinant, or may be partially synthetic or recombinant. Reference to a nucleotide sequence explicitly stated herein encompasses DNA molecules with the specified sequence, but also RNA molecules with the specified sequence, in which T is replaced by U, or a derivative thereof, such as pseudouridine, unless the context requires otherwise. Other nucleotide derivatives or modified nucleotides may also be incorporated into artificial nucleic acid molecules that code for the disclosed modified monomer N2.

[0133] The present disclosure also provides constructs in the form of vectors (e.g., plasmids, phagemids, cosmids, transcription or expression cassettes, artificial chromosomes, etc.) that contain artificial nucleic acid molecules encoding modified monomeric N2. The present disclosure further provides host cells that contain one or more of the above constructs.

[0134] Also provided are methods for producing modified monomeric N2 encoded by these artificial nucleic acid molecules. Modified N2 polypeptides can be produced using recombinant methods. Recombinant protein production and expression is well known in the art and is carried out using routine procedures such as those disclosed in Sambrook et al., "Molecular Cloning: A Laboratory Manual" (4th ed., 2012), Cold Spring Harbor Press. For example, expression of modified N2 polypeptides is achieved by culturing host cells containing artificial nucleic acid molecules encoding modified monomeric N2 polypeptides under appropriate conditions. Thus, a method for producing tetrameric NAs can include culturing host cells in a cell culture medium, the host cells containing artificial nucleic acids encoding modified monomeric N2, and expressing modified N2 in the host cells, such that modified N2 is secreted from the host cells as soluble tetrameric NAs. After production by expression, the tetrameric NAs are isolated and / or purified using any suitable technique and then used as needed.

[0135] Systems for cloning and expressing polypeptides in a variety of different host cells are well known in the art. Any protein expression system (e.g., stable or transient) that is compatible with the constructs disclosed in this application can be used to produce the modified N2 described herein.

[0136] Suitable vectors can be chosen or constructed so that they contain appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes, and other sequences as appropriate.

[0137] A further aspect of the disclosure provides a host cell comprising the artificial nucleic acid molecule disclosed herein. Yet a further aspect provides a method comprising the step of introducing such an artificial nucleic acid molecule into a host cell. The introduction can utilize any available technique. For eukaryotic cells, suitable techniques can include calcium phosphate transfection, DEAE-dextran method, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses, such as vaccinia or baculovirus for insect cells. For bacterial cells, suitable techniques can include calcium chloride transformation, electroporation, and transfection using bacteriophages. These techniques are well known in the art (see, for example, "Current Protocols in Molecular Biology", Ausubel et al., eds., John Wiley & Sons, 2010). The introduction of DNA is followed by a selection method (e.g., antibiotic resistance) to select cells containing the vector.

[0138] The host cell can be a plant cell, a yeast cell, or an animal cell. Animal cells include non-vertebrate (e.g., insect cells), non-mammalian vertebrates (e.g., birds, reptiles, and amphibians), and mammalian cells. In one embodiment, the host cell is a mammalian cell. Examples of mammalian cells include, but are not limited to, COS-7 cells, HEK293 cells; baby hamster kidney (BHK) cells; Chinese hamster ovary (CHO) cells; mouse Sertoli cells; African green monkey kidney cells (VERO-76); human cervical carcinoma cells (e.g., HeLa); canine kidney cells (e.g., MDCK), and the like. In one embodiment, the host cell is a CHO cell.

[0139] D. Production method The truncated NA stalk design disclosed herein allows for large-scale production and purification of soluble tetrameric NA. As described above, a vector containing an artificial nucleic acid molecule encoding the disclosed modified monomeric N2 is inserted into a host cell suitable for producing soluble tetrameric NA.

[0140] In some embodiments, the method for producing soluble tetrameric NAs uses large-scale production conditions. As used herein, "large-scale production conditions" refers to a working volume of between 10 and 10,000 liters, between 25 and 5000 liters, between 25 and 2000 liters, between 50 and 1000 liters, between 50 and 500 liters, between 50 and 250 liters, between 50 and 200 liters, between 100 and 200 liters, between 100 and 5000 liters, between 500 and 8000 liters, between 1500 and 6500 liters, between about 1500 and 16 ... This refers to a process of culturing host cells in a culture tank, typically a bioreactor, of 25 liters or more, such as 0 liter, about 3000 to 3200 liters, about 6000 to 6400 liters, 100 liters or more, such as at least 100 liters and not more than 10,000 liters, such as at least 100 liters and not more than 8000 liters, such as at least 100 liters and not more than 4000 liters, or at least 100 liters and not more than 2000 liters.

[0141] As described herein, the soluble tetrameric NA described herein is purified from cell culture supernatant using chromatography methods including, for example, ion exchange chromatography, affinity chromatography, and size exclusion chromatography.For example, when modified N2 contains a charged tag such as a histidine tag, the soluble NA is purified using ion exchange chromatography.In some embodiments, the soluble tetrameric NA described herein is purified using size exclusion chromatography (SEC).In addition, SEC-multi-angle light scattering (SEC-MALS) is also used, for example, to determine both the molecular weight and purity of soluble NA molecules.

[0142] In some N2 influenza strains, when modified monomeric N2 is expressed in host cells, the host cell supernatant contains a mixture of modified NA, including monomeric form of modified NA, tetrameric form of modified NA, and / or other oligomeric forms of modified NA, such as dimeric NA, trimeric NA, and / or higher oligomeric NA (e.g., aggregates or other multimers of modified monomeric, dimeric, trimeric, or tetrameric NA). When the host cell supernatant is applied to a chromatography column, in some N2 influenza strains, the soluble NA may elute in two major peaks or pools, whereas for other N2 influenza strains, the soluble NA may elute as a single peak or pool. For some N2 influenza strains, when modified monomeric N2 is expressed in cells and the cell supernatant is applied to a chromatography column, the soluble NA may elute in more than two major peaks or pools. When soluble NAs elute in two peaks or pools, the first peak or pool contains mainly monomeric NAs with a molecular weight of about 60 kD, while the second peak contains mainly tetrameric NAs with a molecular weight of about 210 kD. When soluble NAs elute as a single peak or pool, the pool typically contains a mixture of monomeric and tetrameric NAs. The purity of tetrameric NAs in the cell supernatant may vary between different N2 strains, with certain strains, such as PERT09 and BELG17, eluting with a single peak or pool containing about 70-100% and 20-25% tetrameric NAs, respectively, in a single peak or pool. In some embodiments, the soluble tetrameric NA represents at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the modified N2 in the host cell supernatant as measured using SEC.In some embodiments, the soluble tetrameric NA is present in the host cell supernatant in an amount of about 50-150 mg / L, including, for example, about 75-140 mg / L, about 80-135 mg / L, or about 80 mg / L-120 mg / L. If desired, the soluble tetrameric NA obtained from the host cell supernatant is further purified.

[0143] In some embodiments, the soluble tetrameric NA described herein is purified using affinity chromatography. For example, the soluble tetrameric NA is purified using the novel TAMIFLU® binding assay described in the Examples. The purification method of TAMIFLU® is also described in U.S. Provisional Patent Application No. 63 / 231,795, entitled "METHODS AND RELATED ASPECTS OF DETECTING AND PURIFYING INFLUENZA NEURAMINIDASE," filed on August 11, 2021, and incorporated herein by reference in its entirety. Since it was found that recombinant monomeric NA does not bind to TAMIFLU®, the purification method of TAMIFLU® provides a convenient way to purify soluble tetrameric NA in a mixture containing soluble tetramers and monomeric NA. In some embodiments, TAMIFLU®-linked conjugate is used to purify soluble tetrameric NA from cell culture supernatant.In some embodiments, the cell culture supernatant is subjected to a first purification method, such as ion exchange chromatography or SEC, to obtain a partially purified peak or partially purified pool containing a mixture of monomeric and tetrameric NA, and then subjected to a second purification method, which is a TAMIFLU®-based purification method, comprising incubating a mixture containing monomeric and tetrameric NA with TAMIFLU® conjugate, isolating the tetrameric NA bound to TAMIFLU® conjugate, and purifying the tetrameric NA.

[0144] In some embodiments, after purification, a purified composition is obtained, which is a composition containing at least 90% tetrameric NA. In some embodiments, the purified sample contains at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% tetrameric NA. In some embodiments, after purification, a purified composition is obtained, which is a composition containing tetrameric NA at a concentration of at least 0.01, 0.2, 0.5, 1, 2, 3, 4, or 5 mg / mL, such as 0.5 mg / mL to 10 mg / mL, 2 mg / mL to 10 mg / mL, 0.5 mg / mL to 20 mg / mL, 2 mg / mL to 20 mg / mL, 0.5 mg / mL to 15 mg / mL, or 2 mg / mL to 15 mg / mL.

[0145] E. Usage The present disclosure provides a method of administering the vaccine composition described herein to a subject. The method is used to vaccinate the subject against influenza virus. In some embodiments, the vaccination method comprises administering to a subject in need thereof a vaccine composition comprising an artificial nucleic acid molecule encoding a modified monomeric N2 described herein or a tetrameric NA comprising four copies of a modified monomeric N2 described herein, and an appropriate adjuvant, in an amount effective to vaccinate the subject against influenza virus. Similarly, the present disclosure provides a vaccine composition comprising an artificial nucleic acid molecule encoding a modified monomeric N2 described herein or a tetrameric NA comprising four copies of a modified monomeric N2 described herein, and an appropriate adjuvant, for use in vaccinating a subject against influenza virus.

[0146] The present disclosure also provides a method of immunizing a subject against influenza virus, comprising administering to the subject an immunologically effective amount of a vaccine composition comprising an artificial nucleic acid molecule encoding a modified monomeric N2 as described herein, or a tetrameric NA comprising four copies of a modified monomeric N2 as described herein, and an appropriate adjuvant. Similarly, the present disclosure provides a vaccine composition comprising an artificial nucleic acid molecule encoding a modified monomeric N2 as described herein, or a tetrameric NA comprising four copies of a modified monomeric N2 as described herein, and an appropriate adjuvant, for use in immunizing a subject against influenza virus.

[0147] In some embodiments, the method or use prevents influenza virus infection or disease in a subject. In some embodiments, the method or use elicits a protective immune response in the subject. In some embodiments, the protective immune response is an antibody response.

[0148] The immunization methods provided herein may broadly induce a neutralizing immune response against one or more influenza viruses. Thus, in various embodiments, the compositions described herein may provide broad cross-protection against different types of influenza viruses. In some embodiments, the compositions provide cross-protection against avian, swine, seasonal, and / or pandemic influenza viruses. In some embodiments, the immunization methods may induce an improved immune response against one or more seasonal influenza strains (e.g., standard of care strains). For example, the improved immune response may be an improved humoral immune response. In some embodiments, the immunization methods may induce an improved immune response against one or more pandemic influenza strains. In some embodiments, the immunization methods may induce an improved immune response against one or more swine influenza strains. In some embodiments, the immunization methods may induce an improved immune response against one or more avian influenza strains.

[0149] Also provided is a method of preventing influenza virus disease in a subject, comprising administering to the subject a vaccine composition comprising an artificial nucleic acid molecule encoding a modified monomeric N2 as described herein or a tetrameric NA comprising four copies of a modified monomeric N2 as described herein, and an appropriate adjuvant, in an amount effective to prevent influenza virus disease in the subject. Similarly, the present disclosure provides a vaccine composition comprising an artificial nucleic acid molecule encoding a modified monomeric N2 as described herein or a tetrameric NA comprising four copies of a modified monomeric N2 as described herein, and an appropriate adjuvant, for use in preventing influenza virus disease in a subject.

[0150] Also provided is a method of inducing an immune response against influenza virus NA in a subject, comprising administering to the subject a vaccine composition comprising an artificial nucleic acid molecule encoding a modified monomeric N2 as described herein or a tetrameric NA comprising four copies of a modified monomeric N2 as described herein, and an appropriate adjuvant.Similarly, the present disclosure provides a vaccine composition comprising an artificial nucleic acid molecule encoding a modified monomeric N2 as described herein or a tetrameric NA comprising four copies of a modified monomeric N2 as described herein, and an appropriate adjuvant, for use in inducing an immune response against influenza virus NA in a subject.

[0151] A vaccine composition comprising an artificial nucleic acid molecule encoding a modified monomeric N2 as described herein or a tetrameric NA comprising four copies of a modified monomeric N2 as described herein and an appropriate adjuvant may be administered prior to or after the onset of one or more symptoms of influenza infection. That is, in some embodiments, the vaccine composition as described herein is administered prophylactically to prevent influenza infection or ameliorate symptoms of potential influenza infection. In some embodiments, a subject is at risk of influenza virus infection when in contact with other individuals or livestock (e.g., pigs) known or suspected to be infected with a pandemic influenza virus and / or is in a location where influenza infection is known or considered to be endemic. In some embodiments, the vaccine composition is administered to a subject suffering from influenza infection or the subject is exhibiting one or more symptoms generally associated with influenza infection. In some embodiments, the subject is known or considered to have been exposed to influenza virus. In some embodiments, a subject is at risk or susceptible to influenza infection when it is known or believed to have been exposed to influenza virus. In some embodiments, a subject is known or believed to have been exposed to influenza virus when it comes into contact with other individuals or livestock (e.g., pigs) known or suspected to be infected with pandemic influenza virus, and / or is present in a place where influenza infection is known or believed to be endemic or prevalent. The vaccine compositions disclosed herein are used to treat or prevent disease caused by one or both of seasonal and / or pandemic influenza strains.

[0152] Vaccine compositions according to the present disclosure are administered in any amount or dose appropriate to achieve a desired outcome. In some embodiments, the desired outcome is induction of a sustained adaptive immune response against a broad spectrum of influenza strains, including both seasonal and pandemic strains. In some embodiments, the desired outcome is reduction in the intensity, severity, and / or frequency of, and / or delay in onset of, one or more symptoms of influenza infection. The required dose may vary from subject to subject, depending on the species, age, weight, and general condition of the subject, the severity of the infection being treated, the particular composition used, and its mode of administration.

[0153] In various embodiments, the vaccine compositions described herein are administered to a subject, where the subject can be any member of the animal kingdom. In some embodiments, the subject is a non-human animal. In some embodiments, the non-human subject is a bird (e.g., a chicken or bird), a reptile, an amphibian, a fish, an insect, and / or a worm. In some embodiments, the non-human subject is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, a cow, a primate, and / or a pig).

[0154] In some embodiments, the vaccine compositions described herein are administered to a human subject. In certain embodiments, the human subject is 6 months or older, 6 months to 35 months, 36 months to 8 years, or 9 years or older. In some embodiments, the human subject is an infant (under 36 months). In some embodiments, the human subject is a child or juvenile (under 18 years). In some embodiments, the human subject is an elderly (at least 60 years or at least 65 years). In some embodiments, the human subject is a non-elderly adult (at least 18 years and under 65 years).

[0155] In some embodiments, the methods and uses of the vaccine compositions described herein include a prime-boost vaccination strategy. Prime-boost vaccination includes administering a priming vaccine and then administering a boosting vaccine to a subject after a period of time. The immune response is "primed" upon administration of the priming vaccine and "boosted" upon administration of the boosting vaccine. The priming vaccine may include a vaccine composition comprising an artificial nucleic acid molecule encoding a modified monomeric N2 as described herein, or a tetrameric NA comprising four copies of a modified monomeric N2 as described herein, and an appropriate adjuvant. Similarly, the boosting vaccine may include a vaccine composition comprising an artificial nucleic acid molecule encoding a modified monomeric N2 as described herein, or a tetrameric neuraminidase comprising four copies of a modified monomeric N2 as described herein, and an appropriate adjuvant. The priming vaccine composition may be the same as the boosting vaccine, but need not be the same. Administration of the boosting vaccine generally occurs several weeks or months after administration of the priming composition, preferably about 2-3 weeks, or 4 weeks, or 8 weeks, or 16 weeks, or 20 weeks, or 24 weeks, or 28 weeks, or 32 weeks.

[0156] The vaccine composition is administered using any suitable route of administration, including, for example, parenteral delivery, as discussed above.

[0157] Typically, the artificial nucleic acid molecule encoding the modified monomer N2 described herein or the tetrameric NA comprising four copies of the modified monomer N2 described herein and the adjuvant are administered together as components of the same vaccine composition.However, the artificial nucleic acid molecule or the tetrameric NA and the adjuvant do not have to be administered as part of the same vaccine composition.That is, if desired, the artificial nucleic acid molecule or the tetrameric NA and the adjuvant are administered sequentially to the subject.

[0158] The present disclosure will be more fully understood with reference to the following examples. EXAMPLES

[0159] Design of NA with truncated stalk region An engineered subtype 2 influenza neuraminidase ("N2") with a truncated stalk region was designed as shown in Figure 1, in which amino acids 1-74 of wild-type neuraminidase, including substantially all of the cytoplasmic domain, transmembrane region, and stalk region, have been replaced with a secretion signal and an appropriate histidine tag that is used to purify the truncated N2. This engineered N2 is referred to as dTM75. Because truncated N2 is purified by methods that do not involve the histidine tag, it is not necessary for the recombinant truncated stalk construct to contain this histidine tag. Another truncated N2 was also designed in which the cytoplasmic domain and transmembrane region (amino acids 1-35) have been replaced with a secretion signal and a histidine tag. This construct is referred to as dTM36 in Figure 1 and contains the entire stalk region. Also depicted in Figure 1 are 1) an engineered N2 in which substantially all of the cytoplasmic domain, transmembrane region, and stalk region have been replaced with a secretion signal, a histidine tag, and a full-length heterologous tetrabrachion tetramerization domain ("tet-NA"); and 2) a wild-type N2 ("WT") with a short N-terminal cytoplasmic domain, transmembrane region, stalk region, and head region. In initial experiments, the N2 sequence from A / Singapore / INFIMH-16-0019 / 2016 (SEQ ID NO: 72) was used as a representative N2 sequence. The amino acid sequence of the tet-NA construct is set forth in SEQ ID NO: 116.

[0160] Constructs dTM36, dTM75, and tet-NA were expressed in CHO cells and purified to near homogeneity for further characterization. Enzyme activity was measured by MUNANA assay, and specific activity was expressed in nanomoles per minute per μg protein. The MUNANA assay is based on a previously described method (Potier et al., Anal. Biochem., 94, 287-296, 1979), but with modifications. Briefly, 2'-(4-methylumbelliferyl)-alpha-DN-acetylneuraminic acid (MUNANA) was used as a substrate. Neuraminidase contained in the sample cleaves the MUNANA substrate, releasing the fluorescent compound, 4-methylumbelliferone (4-MU). The neuraminidase activity of the test sample is determined by measuring the fluorescence intensity (RFU: relative fluorescence units). In addition, a calibration curve for NA samples of known concentration is used to determine the NA concentration (μg / ml) in the test sample.

[0161] The MUNANA reaction was initiated by adding buffer [33.3 mM 2-(N-morpholino)ethanesulfonic acid (MES, pH 6.5), 4 mM CaCl2, 50 mM BSA] and substrate (100 μM MUNANA). After 1 h of incubation (37° C. with shaking), the reaction was stopped by adding an alkaline pH solution (0.2 M Na2CO3). Fluorescence intensity was detected on a SpectraMax M5 (Molecular Devices) using excitation and emission wavelengths of 355 and 460 nm, respectively. Enzyme activity was calculated against a 4MU reference, and results were expressed in μM per 60 min for total NA activity and in nanomoles per min per μg for NA specific activity.

[0162] The dTM36 mutant had much less enzymatic activity than tet-NA, whereas the dTM75 mutant had similar / higher activity than tet-NA. Size-exclusion chromatography / multi-angle light scattering (SEC-MALS) analysis (described in Example 4) showed that tet-NA was a tetramer, dTM36 was mostly monomeric (90%) with less than 10% being tetramers, and dTM75 was at least 90% tetrameric (Table 1). Based on this result, the dTM75 design was selected for further evaluation in other N2 strains.

[0163] [Table 3] EXAMPLES

[0164] High-throughput (HT) screening of dTM75 from 37 different N2 strains Next, dTM75 constructs were synthesized using N2 sequences from 37 naturally occurring subtype 2 influenza strains and screened for NA activity and tetramer formation. The dTM75 constructs were inserted into plasmids and transfected into ExpiCHO cells using the Expifectamine CHO Transfection Kit (ThermoFisher Scientific, Waltham, MA) according to the manufacturer's protocol. After 4-5 days in culture, the supernatants containing the soluble truncated NA proteins were clarified, filtered, and aliquoted for further characterization, including NA activity and tetramer formation.

[0165] Clarified supernatants were assayed for neuraminidase activity levels using the NA-Star® assay (ThermoFisher Scientific, Waltham, MA) according to the manufacturer's protocol. Samples were collected into 96-well plates and serial 3-fold dilutions were performed. Samples were transferred to the assay plate and once the assay was complete, the plate was read in an Envision plate reader. Data was analyzed and plotted using Prism software.

[0166] A novel high-throughput TAMIFLU® binding assay was developed to measure the formation of tetrameric NA. Oseltamivir phosphate (TAMIFLU®) is a competitive inhibitor of the enzymatic activity of NA. The substrate of oseltamivir is specific for the enzymatic site of the tetrameric NA head. Oseltamivir phosphate was used as a ligand to determine the binding kinetics of recombinant NA protein. The binding of recombinant NA to TAMIFLU® indicated the presence of an active site on the head region of recombinant NA and was used to measure tetrameric NA concentration. First, the validity of the TAMIFLU® binding assay was confirmed by using Tet-NA constructs to demonstrate that tetrameric NA of various influenza strains bind to TAMIFLU® in a dose-dependent manner. In contrast, monomeric NA extracellular domain mutants that are enzymatically inactive do not bind to TAMIFLU®. Therefore, the binding level of recombinant NA in the TAMIFLU® binding assay is proportional to the concentration of recombinant NA assembled as a tetramer.

[0167] For the TAMIFLU® binding assay, oseltamivir phosphate-biotin conjugate (5-10 μg / ml in 1× KB buffer (1% BSA + 0.02% Tween in PBS)) was captured on the surface of a streptavidin-coated biosensor and the binding kinetics of neuraminidase to oseltamivir phosphate was measured using Biolayer Interferometry (BLI) on an Octet instrument (ForteBio, Molecular Devices, LLC, Fremont, California). The biosensor was immersed into wells containing two-fold serial dilutions of recombinant NA samples (0.16-10 ug / ml in 1× KB). Any change in the number of bound molecules results in a measured pattern shift in the detector.

[0168] As shown in FIG. 2, many of the 37 dTM75 constructs had substantial NA activity and / or binding to TAMIFLU® (tetramer formation), including, for example, dTM75 constructs from the following strains: A / Nevada / 32 / 2013, A / Washington / 60 / 2014, A / Nigeria / 120 / 2014, A / Bangkok / 1 / 1979, A / Kannur / MCVR5404 / 2010, A / HongKong / 1774 / 99, A / Marrakech / 79 / 2014, A / Albany / 42 / 1975, A / Yamagata / 62 / 1993, A / Peru / 3216 / 2016, A / HongKong / CUHK13510 / 2001, and A / Illinois / 34 / 2012. A modified A / Singapore / INFIMH160019 / 2016 NA containing a full-length heterologous tetrabrachion tetramerization domain was used as a positive control. Although the dTM75 construct does not form active tetramers in all influenza subtype 2 tested strains, the TAMIFLU® binding assay provides a convenient high-throughput screening assay that can be used to identify which truncated N2 molecules form soluble tetramers when expressed in cells.

[0169] A second high throughput screen was performed on dTM75 constructs from approximately 100 additional subtype 2 influenza strains. Partial results are shown in Figures 3C-3D. When expressed intracellularly, the dTM75 construct from the A / PERTH / 16 / 2009 (PERT09) strain forms approximately 70% tetramers. Approximately 20% of the dTM75 constructs from the additional test strains in the second high throughput screen exhibited higher binding to TAMIFLU® than the dTM75 constructs from PERT09. Approximately 38% of the test strains bound TAMIFLU® within 3-fold of the binding observed for the dTM75 construct from PERT09. See Figure 3C. Additional strains tested that did not show binding to TAMIFLU® within 3-fold of PERT09 were A / TEXAS / 50 / 2012 (SEQ ID NO: 87), A / PORTOALEGRE / LACENRS2376 / 2014 (SEQ ID NO: 69), A / MEMPHIS / 18 / 1978 (SEQ ID NO: 49), A / BELGIUM / 4217 / 2015 (SEQ ID NO: 5), A / TAIWAN / 1 / 1969 (SEQ ID NO: 81), A / SIENA / 3 / 1991 (SEQ ID NO: 71), A / BRISBANE / 273 / 2016 (SEQ ID NO: 7), A / ONTARIO / RV3236 / 2016 (SEQ ID NO: 63), A / TEHRAN / 996 / 2012 (SEQ ID NO: 85), A / ANKARA / 2396 / 2015 (SEQ ID NO: 3), A / KANSAS / 14 / 2017 (SEQ ID NO: 45), A / TEXAS / 71 / 2017 (SEQ ID NO: 88), A / HONGKONG / 4801 / 2014 (SEQ ID NO: 30), A / HONGKONG / 3089 / 2017 (SEQ ID NO: 29), A / FUKUOKA / DS729 / 2016 (SEQ ID NO: 12), A / TOKUSHIMA / DS5288 / 2015 (SEQ ID NO: 89) ), A / HELSINKI / 823 / 2013 (SEQ ID NO: 22), A / ALASKA / 251 / 2015 (SEQ ID NO: 1), A / HATAY / 4990 / 2016 (SEQ ID NO: 20), A / HANOI / ELI15597 / 2015 (SEQ ID NO: 19), A / MISSISSIPPI / 1 / 1985 (SEQ ID NO: 51), A / INDIANA / 18 / 2017 (SEQ ID NO: 36),A / POLAND / 19B / 2017 (SEQ ID NO: 68), A / TENNESSEE / 18 / 2017 (SEQ ID NO: 86), A / HONGKONG / 1774 / 1999 (SEQ ID NO: 28), A / DAKAR / 14 / 2014 (SEQ ID NO: 10), A / SAUDIARABIA / 21 / 1999 (SEQ ID NO: 70), A / GIFU / DS7388 / 2017 (SEQ ID NO: 15), A / NEWYORK / 581 / 1997 (SEQ ID NO: 57), A / HELSINKI / 941 / 2013 (SEQ ID NO: 23), A / ISHIKAWA / DS7294 / 2017 (SEQ ID NO: 39) ), A / GUANGDONGDUANZHOU / 1227 / 2017 (SEQ ID NO: 16), A / AUCKLAND / 5 / 1996 (SEQ ID NO: 4), A / ISHIKAWA / DS7215 / 2016 (SEQ ID NO: 38), A / WESTVIRGINIA / 17 / 2012 (SEQ ID NO: 98), A / SINGAPORE / INFIMH160019 / 2016 (SEQ ID NO: 72), A / KAGAWA / DS7115 / 2016 (SEQ ID NO: 42), A / BILTHOVEN / 21801 / 1971 (SEQ ID NO: 6), A / FUKUOKA / DS72 / 2016 (SEQ ID NO: 1 1), A / MORAMANGA / 1907 / 2017 (SEQ ID NO: 52), A / HUNAN / 01 / 2014 (SEQ ID NO: 33), A / WISCONSIN / 16 / 2015 (SEQ ID NO: 100), A / CANBERRA / 13 / 2015 (SEQ ID NO: 8), A / HONGKONG / 107 / 1971 (SEQ ID NO: 27), A / LYON / 1242 / 2000 (SEQ ID NO: 48), A / NAGANO / 2153 / 2017 (SEQ ID NO: 53), A / NANJING / 49 / 1977 (SEQ ID NO: 54), A / ALBANY / 6 / 1970 (SEQ ID NO: 2), A / KAGAWA / DS769 / 2016 (SEQ ID NO: 41), A / TAIWAN / 4183 / 2004 (SEQ ID NO: 83), A / KOREA / KUMCGR570 / 2011 (SEQ ID NO: 47), A / SYDNEY / 24 / 2015 (SEQ ID NO: 80), A / KOREA / KUMCGR99 / 2011 (SEQ ID NO: 46), A / TOKYO / DS7277 / 2017 (SEQ ID NO: 91), A / HONGKONG / CUHK18194 / 1998 (SEQ ID NO: 31), A / GUNMA / DS7107 / 2016 (SEQ ID NO: 18), A / HONGKONG / 8 / 1968 (SEQ ID NO: 26),The viruses were A / TOKYO / DS7448 / 2017 (sequence number 93), A / PERTH / 61 / 2015 (sequence number 65), A / TW / 875 / 2004 (sequence number 95), A / TAIWAN / 2332 / 2001 (sequence number 82), and A / INDIA / C058671 / 2005 (sequence number 34).

[0170] About 25% of the strains tested in the second high-throughput screen were below the detection level.Thus, the dTM75 construct did not form tetramers in all subtype 2 influenza strains tested, but the majority of the strains tested formed soluble NA tetramers when the dTM75 construct was expressed in cells, indicating that the dTM75 construct derived from subtype 2 influenza strains offers a high possibility of producing soluble tetrameric NA.Furthermore, the TAMIFLU® binding assay provides a convenient way to rapidly screen truncated NA constructs to determine which constructs form the desired tetrameric NA. EXAMPLES

[0171] Sequential deletion of the N2 stalk to identify additional soluble designs To evaluate whether other modified N2 proteins with smaller truncations of the stalk could result in soluble tetrameric NA, a panel of constructs from the PERT09 background was synthesized and screened for NA activity and binding to TAMIFLU® as described above. One dTM36 construct lacked the cytoplasmic and transmembrane domains (i.e., amino acids 1-35 of PERT09) but contained the entire stalk domain. See FIG. 1. Starting at amino acid 60 of the wild-type PERT09 NA sequence, one amino acid at a time was deleted to create a panel of stalk truncation mutants that were tested for NA activity and binding to TAMIFLU® and compared to the results obtained with the PERT09 dTM75 mutant. The PERT09 stalk truncation mutants and the results of the high-throughput screen are shown in the table below. See also FIG. 4.

[0172] [Table 4]

[0173] Results of the high-throughput screen show that one truncation, dTM73, had significantly higher binding activity to TAMIFLU® than the dTM75 control. Other designs, including dTM74, dTM72, and dTM71, showed similar values ​​to dTM75. These results suggested that other stalk truncation mutants of comparable size to dTM75 could be used to successfully generate soluble tetrameric N2.

[0174] For two additional subtype 2 influenza strains, sequential deletion analysis was performed, extending beyond amino acid 74 of the stalk region into the putative NA head region, beginning near amino acid 83 of the N2 sequence. The two additional strains were A / BELGIUM / 4217 / 2015 (BELG15) and A / KANSAS / 14 / 2017 (KANS17). For PERT09, BELG15, and KANS17, sequential deletion mutants beginning at amino acid 60 and extending to amino acid 90 were synthesized and subjected to high-throughput screening as described above. A truncated N2 stalk mutant lacking the entire stalk region formed soluble tetrameric NA in all three tested strains. This screening assay also showed that deletion of the stalk extending into the putative head region was still capable of forming tetrameric NA. Whereas a truncation mutant lacking the first 84 amino acids of the N2 protein was able to form tetramers, extension of the truncation beyond amino acid 84 did not result in the formation of soluble tetrameric NA in the three N2 influenza test strains. Screening analysis also showed that truncation stalk mutants of variable length, in which amino acid 36 of the N2 stalk region was deleted from at least near amino acids 70-84, allowed the formation of soluble tetrameric NA among all three test strains. For example, for PERT09, BELG15, and KANS17, a number of different stalk truncation mutants formed tetrameric NAs, with constructs dTM73 and dTM74 showing the highest amount of binding to TAMIFLU® for PERT09, constructs dTM76, dTM78, and dTM82 showing the highest amount of binding to TAMIFLU® for BELG15, and constructs dTM78 and dTM83 resulting in the highest amount of binding to TAMIFLU® for KANS17. The results are shown in FIG. 4. EXAMPLES

[0175] Large-scale production and purification of dTM75 For detailed characterization of yield, enzyme activity, and conformation, dTM75 constructs from five subtype 2 influenza strains: PERT09, BELG15, KANS17, A / Peru / 4617 / 2017 (PERU17), and A / Texas / 71 / 2017 (TEX17) were expressed and purified at large scale (typically 200 ml). Recombinant NA constructs ("tetNA") from each of these strains containing the full-length heterologous tetrabrachion tetramerization domain described above were also expressed and purified at large scale.

[0176] Briefly, CHO-S cells were transfected with plasmids encoding dTM75 constructs at 1 mg / mL. Transfection was performed using Gibco ExpiCHO transfection reagent (ThermoFisher Scientific, Waltham, MA). One day after transfection, cells were fed and the temperature was shifted from 36.5°C to 32°C. Four days after transfection, cells were centrifuged at 10,500×g for 30 minutes and the supernatant was filtered through a 0.2 μm vacuum filter. The clarified supernatant was purified by immobilized metal ion chromatography (IMAC) using the 6His tag (SEQ ID NO: 135) incorporated into the dTM75 protein. A HisTrap™ HP (Millipore Sigma, Burlington, MA) column pre-packed with Ni Sepharose High Performance and designed for simple one-step purification of histidine-tagged proteins was equilibrated with 20 mM phosphate buffered saline (PBS) with 500 mM sodium chloride (NaCl). The clarified supernatant sample was directly loaded onto the HisTrap™ HP (Millipore Sigma, Burlington, MA) column (5 mL column / 200 mL supernatant). The column was washed with equilibration buffer and the target protein was eluted using a linear gradient (0-100% elution buffer over 10 column volumes) with elution buffer 20 mM PBS with 500 mM NaCl and 1 M imidazole buffer. This initial Ni affinity chromatography step captures both monomeric and tetrameric NA.

[0177] Overall yields of purified dTM75 samples from BELG15, PERT09, PERU17, TEX17, and KANS17 were calculated by multiplying the appropriate molar extinction coefficient by the volume of each pool collected and measuring the protein concentration at A280. Pool refers to the elution peak. KANS17 eluted in two peaks, the larger one representing monomeric NA and the other peak representing tetrameric NA. For the remainder of the dTM75 constructs, no discernible degradation was observed between the peaks, so a single pool was collected representing a mixture of monomeric and tetrameric NA. High overall yields were observed for all dTM75 constructs from BELG15, PERT09, PERU17, TEX17, and KANS17, ranging from approximately 25 to 250 mg / L.

[0178] The sample obtained from the initial Ni affinity chromatography step was subjected to size exclusion chromatography / multi-angle light scattering (SEC-MALS) to determine the purity and molecular weight of the purified dTM75 pool and for further characterization (thermostability, binding to TAMIFLU®, and NA activity). For SEC-MALS, a TSK-GEL G4000 PWXL (7.8 mm×30 cm) column from Tosoh Bioscience was used. The mobile phase for the column was 1× PBS, pH 7.4, with 0.02% sodium azide. Empower® (Waters Corporation, Milford, MA) software was used to integrate the UV peak areas on the chromatogram. The purity of the sample was calculated based on the percentage of specific peak area / total peak area. The molecular weight (MW) of the proteins within the peak was determined by ASTRA (Wyatt Technologies, Santa Barbara, CA) software, which uses light scattering signals together with a concentration detector (RI or UV). The recombinant N2 monomer (extracellular domain) has a MW of approximately 74 kD, whereas the tetrameric NA formed from the tet-NA construct has a MW of approximately 262 kD, which is approximately four times that of the monomeric NA.

[0179] The results of SEC-MALS analysis are shown in FIG. 5. For the dTM75 construct derived from KANS17, the MW of sample pool 1 was about 60 kD, which is consistent with the predicted size of stalk-truncated NA monomers, whereas the MW of pool 2 was about 210 kD, which is consistent with the predicted size of tetrameric NAs formed by stalk-truncated NAs (without the full-length tetrabrachion domain) (FIG. 5, lower panel). The pool 2 fraction of KANS17 dTM75 contained nearly 100% tetrameric NAs (FIG. 5, upper panel). For BELG15, PERT09, PERU17, and TEX17, single pools were collected and analyzed by SEC-MALS. The calculated MW value for each of these single pools was about 210 kD, indicating tetrameric NAs (FIG. 5, lower panel). The main peak of each of these single pools was also analyzed to determine the purity of tetrameric NAs within the single pools, which varied among the different tested strains. The dTM75 construct from PERT09 yielded about 70% tetrameric NAs; the dTM75 construct from PERU17 yielded about 40-45% tetrameric NAs; the dTM75 construct from TEX17 yielded about 30% tetrameric NAs; and the dTM75 construct from BELG15 yielded about 20% tetrameric NAs (Figure 5, upper panel; and Figure 6). The tetNA constructs from all five strains yielded about 90-100% tetrameric NAs (Figure 6).

[0180] The purified tetrameric NA was subjected to further characterization. The tetrameric NA was examined for binding to TAMIFLU® and enzymatic activity (MUNANA assay) as described above. All purified dTM75 constructs exhibited binding to TAMIFLU® (Figure 6). The dTM75 construct derived from PERT09 showed the highest binding to TAMIFLU®, which was equivalent to the binding of PERT09 tet-NA to TAMIFLU®, followed by the dTM75 constructs derived from KANS17 and PERU17, which also showed binding to TAMIFLU®, which was equivalent to the PERU17 tet-NA construct (Figure 6). All constructs forming tetrameric NA were enzymatically active (Figure 6). Only the pool 1 fraction (monomeric NA) from KANS17 was enzymatically inactive (Figure 6).

[0181] Tetrameric NAs were also calculated for thermal stability using an Applied Biosystems 7500 Fast Real-Time PCR instrument and Protein Thermal Shift™ (Thermo Fisher Scientific, Waltham, MA) software to calculate protein melting temperatures (Tm) by the Boltzmann or derivative curve methods. For the KANS17 dTM75 construct, the pool 2 (tetrameric) purified fraction had a higher Tm than the pool 1 (monomer) fraction, indicating that tetrameric NAs are more stable molecules than monomeric NAs (Figure 6).

[0182] These large-scale production and purification experiments show that the truncated N2 stalk design can be successfully applied to various subtype 2 influenza strains to produce soluble and enzymatically active tetrameric NA. Some strains, such as PERT09 and PERU17, can produce a higher proportion of tetrameric NA. Some strains, such as PERT09 and KANS17, can produce tetrameric NA with higher binding affinity to TAMIFLU®. However, the design strategy is consistently used to obtain tetrameric NA in subtype 2 influenza strains. EXAMPLES

[0183] Large-scale generation and purification of additional truncated stalk mutants Further truncated stalk mutants in the PERT09 N2 background were expressed in large scale and purified by Ni affinity purification as described above. Specifically, the following PERT09-based constructs were synthesized, inserted into plasmids, transfected into CHO-S cells, and purified. All of the truncated stalk mutants produced purifiable tetrameric NAs, confirming by SEC-MALS that other truncated stalk mutants identified by the high-throughput TAMIFLU® binding assay form tetrameric NAs, and supporting the fact that large quantities of highly purified tetrameric NAs formed by these truncated stalk mutants can be obtained after large-scale production. As shown in Table 3 below, the estimated tetramer yields from the truncated stalk mutants ranged from about 50 mg / L to about 135 mg / L, and all of the constructs were enzymatically active and had binding activity to TAMIFLU®, and thermal stability with a Tm of about 48-52° C. See also FIG.

[0184] [Table 5] EXAMPLES

[0185] Immunogenicity of dTM75 constructs in mice The immunogenicity of recombinant truncated N2 stalk mutants was evaluated using a naive mouse model (Sultana et al., Vaccine, 29(2011), 2601-2606; Eichelberger et al., Current Opinion in Immunology, 2018, 53:38-44). 6-8 week old female BALB / c mice (n=6 per group) were immunized twice intramuscularly (IM) on days 0 and 21 with Ni affinity purified recombinant modified N2 at a dose of 1 microgram formulated with squalene adjuvant in water (AF03). The experimental schedule is illustrated in Figure 8A. Serum was collected 14 days after the second immunization.

[0186] The immunized mice were examined for the presence of NA-specific antibodies using a neuraminidase-specific enzyme-linked immunosorbent assay (NA ELISA). His-tagged tet-NA from the H3N2 strain of interest was immobilized on nickel-coated plates. After washing, the plates were blocked with 5% Milk BLOT-QuickBlocker™ (G-BioSciences, St. Louis, MO) to eliminate non-specific binding. The plates were then incubated with serially diluted sera from immunized mice to allow binding of anti-NA antibodies, followed by incubation with HRP-linked Goat anti-mouse IgG-detecting antibody (ABCAM, Cambridge, UK) and introduction of a mixture containing TMB (3,3',5,5'-tetramethylbenzidine) and hydrogen peroxide. The reaction was stopped by adding an acidic stop buffer. The absorbance at 450 nm was read using a plate reader SpectraMaxi3 (Molecular Devices, San Jose, CA). OD450 values ​​were fitted with a nonlinear four-parameter logistic (4PL) curve using GraphPad Prism (San Diego, CA) software to calculate the half-maximal effective concentration (EC50).

[0187] Neuraminidase inhibitory antibody response was measured against H6N2 reassortant viruses containing NA from H3N2 strains of interest by enzyme-linked lectin assay (ELLA). Briefly, H6N2 reassortant viruses containing NA from H3N2 strains of interest were titrated in fetuin-coated plates to determine the standard amount of virus that resulted in 70% of the maximum enzymatic activity of NA. Titration of NA inhibitory (NAI) antibodies present in the serum of immunized mice was achieved by performing serial dilutions of heat-activated serum and adding up to a standard amount of virus in fetuin-coated plates. The serum-virus mixture was incubated overnight. The plate was washed and developed after incubation with peroxidase-conjugated peptide nucleic acid (PNA). Low or no signal compared to the virus control indicates inhibition of NA activity due to the presence of NA-specific antibodies. NAI potencies were fitted with a non-linear 4PL curve using GraphPad Prism software and half-maximal inhibitory concentrations (IC50) were calculated.

[0188] Naive BALB / c mice were immunized with vaccine compositions containing squalene adjuvant in water (AF03) and Ni affinity purified dTM75 mutants from KANS17, BELG15, and PERT09, or tet-NA positive controls from each strain, according to the schedule illustrated in Figure 8A. For KANS17, two vaccine compositions were administered: a vaccine composition containing monomeric dTM75 NA eluate (obtained from pool 1 eluate) and a vaccine composition containing tetrameric dTM75 NA eluate (obtained from pool 2 eluate). As measured by ELISA, KANS17 dTM75 from pool 2 induced NA-specific IgG responses that were comparable in magnitude to the KANS17 tet-NA positive control (Figure 8B). Monomeric NA from pool 1 fraction of KANS17 dTM75 induced lower ELISA titers compared to the tetrameric pool 2 fraction (Figure 8B). Similarly, the KANS17 dTM75 fraction from pool 2 induced a lower, but much higher, NAI response compared to the KANS17 tet-NA control as determined by ELLA (Figure 8B).

[0189] Tetrameric NA formed by the constructs PERT09 dTM75 and BELG15 dTM75 induced highly homologous ELISA titers similar to those obtained with tetrameric dTM75 derived from KANS17 (FIG. 8B). The sum of NA-specific IgG responses was comparable among all three tested dTM75 constructs and approached the sum of NA-specific IgG responses from the tet-NA control. Similar to tetrameric dTM75 derived from KANS17, tetrameric dTM75 derived from PERT09 and BELG15 also induced NAI responses, albeit at lower levels than those induced by tet-NA (FIG. 8B).

[0190] Next, mouse immunogenicity studies were extended to assess other N2 stalk truncation mutants of varying lengths and compare these to the dTM75 construct. Specifically, the following PERT09 dTM mutants were assessed for immunogenicity and compared to the constructs PERT09 dTM75 and tet-NA: dTM74, dTM73, dTM72, and dTM71. Naive BALB / c mice were immunized on days 0 and 21 with dTM mutants + squalene in water adjuvant (AF03) or tet-NA control + adjuvant (AF03) according to the schedule illustrated in Figure 9A.

[0191] NA-specific IgG responses were measured by ELISA on day 35 as described above and expressed as ELISA titers by EC50. NAI responses were measured against H6N2 reassortant viruses expressing homologous full-length NA by ELLA as described above and expressed as NAI titers by IC50. Individual animal titers were normalized (Log2) and graphed as box-and-whisker plots. The dashed line indicates the lower limit of detection (LLD) of the assay, while the grey shaded area represents titers within 4-fold of the tet-NA control protein. As shown in Figure 9B, the immunogenicity of the PERT09 mutants dTM75, dTM74, dTM73, dTM72, and dTM71 in naive mice was comparable to the tet-NA control.

[0192] Overall, these mouse data indicate that tetrameric NA formed by a number of different truncated N2 stalk mutants of diverse lengths and derived from different N2 strains induces NA-specific antibody responses in mice. EXAMPLES

[0193] Immunogenicity of dTM75 constructs in preimmunized ferrets Although most studies focus on naive responses to vaccines or responses to natural infection, ferrets have already been used to evaluate the immunogenicity of influenza neuraminidase (Bosch et al., J. Virol., October 2010, pp. 10366-10374). In this example, rather than using naive or naturally infected ferrets, a novel immunogenicity model was developed in which ferrets were pre-immunized via the intranasal route with an influenza H1N2 reassortant virus expressing a wild-type NA of interest, e.g., a wild-type NA from one of the subtype 2 influenza strains used to develop the truncated stalk mutant, such as the KANS17 H3N2 strain. This pre-immunized ferret model measures the ability of recombinant NA antigens to recall or boost responses induced by a previous viral infection.

[0194] Fitch ferrets were immunized according to the schedule shown in Figure 10A. Specifically, ferrets were immunized on day 0 with a single intranasal (IN) dose of H1N1 virus reassortant containing wild-type KANS17 NA, and on day 21 with a single intramuscular (IM) dose of unadjuvanted purified KANS17 dTM75 or tet-NA, and 21 days later, animals were bled to assess NA-specific responses by ELISA and ELLA as described above.

[0195] N2 with KANS17 dTM75 mutants efficiently boosted NA-specific IgG responses in pre-immunized ferrets as measured by ELISA, with N2 with dTM75 mutants yielding slightly higher ELISA titers than the tet-NA control (Figure 10B). dTM75 mutants derived from BELG15 and KANS17 yielded ELISA titers comparable to the tet-NA control. A single IM vaccination with the dTM75-NA design was able to boost NAI responses in pre-immunized ferrets by 3-fold (PERTH09) to 5-fold (BELG15 and KANS17) compared to mock-vaccinated animals (groups only pre-immunized with H1N2 virus) (Figure 10B).

[0196] Overall, these pre-immunized ferret data indicate that the tetrameric NAs formed using the N2 truncated stalk design disclosed in this application are highly immunogenic. High NA-specific antibody responses were observed in ferrets pre-exposed to the virus after a single immunization and even in the absence of adjuvant. EXAMPLES

[0197] Immunogenicity and protection against viral infection and / or disease severity in naive ferrets immunized with recombinant truncated NA mutants Naive ferrets immunized with recombinant truncated NA mutant (PERT09 dTM75) were evaluated for immunogenicity, protection, and / or disease severity against viral infection (Figure 12A). For this study, 17-21 week old outbred naive male Fitch ferrets (n=9 per group) were immunized twice intramuscularly (IM) on days 0 and 21 with Ni affinity purified recombinant PERT09 dTM75, or PERT09 tet-NA as a positive control, using a dose of 5 μg formulated with AF03 adjuvant, or a dose of 45 μg with or without adjuvant. Negative control groups were injected IM with phosphate buffered saline (mock), or mock+adjuvant, on days 0 and 21. Additional control groups received a low dose (10 3 The animals were inoculated intranasally with 100 plaque forming units (PFU) of homologous H3N2 virus (previously infected with A / PE / 09) followed by mock inoculation intranasally on day 21. A group of ferrets immunized with a dose of 45 μg of recombinant HA (rHA) was also used as a control. See Table 4.

[0198] All groups were bled one day (day 20) before the second (booster) dose, and then three weeks later (day 42) prior to virus challenge (FIG. 12B). To assess immunogenicity, serum samples (stored at −20° C. until required) were assayed for NAI using ELLA (IC50) or NA-specific IgG titers using ELISA (EC50), as described in Example 6 above.

[0199] On day 43, all ferret groups were 6PFU of PERT09 H3N2 wild type influenza A challenge virus (1,000 μL per dose, divided equally between the nostrils) was inoculated intranasally. An additional control group received TAMIFLU® (10 mg per kg per day) via intragastric route, with the first dose beginning 6 hours after challenge and divided into two daily doses. Animals were monitored for clinical symptoms, including changes in body weight (once daily) and temperature (twice daily), for 14 days after challenge. Serum was collected 57 days after challenge and stored at −20° C. until ELLA or ELISA testing, as described above ( FIG. 12B ).

[0200] To assess total viral shedding, nasal washes were collected from all challenged animals daily on days 1-7 post-challenge and samples were stored at -65°C or below. Ferrets were anesthetized with a mixture of ketamine (25 mg / kg) and xylazine (2 mg / kg), 0.5 mL of sterile PBS containing penicillin (100 U / mL), streptomycin (100 μg / mL), and gentamicin (50 μg / mL) was injected into each nasal cavity, and samples were collected and stored at -65°C or below. Virus in nasal wash specimens was detected using a standard TCID 50 The antibodies were titrated by 50% tissue culture infectious dose (TDC) assay. The nasal washes were thawed and then clarified by centrifugation. The resulting supernatants were serially diluted 10-fold and then transferred to each well of a 96-well plate containing monolayer MDCK (Madin-Darby Canine Kidney) cells for titration.

[0201] As shown in Figure 13, dTM75 based on PERT09 induced higher levels of NAI titers compared to mock (with or without adjuvant) before challenge (days 20 and 42) and after challenge (day 57). NAI titers were boosted by adjuvant. dTM75 with adjuvant (5μg and 45μg) induced higher levels of NAI titers after the second immunization than those induced by previous infection. Adjuvants were dose-sparing (e.g., 5μg+AF03 is more immunogenic than 45μg without adjuvant). A similar trend was observed for NA ELISA titers (Figure 14).

[0202] As shown in Figures 15-18, vaccination with PERT09 dTM75 reduced viral shedding and disease severity, as indicated by reduced peak body temperature (fever) change and reduced peak body weight change, in naive ferrets after challenge with homologous H3N2, compared to mock. NA-mediated protection for reducing viral shedding was characterized by adjuvant dependency, with a 5 μg dose in the presence of adjuvant resulting in the highest reduction, comparable to that in the rHA group and significantly higher than that in the TAMIFLU® group (Figures 15 and 16). PERT09 dTM75 does not appear to be as effective as previous infection in reducing viral shedding, but this result is expected since infection provides both anti-NA and anti-HA immunity in addition to T cell immunity against conserved epitopes.

[0203] NA-mediated protection against the increase in peak body temperature was characterized by dose and adjuvant dependency. Only the doses of PERT09 dTM75 or tet-NA adjuvanted with AF03 (5 μg and 45 μg) were significantly reduced compared to mock (Figures 15 and 17). Although not significant, NA-mediated protection against the reduction in peak body weight was observed across all treatment groups compared to mock. However, these differences were not significant (Figures 15 and 18).

[0204] Overall, the naive ferret data indicate that PERT09 dTM75 induced NA-specific antibody responses that correlated with protection from infection and disease severity.

[0205] [Table 6] EXAMPLES

[0206] Immunogenicity and protection against viral infection and / or disease severity in naive ferrets immunized with recombinant neuraminidase in the context of mismatched hemagglutinin Using a naive ferret challenge model, we demonstrated the benefit of a vaccine strategy that combines recombinant NA with HA derived from a mismatched influenza strain. The general vaccination schedule is outlined in FIG. 12B, while further details are summarized in Table 5.

[0207] [Table 7]

[0208] For this study, 17-21 week old outbred naive male Fitch ferrets (n=9 per group) were immunized intramuscularly (IM; 500 μL per dose) on days 0 and 21 with 5 μg or 45 μg of 1) A / Perth / 16 / 2009 recombinant N2 dTM75, 2) mismatched A / Singapore / INFIMH160019 / 2016 recombinant hemagglutinin (rHA), or 3) a combination of both PERT09 dTM75-NA and SING16 rHA on two occasions. A dose of 5 μg was administered in a 1:1 ratio with AF03 adjuvant. Three weeks after the booster vaccination, ferrets were given 10 3Animals were challenged intranasally with PFU of A / Perth / 16 / 2009 H3N2 wild type influenza A virus (1,000 μL per dose, divided equally between the nostrils). Animals were monitored for clinical signs and changes in body weight once daily and temperature twice daily for 14 days post-challenge. Nasal washes were collected from all challenged animals on days 1-7 post-challenge and samples were stored at ≦-65°C for viral shedding assessment. Group 1 was immunized with PBS diluent only and served as a negative control, demonstrating the most severe symptoms following challenge / infection. Group 2 received a live intranasal challenge (previous infection) with A / Perth / 16 / 2009 H3N2 virus on day 0, rather than vaccination with PERT09 dTM75-NA or SING16-rHA, and served as a positive control, providing the best protection from challenge. Study endpoints were assessed as summarized in Table 6.

[0209] [Table 8]

[0210] All ferrets were bled under sedation at baseline (day 0), 3 weeks after primer vaccination (1 day or immediately before booster, day 20) and booster vaccination (day 42), and 2 weeks after challenge (day 57), and serum samples (stored at -20°C until required) were examined by ELLA to assess NAI activity as described above, by NA ELISA to assess binding antibody levels as described above, and by hemagglutinin inhibition assay (HAI) to assess antibody responses to the hemagglutinin antigen of A / Perth / 16 / 2009 virus or SING16-rHA, as described above. Animals were monitored for clinical symptoms, as well as changes in body weight once daily and temperature twice daily (morning and afternoon) for 14 days after challenge. Nasal wash specimens were collected from experimentally infected ferrets daily after intranasal challenge. Virus in nasal wash samples was detected in standard TCID 50 The antibodies were titrated by 50% tissue culture infectious dose (TDC) assay. The nasal washes were thawed and then clarified by centrifugation. The resulting supernatants were serially diluted 10-fold and then transferred to monolayers of MDCK (Madin-Darby Canine Kidney) cells in each well of a 96-well plate for titration.

[0211] NAI antibody responses were measured against H6 reassortant viruses containing NA from the strains of interest by ELLA essentially as described in Example 6 above. Sera were collected 3 weeks after the first dose or 3 weeks after prime and booster vaccinations, respectively, and examined via ELLA to assess NAI antibody activity using H6N2 A / Perth / 16 / 2009 virus as a source of sialidase. Comparison of NAI responses between groups immunized with PERT09 dTM75-NA alone or a combination of dTM75NA and rHA did not reveal any statistically significant differences (Figure 19). A robust induction of anti-NA titers (ELLA) against A / Perth / 16 / 2009 over time (days 20, 42, and 57) was observed in all ferrets administered live virus (previously infected with PERT09) or immunized with low (5 μg+AF03) or high (45 μg) doses of PERT09 dTM75-NA (FIG. 19). Vaccine formulations containing low doses (5 μg+AF03) of dTM75-NA (dTM75-NA alone or dTM75-NA+rHA) induced higher NAI titers than vaccine formulations containing high doses (45 μg) of dTM75-NA (FIG. 19).

[0212] A robust induction of anti-NA binding titers (ELISA) against A / Perth / 16 / 2009 over time (days 20, 42, and 57) was observed in all ferrets administered live virus (previous infection with PERT09) or immunized with low dose (5 μg+AF03) or high dose (45 μg) of PERT09 dTM75-NA (FIG. 20). Sera were collected 3 weeks after the first dose or 3 weeks after prime vaccination and 3 weeks after booster vaccination and examined via ELISA to assess NA binding titers essentially as described in Example 6 above. Comparison of ELISA NA binding titers between groups immunized with PERT09 dTM75-NA alone or a combination of dTM75-NA and rHA did not reveal any statistically significant differences consistent with the ELLA data. Similar to the NAI titers, vaccine formulations containing a low dose (5 μg + AF03) of dTM75-NA (dTM75-NA alone or dTM75-NA + rHA) induced higher ELISA titers than vaccine formulations containing a high dose (45 μg) of dTM75-NA (Figure 20).

[0213] Robust HAI antibody titers against SING16 were induced after the second immunization (day 42) with vaccine formulations containing rHA or dTM75-NA+rHA without further boosting (Figure 21). Sera were collected 3 weeks after the first dose or 3 weeks after prime vaccination and 3 weeks after booster vaccination and examined via HAI to assess response to A / Singapore / INFIMH160019 / 2016 rHA. Comparison of HAI titers between groups immunized with SING16-rHA alone or the combination of dTM75NA and rHA did not reveal any statistically significant differences consistent with the ELLA and ELISA data (Figure 21). Interestingly, a further boost (4-10 fold) in HAI titers against SING16 was observed on day 57 after challenge with A / Perth / 16 / 2009 virus, pointing to an increase in cross-reactive HAI titers after heterologous challenge (Figure 21). Vaccine formulations containing low doses (5μg+AF03) of dTM75-NA (dTM75-NA alone or dTM75-NA+rHA) induced higher HAI titers against SING16 than vaccine formulations containing high doses (45μg), consistent with the NAI and ELLA titers (Figure 21).

[0214] To measure HAI titers, serum was treated with receptor-destroying enzyme (RDE; Denka Co., Japan) to inactivate nonspecific inhibitors prior to the HAI assay. Briefly, RDE was added at a volume ratio of 3 to serum at a volume ratio of 1 and incubated overnight at 37°C. RDE was inactivated by incubation with 6 volumes of serum for 30 min at 56°C and added to two-fold serial dilutions of RDE-treated serum in 0.9% saline in a v-bottom 96-well microtiter plate. Equal volumes of each virus from the panel adjusted to 4 agglutination units (HAU) per 50 μL were added to each well. For this study, the homologous virus panel included A / Singapore / INFIMH-16-0019 / 2016(H3N2) virus grown in chicken eggs. Plates were covered and incubated for 20 minutes at room temperature followed by the addition of 1% chicken red blood cells (CRBCs) (Lampire Biologicals) in PBS. Plates were mixed by shaking, covered and the RBCs were allowed to settle for 1 hour at room temperature. HAI titers were determined by the reciprocal dilution of the last well containing non-agglutinated RBCs.

[0215] Vaccine formulations containing rHA or dTM75-NA+rHA without further boosting induced robust HAI antibody titers to PERT09 following challenge with A / Perth / 16 / 2009 virus (day 57) (Figure 22). Sera were collected 3 weeks after the first dose or 3 weeks after prime vaccination and 3 weeks after booster vaccination and examined via HAI to assess for antibody titers capable of neutralizing A / Perth / 16 / 2009 virus. Comparison of HAI titers between groups immunized with SING16-rHA alone or a combination of dTM75-NA and SING16-rHA revealed no statistically significant differences in agreement with the ELLA and ELISA data (Figure 22). Interestingly, low levels of cross-reactivity were observed in groups immunized with SING16-rHA or the combination of SING16-rHA and dTM75-NA on day 42, confirming detectable heterologous titers (HAI titers of >50) against PERT09 virus (Figure 22).

[0216] Immunization with two doses of PERT09 dTM75-NA or SING16 rHA administered individually or in combination provided protection against weight loss induced via challenge with live A / Perth / 16 / 2009 virus (Figures 23A-23C). Body weight changes were observed for 14 days after challenge (Figures 23A-23B). Non-vaccinated animals (PBS control group) lost a maximum of 9.8% body weight by day 6, whereas SING16 rHA+AF03 5μg or SING16 rHA 5μg+PERT09 dTM75-NA+AF03 5μg combinations lost 5.3% and 3.9%, respectively (Figure 23C). Vaccinated animals appeared to regain weight more quickly than the non-vaccinated PBS group. A vaccine formulation containing 5 μg of dTM75NA in combination with 5 μg of rHA and AF03 provided protection from weight loss similar to that in ferrets previously infected with PERT09 (FIGS. 23A and 23C).

[0217] Viral shedding was assessed daily for 7 days post-challenge and the change in titer, in TCID50 per ml, is presented in Figures 24A-24B. The duration and intensity of viral shedding (AUC) is particularly reduced for vaccine formulations containing 5 μg dTM75NA in combination with 5 μg rHA and AF03 (Figures 24A-24C). AUC was calculated for all groups and pairwise comparison analysis confirmed that all low dose (5 μg + AF03) groups (dTM75-NA, rHA, or combination of dTM75-NA and rHA) induced a statistically significant reduction in viral shedding compared to the PBS control group (Figure 24C). In addition, log10 TCID50 values ​​per ml 2 days after challenge confirmed the lowest viral shedding titers in the low dose (5μg+AF03) formulation group (1.3 Log10) and the rHA alone group (1.6 Log10), although these titer reductions were not statistically significantly different from the non-protected PBS control group (2.8 Log10).

[0218] In all groups vaccinated with the low dose (5 μg+AF03), the duration of infection was reduced from 6 to 3 days compared to non-vaccinated (PBS) animals, and the number of infected ferrets over time was also reduced (Figure 25A). The high dose (45 μg) group showed a reduction in the duration of viral shedding only in the rHA group, but did not reduce the length of infection (up to 6 days) in any of the groups vaccinated with high dose dTM75-NA or high dose dTM75-NA+rHA (Figure 25A). Body temperature (BT) monitored daily over 14 days confirmed a reduction in fever, presented as an increase in peak body temperature in the afternoon (PM) in all vaccinated groups compared to PBS controls (Figure 25B). Statistically significant differences in morning peak body temperature were identified only between the PBS control group and the PERT09 previously infected ferret group, with overall peak body temperature of 1.6 and 0.9 degrees Celsius, respectively (Figure 25B).

[0219] In summary, in the ferret challenge study, serological data for vaccine formulations containing rNA mutants and rHA combinations were similar to those for vaccine formulations containing rNA mutants or rHA alone, with the low dose formulation (5 μg + adjuvant) consistently inducing higher titers than the high dose formulation (45 μg without adjuvant). Despite similar serological data, the addition of recombinant NA mutants with recombinant HA derived from mismatched influenza strains results in increased protection in the ferret challenge model. The weight loss for animals vaccinated with low dose dTM75-NA and rHA combination (5 μg + AF03) was significantly different from non-vaccinated animals. In addition, animals vaccinated with low dose (5 μg + AF03) rHA or low dose (5 μg + AF03) dTM75-NA and rHA combination exhibited significant reductions in viral shedding (AUC) and length of viral clearance.

[0220] Although the foregoing disclosure has been described in some detail for purposes of illustration and example, for clarity and understanding, it will be apparent to those skilled in the art from reading the disclosure that various changes in form and detail may be made and practiced within the scope of the appended claims without departing from the true scope of the disclosure, e.g., all of the protein constructs, methods, and / or component features, steps, elements, or other aspects thereof may be used in various combinations.

[0221] Unless indicated to the contrary or otherwise clear from the context, a claim or description containing "or" between one or more members of a group is considered to be satisfied if one, more than one, or all of the members of the group are present in, utilized in, or otherwise suitable for a given product or process. The invention includes embodiments in which exactly one member of a group is present in, utilized in, or otherwise suitable for a given product or process. The invention also includes embodiments in which more than one or all of the members of a group are present in, utilized in, or otherwise suitable for a given product or process. Furthermore, unless indicated otherwise or unless it would be apparent to one of ordinary skill in the art that a contradiction or inconsistency would result, the disclosure is also to be understood to encompass all variations, combinations, and permutations of the introduction of one or more limitations, elements, clauses, descriptive terms, and the like, from one or more of the enumerated claims into another claim that is dependent on the same base claim (or any other claim, if appropriate). When elements are presented as a list (e.g., in a Markush group or similar format), it is to be understood that each subgroup of elements is also disclosed, and that any element(s) may be excluded from the group. In general, when an embodiment or aspect of the disclosure is referred to as including certain elements, features, etc., it is to be understood that a particular embodiment or aspect consists of or consists essentially of such elements, features, etc. For the sake of brevity, these embodiments are not specifically and literally set forth in this specification in every instance. It is also to be understood that any embodiment or aspect of the disclosure may be expressly excluded from the claims, regardless of whether the specific exclusion is recited herein.

[0222] All patents, patent applications, websites, other publications, or documents, accession numbers, etc., used herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual reference was specifically and individually indicated to be so incorporated by reference. Where different sequence variants are associated with accession numbers at different times, the variants associated with the accession number at the effective filing date of this application are intended. By effective filing date, we mean the earlier of the actual filing date or, where appropriate, the filing date of the priority application that references the accession number. Similarly, where different versions of publications, websites, etc. are published at different times, the most recently published version at the effective filing date of this application is intended unless otherwise indicated.

Claims

1. 1. A modified monomeric influenza virus subtype 2 neuraminidase, comprising: a signal peptide; and Influenza virus subtype 2 neuraminidase head region Including, amino acid 1 through at least amino acids 70-82 of influenza virus subtype 2 neuraminidase are replaced by a signal peptide; The modified monomeric influenza virus subtype 2 neuraminidase does not contain a heterologous oligomerization domain; Expression of the modified monomeric influenza virus subtype 2 neuraminidase in a host cell results in the secretion of tetrameric neuraminidase from the host cell. The modified monomeric influenza virus subtype 2 neuraminidase.

2. An artificial nucleic acid encoding the modified monomeric influenza virus subtype 2 neuraminidase of claim 1.

3. A tetrameric neuraminidase comprising four copies of a modified monomeric influenza virus subtype 2 neuraminidase, the modified monomeric influenza virus subtype 2 neuraminidase comprising: Influenza virus subtype 2 neuraminidase head region Including, the modified monomeric influenza virus subtype 2 neuraminidase does not contain amino acid 1 to at least amino acids 70-82 of influenza virus subtype 2 neuraminidase; The modified monomeric influenza virus subtype 2 neuraminidase does not contain a heterologous oligomerization domain. The tetrameric neuraminidase.

4. After expression of the modified monomeric influenza virus subtype 2 neuraminidase in the host cell, the host cell supernatant contains at least the modified monomeric influenza virus subtype 2 neuraminidase, including tetrameric neuraminidase and a monomeric form of the modified monomeric influenza virus subtype 2 neuraminidase. The modified monomeric influenza virus subtype 2 neuraminidase of claim 1, the artificial nucleic acid molecule of claim 2, or the tetrameric neuraminidase of claim 3, comprising virus subtype 2 neuraminidase, wherein the tetrameric neuraminidase represents at least 40%, at least 60%, at least 80%, or at least 90% of the modified monomeric influenza virus subtype 2 neuraminidase in a host cell supernatant as measured by size exclusion chromatography.

5. i) amino acids 1-70, 1-71, 1-72, or 1-73 of influenza virus subtype 2 neuraminidase are replaced by a signal peptide in a modified monomeric influenza virus subtype 2 neuraminidase; ii) amino acids 1-74 of influenza virus subtype 2 neuraminidase are replaced by a signal peptide in a modified monomeric influenza virus subtype 2 neuraminidase; iii) amino acids 1-75, 1-76, 1-77, 1-78, or 1-79 of influenza virus subtype 2 neuraminidase are replaced by a signal peptide in the modified monomeric influenza virus subtype 2 neuraminidase; iv) amino acids 1-80, 1-81, or 1-82 of influenza virus subtype 2 neuraminidase are replaced by a signal peptide in a modified monomeric influenza virus subtype 2 neuraminidase; or v) amino acids 1-82 of influenza virus subtype 2 neuraminidase are replaced by a signal peptide in a modified monomeric influenza virus subtype 2 neuraminidase, and the first amino acid, or the first and second amino acids, of the head region are deleted; The modified monomeric influenza virus subtype 2 neuraminidase or artificial nucleic acid molecule according to claim 4; or a) the modified monomeric influenza virus subtype 2 neuraminidase lacks amino acids 1-70, 1-71, 1-72, or 1-73 of influenza virus subtype 2 neuraminidase; b) the modified monomeric influenza virus subtype 2 neuraminidase lacks amino acids 1-74 of influenza virus subtype 2 neuraminidase; c) the modified monomeric influenza virus subtype 2 neuraminidase lacks amino acids 1-75, 1-76, 1-77, 1-78, or 1-79 of influenza virus subtype 2 neuraminidase; d) the modified monomeric influenza virus subtype 2 neuraminidase lacks amino acids 1-80, 1-81, or 1-82 of influenza virus subtype 2 neuraminidase; or e) the modified monomeric influenza virus subtype 2 neuraminidase lacks amino acids 1-83 or 1-84 of influenza virus subtype 2 neuraminidase; The tetrameric neuraminidase of claim 4.

6. The modified monomeric influenza virus subtype 2 neuraminidase of claim 1 or the artificial nucleic acid molecule of claim 2, wherein the signal peptide is a mammalian signal peptide.

7. The modified monomeric influenza virus subtype 2 neuraminidase of claim 1, the artificial nucleic acid molecule of claim 2, or the tetrameric neuraminidase of claim 3, wherein the modified monomeric influenza virus subtype 2 neuraminidase further comprises a linker sequence connecting the head region to a signal peptide or a C-terminal amino acid of the stalk region.

8. The linker sequence contains glycine and / or serine residues and is 2 to 10 amino acids in length. The modified monomeric influenza virus subtype 2 neuraminidase, artificial nucleic acid molecule, or tetrameric neuraminidase according to claim 7, wherein:

9. i) the host cell is a mammalian cell; ii) the influenza virus subtype 2 neuraminidase is derived from a standard therapeutic influenza strain; or iii) influenza virus subtype 2 neuraminidase is derived from influenza A virus; The modified monomeric influenza virus subtype 2 neuraminidase, artificial nucleic acid molecule, or tetrameric neuraminidase according to claim 4.

10. The modified monomeric influenza virus subtype 2 neuraminidase of claim 1, the artificial nucleic acid molecule of claim 2, or the tetrameric neuraminidase of claim 3, wherein the head region of the influenza virus subtype 2 neuraminidase is a head region of an influenza virus subtype 2 neuraminidase according to any one of SEQ ID NOs: 1 to 115, and / or the modified monomeric influenza virus subtype 2 neuraminidase comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 131.

11. A host cell comprising the artificial nucleic acid molecule of claim 2.

12. The host cell of claim 11 , which is a mammalian cell.

13. The host cell of claim 12, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.

14. A vaccine composition comprising the tetrameric neuraminidase of claim 3.

15. A vaccine composition comprising the artificial nucleic acid molecule of claim 2.

16. 16. The vaccine composition of claim 14 or 15, further comprising an adjuvant.

17. 16. The vaccine composition of claim 14 or 15, further comprising influenza virus hemagglutinin.

18. 12. An in vitro method for producing a tetrameric neuraminidase, comprising culturing the host cell of claim 11 in a cell culture medium and expressing a modified monomeric influenza virus subtype 2 neuraminidase in the host cell, wherein after expression of the modified monomeric influenza virus subtype 2 neuraminidase in the host cell, a host cell supernatant contains at least the modified monomeric influenza virus subtype 2 neuraminidase, the modified monomeric influenza virus subtype 2 neuraminidase comprising a tetrameric neuraminidase and a monomeric form of the modified monomeric influenza virus subtype 2 neuraminidase.

19. 19. The method of claim 18, wherein the tetrameric neuraminidase represents at least 20%, at least 50%, at least 70%, or at least 90% of the modified monomeric influenza virus subtype 2 neuraminidase that is secreted upon expression of the modified monomeric influenza virus subtype 2 neuraminidase in a host cell, as measured by size exclusion chromatography.

20. 16. A vaccine composition according to claim 14 or 15 for use in a method for immunising a subject against influenza virus.

21. 16. A vaccine composition according to claim 14 or 15 for use in a method for reducing one or more symptoms of influenza virus infection.

22. 22. The vaccine composition of claim 21, wherein the one or more symptoms of influenza virus infection is weight loss or the one or more symptoms of influenza virus infection is elevated body temperature.

23. 16. A vaccine composition according to claim 14 or 15 for use in a method of vaccinating a subject against influenza virus.

24. 21. The vaccine composition for use according to claim 20, wherein the subject is a human.

25. 25. The vaccine composition for use according to claim 24, wherein the human subject is at least 6 months and under 18 years of age, at least 60 years of age, at least 65 years of age, at least 6 months and under 18 years of age, or at least 18 years of age and under 65 years of age.

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