Vaccine composition having improved stability and immunogenicity

Abstract

To provide nanoparticles suitable for use in vaccines.SOLUTION: Disclosed herein is a nanoparticle that presents a pathogen-derived antigen that is surrounding and associated with a surfactant core to give enhanced stability and good immunogenicity. Dosages, formulations, methods for preparing vaccines and nanoparticles are also disclosed. In one aspect, the invention provides a vaccine composition comprising: (i) a nanoparticle comprising a nonionic surfactant core present in an amount about 0.03% to about 0.05% and a viral glycoprotein associated with the core; and (ii) a pharmaceutically acceptable buffer, where the nonionic surfactant is selected from the group consisting of PS20, PS40, PS60, PS65 and PS80.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - References to Related Applications This application incorporates by reference in its entirety for all purposes the disclosures of U.S. Provisional Application No. 62 / 213,947, filed on September 3, 2015; U.S. Provisional Application No. 62 / 255,786, filed on November 16, 2015; U.S. Provisional Application No. 62 / 309,216, filed on March 16, 2016; and U.S. Provisional Application No. 62 / 350,973, filed on June 16, 2016.

[0002] Description of Electronically Submitted Text Files The content of the text file electronically submitted with this specification is incorporated herein by reference in its entirety: a computer - readable format copy of the sequence listing (file name: NOVV_060_03US_SeqList_ST25.txt, recording date: September 6, 2016; file size: 91 kilobytes). Technical Field The present disclosure generally relates to nanoparticles useful for stimulating an immune response. The nanoparticles provide an antigen, such as a glycoprotein antigen, associated with a surfactant core and are typically produced using a recombinant approach. The nanoparticles have improved stability and enhanced epitope presentation. The present disclosure also provides compositions containing the nanoparticles, methods for producing them, and methods for stimulating an immune response.

Background Art

[0003] Infectious diseases remain a global challenge. While there has been progress in vaccine development against some pathogens, many still pose a threat to human health. The most notorious, HIV, still lacks an effective vaccine. Attempts to produce vaccines against certain pathogens have ended in failure, resulting in further pathologies. Other pathogens, including Ebola, which occurs sporadically, particularly in Africa, causing loss of life and global economic impact, also remain a problem. Influenza virus, while existing vaccines provide some protection, is yet another virus where technical hurdles in virus production mean that seasonal influenza vaccines may provide inadequate protection.

[0004] Deploying an effective vaccine depends on a combination of factors. The vaccine must stimulate an effective immune response that reduces infection or disease by a sufficient amount to be beneficial. The vaccine must also be stable enough to be used in difficult environments where refrigeration may not be available. SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0005] Accordingly, there is a continuing interest in producing vaccines against viruses that represent a public health problem worldwide, and a continuing need to produce effective vaccines with good stability. MEANS FOR SOLVING THE PROBLEMS

[0006] The present disclosure provides nanoparticles suitable for inducing an immune response against a pathogen. The nanoparticles provide improved stability and effective immunogenicity. In certain embodiments, the pathogen is a virus, and typically, the antigen used to produce the viral nanoparticles is a viral glycoprotein.

[0007] In one aspect, the present disclosure provides nanoparticles containing a virus protein with enhanced stability. In some embodiments, the present disclosure includes a vaccine composition comprising nanoparticles comprising a nonionic surfactant, a viral glycoprotein, and a pharmaceutical buffer. In typical embodiments, the nonionic surfactant may be selected from the group consisting of PS20, PS40, PS60, PS65, and PS80. In some embodiments, the composition does not contain any free nonionic surfactant. One or more glycoprotein antigen molecules surround a surfactant core containing a nonionic surfactant, which provides a nanoparticle structure that promotes immunogenicity and inhibits antigen degradation.

[0008] In some embodiments, the antigen is selected from the group consisting of RSV F protein, influenza HA protein, influenza NA protein, and combinations thereof. Other antigens, including Ebola, may also be used. Typically, the antigen is a glycoprotein.

[0009] Optionally, the RSV F protein is a trimeric RSV F protein. The RSV F protein induces the production of neutralizing antibodies. In further embodiments, the neutralizing antibodies recognize the RSV F protein in the post-fusion state and / or the pre-fusion state. In a further aspect, each PS80 particle may contain between 4 and 7 RSV F proteins.

[0010] In some embodiments, the RSV F composition may contain sodium phosphate at a concentration between 15 mM and 25 mM; NaCl at a concentration between 125 mM and 175 mM; and histidine between 0.25% and 2% w / v; and the pH of the composition is between 5.8 and 7.2.

[0011] In some embodiments, the HA or NA influenza composition may contain sodium phosphate at a concentration between 15 mM and 25 mM; NaCl at a concentration between 125 mM and 300 mM; and histidine between 0.25% and 2% w / v; and the pH of the composition is greater than pH 6.8 and typically less than about pH 8.0.

[0012] In some embodiments, the composition comprises an adjuvant. In further embodiments, the adjuvant is alum or Martrix M™. In some embodiments, the composition does not comprise an adjuvant.

[0013] In some embodiments, a method of preventing infection comprises administering one or more doses of a vaccine composition. In some embodiments of the method, a single dose of the composition is administered to induce a protective immune response. In some embodiments of the method, each dose consists of a protein antigen between about 100 μg and about 150 μg. In further embodiments of the method, the one or more doses are administered subcutaneously. In some embodiments of the method, the composition comprises an adjuvant. In further embodiments of the method, the adjuvant is alum. In some embodiments of the method, the composition does not comprise an adjuvant.

[0014] In some embodiments of the method, the one or more doses of the composition are administered to an adult. In further embodiments of the method, the adult is a female, and the female may be pregnant. In further embodiments of the method, the adult is over 65 years old, or over 60 years old. In some embodiments of the method, the one or more doses of the composition are administered to a pediatric patient. In further embodiments of the method, the pediatric patient is a neonate or an infant.

[0015] For an RSV vaccine, in some embodiments, the composition comprises a heterogeneous population of at least three RSV F nanoparticle types, each nanoparticle comprising at least one RSV F protein trimer surrounded by a surfactant-containing core comprising PS80, and wherein a first RSV F nanoparticle type comprises anisotropic rods, a second RSV F nanoparticle type comprises spherical oligomers and a third RSV F nanoparticle type comprises an intermediate between anisotropic rods and spherical oligomers.

[0016] In some embodiments, a method of manufacturing RSV F protein nanoparticles includes preparing an RSV F protein extract from host cells using a first surfactant and replacing the first surfactant with a second surfactant, where the second surfactant is PS80 and the nanoparticles exhibit enhanced stability. In further embodiments of the method, the first surfactant is NP-9. In some embodiments of the method, the enhanced stability is selected from protease resistance, oxidative stress resistance, heat stress resistance, and resistance to agitation. In some embodiments of the method, the molar ratio of PS80:RSV F protein is from about 35 to about 65.

[0017] In some embodiments, the RSV F nanoparticles comprise one or more RSV F protein trimers associated with a PS80 surfactant core. The RSV F nanoparticles have an average diameter of from about 20 nm to about 60 nm as measured by dynamic light scattering. RSV In some embodiments of the RSV F nanoparticles, each RSV F protein trimer comprises an RSV F protein selected from the group consisting of RSV F proteins having a deletion of 1 to 10 amino acids corresponding to residues 137-146 of SEQ ID NO: 2. In some embodiments of the RSV F nanoparticles, each RSV F protein trimer comprises an RSV F protein selected from the group consisting of RSV F proteins having a deletion of 1 to 10 amino acids corresponding to residues 137-146 of SEQ ID NO: 2 and an inactivated major fusion cleavage site.

[0018] In some embodiments of the RSV F nanoparticles, the RSV F protein comprises inactivation of the major furin cleavage site by deletion of residues 137-146 or 10 amino acids corresponding to SEQ ID NO: 2, and mutation of arginine residues at positions 133, 135, and 136 to glutamine. In further embodiments of the RSV F nanoparticles, the RSV F protein comprises or consists of SEQ ID NO: 19, which is the mature peptide. In certain embodiments of the RSV F nanoparticles, the RSV F protein comprises or consists of SEQ ID NO: 8. RSV The vaccine formulation containing F nanoparticles consists essentially of a mature peptide (SEQ ID NO: 8) having some full-length peptide. Small amounts of truncated RSV F peptides may occur over time due to proteolysis. Advantageously, however, the RSV F nanoparticles disclosed herein minimize such degradation and provide long-term stability.

[0019] This application also discloses influenza nanoparticles with enhanced thermal stability. Unlike the previous influenza nanoparticles, the methods and compositions provided herein exhibit resistance to trypsin as well as enhanced thermal stability and thereby immunogenicity.

[0020] For Ebola, the Ebola virus nanoparticles include a vaccine composition containing Ebola virus glycoprotein (GP) trimers attached to a non-ionic surfactant core and nanoparticles, optionally in combination with a matrix M saponin adjuvant. In addition, the present disclosure provides a method of inducing an immune response against Ebola virus in humans by administering a composition containing Ebola virus nanoparticles and a saponin adjuvant. A method of protecting against Ebola infection is also provided.

[0021] Similarly, nanoparticles containing either or both of the influenza proteins, HA, NA are provided. HA nanoparticles are provided that exhibit an indicator of proper folding, trypsin resistance. A method of protecting against influenza infection using influenza nanoparticles in a vaccine formulation is also provided. The present invention provides, for example, the following items. (Item 1) (i) Nanoparticles comprising a non-ionic surfactant core and a viral glycoprotein, wherein the viral glycoprotein is associated with the core and the surfactant is present at from about 0.03% to about 0.05%; and (ii) A pharmaceutically acceptable buffer A vaccine composition comprising, wherein the nonionic surfactant is selected from the group consisting of PS20, PS40, PS60, PS65 and PS80. (Item 2) The vaccine composition according to item 1, wherein the viral glycoprotein is selected from the group consisting of RSV F protein, Ebola glycoprotein, rabies virus G protein, influenza HA protein, influenza NA protein, and combinations thereof. (Item 3) The vaccine composition according to item 2, wherein the viral glycoprotein is RSV F protein, and the molar ratio of the nonionic surfactant to the viral glycoprotein is from about 30:1 to about 60:1. (Item 4) The vaccine composition according to item 3, wherein the RSV F protein contains a 10 - amino acid deletion corresponding to amino acids 137 - 146 of SEQ ID NO: 2 and an inactivated major furin cleavage site corresponding to amino acids 131 - 136 of SEQ ID NO: 2, and the major furin cleavage site is inactivated by mutation. (Item 5) The vaccine composition according to item 4, which does not substantially contain any other surfactant. (Item 6) The vaccine composition according to item 4, wherein the RSV - F protein is selected from the group consisting of SEQ ID NO: 1 - 13 and variants of SEQ ID NO: 1 - 13 lacking part or all of the N - terminal signal peptide. (Item 7) The vaccine composition according to item 6, wherein the RSV F protein contains SEQ ID NO: 19. (Item 8) The vaccine composition according to item 6, wherein the RSV F protein consists of SEQ ID NO: 19. (Item 9) The vaccine composition according to item 8, wherein the nonionic surfactant is PS80. (Item 10) The vaccine composition according to item 9, wherein the molar ratio is about 50. (Item 11) The pharmaceutically acceptable buffer is (i) Sodium phosphate from 15 mM to 25 mM; (ii) Approximately 150 mM of NaCl; (iii) Histidine from 0.25% to 2% w / v The vaccine composition according to item 1, comprising, wherein the pH of the composition is between 5.8 and 6.4. (Item 12) The pharmaceutically acceptable buffer is (i) Approximately 22 mM of sodium phosphate; (ii) Approximately 150 mM of NaCl; (iii) Approximately 1% of histidine The vaccine composition according to item 11, comprising, wherein the pH of the composition is approximately 6.2. (Item 13) The vaccine composition according to any one of items 1 to 12, wherein the RSV F protein further comprises a fatty acid linked to the protein. (Item 14) The vaccine composition according to item 1, wherein the nanoparticles have a Z-average diameter of about 20 nm to about 60 nm. (Item 15) The vaccine composition according to item 2, comprising the RSV F protein at a concentration between about 60 μg / mL and about 290 μg / mL. (Item 16) The vaccine composition according to item 1, wherein the shape of the nanoparticles is a spherical oligomer or an anisotropic rod. (Item 17) The vaccine composition according to any one of items 1 to 16, determined by analytical ultracentrifugation and substantially free of any other surfactant. (Item 18) The vaccine composition according to item 10, wherein each nanoparticle contains between 4 and 7 RSV F protein trimers. (Item 19) A method for preventing viral infection, comprising administering to a subject the vaccine composition according to any one of items 1 to 18. (Item 20) The method according to item 19, wherein the composition is administered intramuscularly. (Item 21) The method according to item 19, comprising RSV F protein with each dosage between about 30 μg and about 150 μg. (Item 22) The method according to item 19, wherein a single dosage of said composition is administered. (Item 23) The method according to item 19, wherein the subject is selected from the group consisting of adults, elderly adults, pregnant women, children, neonates, and infants. (Item 24) The method according to item 19, wherein said composition contains an adjuvant. (Item 25) The method according to item 24, wherein the adjuvant is an alum adjuvant and at least 80% of said adjuvant is bound to said nanoparticles. (Item 26) The method according to item 19, wherein said composition does not contain an additional adjuvant. (Item 27) The method according to item 22, wherein the dosage is a volume of about 0.5 mL. (Item 28) A composition comprising a heterogeneous population of at least three RSV F nanoparticle types, each nanoparticle comprising at least one RSV F protein trimer surrounding a surfactant-containing core comprising PS80; and The first RSV F nanoparticle type comprises anisotropic rods, The second RSV F nanoparticle type comprises spherical oligomers, and The third RSV F nanoparticle type comprises an intermediate between anisotropic rods and spherical oligomers. (Item 29) The composition according to item 28, wherein each RSV F trimer is selected from the group consisting of RSV F proteins having a deletion of 1 to 10 amino acids corresponding to residues 137 to 146 of SEQ ID NO: 2 and mixtures thereof. (Item 30) Each RSV F trimer is (i) 1 to 10 corresponding to residues 137 to 146 of SEQ ID NO: 2 The composition according to item 28, comprising an RSV F protein having an amino acid deletion and (ii) an inactivated major fusion cleavage site, and mixtures thereof. (Item 31) The composition according to item 29, wherein the RSV F protein is selected from SEQ ID NOs: 1 to 13 and variants of SEQ ID NOs: 1 to 13 lacking a part or all of the N-terminal signal peptide. (Item 32) The composition according to item 31, wherein the RSV F protein comprises SEQ ID NO: 19. (Item 33) The composition according to item 31, wherein the RSV F protein consists of SEQ ID NO: 19. (Item 34) A method for producing RSV F protein nanoparticles, comprising the steps of preparing an RSV F protein extract from host cells using a first surfactant and replacing the first surfactant with a second surfactant, wherein the second surfactant is PS80 and the nanoparticles exhibit enhanced stability when maintained in an amount of about 0.03% to about 0.05% of PS80. (Item 35) The method according to item 34, wherein the first surfactant is NP-9. (Item 36) The method according to item 34, wherein the enhanced stability is selected from one or more of protease resistance, oxidative stress resistance, heat stress resistance, and resistance to agitation. (Item 37) The method according to item 34, wherein the PS80 is present at a molar ratio of about 30 to about 60 with respect to the RSV F protein. (Item 38) Nanoparticles comprising a viral protein trimer associated with a PS80 surfactant core, wherein the molar ratio of PS80:the viral protein trimer is from about 30 to about 60, preferably about 50. (Item 39) The nanoparticles according to item 38, wherein the viral protein trimer is an RSV F protein trimer. (Item 40) The nanoparticle according to item 38, having an average diameter (Z-average) of about 20 nm to about 60 nm as measured by dynamic light scattering. (Item 41) The nanoparticle according to item 38, comprising an RSV F protein selected from the group consisting of RSV F proteins having a deletion of 1 to 10 amino acids corresponding to residues 137 to 146 of SEQ ID NO: 2. (Item 42) The nanoparticle according to item 41, further comprising an inactivated major fusion cleavage site. (Item 43) The nanoparticle according to item 39, wherein the RSV F protein has any of SEQ ID NOs: 1 to 13, or a sequence of SEQ ID NOs: 1 to 13 lacking a part or all of the N-terminal signal peptide. (Item 44) The nanoparticle according to item 39, wherein the RSV F protein comprises inactivation of the major furin cleavage site by deletion of 10 amino acids corresponding to residues 137 to 146 of SEQ ID NO: 2 and mutation of arginine residues at positions 133, 135, and 136 to glutamine. (Item 45) The nanoparticle according to item 44, wherein the RSV F protein comprises SEQ ID NO: 19. (Item 46) The nanoparticle according to item 44, wherein the RSV F protein consists of SEQ ID NO: 19. (Item 47) A method for producing a nanoparticle comprising a protein antigen surrounding a non-ionic surfactant core, comprising: (i) binding a protein extract containing a first surfactant to a protein purification column; (ii) performing surfactant exchange by substantially replacing the first surfactant with a second surfactant; and (iii) eluting the protein extract from the column in the presence of the second surfactant to provide the nanoparticle. comprising The molar ratio of the second surfactant to the protein is from about 30 to about 60; and the second surfactant is selected from the group consisting of PS20, PS40, PS60, PS65, and PS80, Method. (Item 48) The method according to item 47, wherein the protein antigen is a viral glycoprotein. (Item 49) The method according to item 48, wherein the nonionic surfactant is PS80. (Item 50) The method according to item 47, wherein the first surfactant is NP-9. (Item 51) The method according to item 47, wherein the protein antigen is RSV-F glycoprotein. (Item 52) The method according to item 47, wherein the molar ratio is from about 45 to about 60. (Item 53) The method according to item 52, wherein the molar ratio is about 45. (Item 54) (iv) Further comprising the step of providing a composition comprising nanoparticles, wherein the nanoparticles are combined with an alum adjuvant and at least 80% of the nanoparticles in the composition are bound to the alum, the method according to item 47. (Item 55) The method according to item 54, wherein at least 90% of the nanoparticles are bound to the alum. (Item 56) The protein extract is prepared by steps including recombinantly expressing the protein in Sf9 host cells and treating the host cells with the first surfactant to provide a protein extract, wherein the Sf9 cells are single knockout for cathepsin L or chitinase, or double knockout for both cathepsin L and chitinase, the method according to item 47. (Item 57) The method according to item 47, wherein the protein purification column is a lentil lectin column. (Item 58) A combination nanoparticle comprising a nonionic surfactant core and at least two viral proteins, each viral protein being associated with said core, and having a molar ratio of nonionic surfactant:at least one viral protein of from about 30 to about 60. (Item 59) The combination nanoparticle according to item 58, comprising an influenza HA protein and an RSV F protein. (Item 60) The combination nanoparticle according to item 58, comprising an influenza HA protein and an influenza NA protein. (Item 61) The combination nanoparticle according to item 60, wherein said HA protein and said NA protein are from the same influenza subtype. (Item 62) The combination nanoparticle according to item 60, wherein said HA protein and said NA protein are from different influenza subtypes. (Item 63) The combination nanoparticle according to item 60, wherein at least one of said HA protein and said NA protein is from influenza group 1 or group 2. (Item 64) The combination nanoparticle according to item 60, wherein at least one of said HA protein and said NA protein is from influenza B. (Item 65) A method of inducing an immune response, comprising the step of administering the composition according to item 1 to a subject. (Item 66) The method according to item 65, wherein said subject is a human female. (Item 67) The method according to item 66, wherein said human female is pregnant. (Item 68) A method of boosting an immune response in an infant, comprising the step of administering the composition according to item 1 to an infant having anti-RSV F antibodies. (Item 69) A method for preparing recombinant trypsin-resistant influenza HA nanoparticles, comprising: (i) binding a protein extract containing a first surfactant to a protein purification column; (ii) performing surfactant exchange by substantially replacing the first surfactant with a second surfactant; and (iii) eluting the protein extract from the column in the presence of the second surfactant to provide the nanoparticles wherein the transition midpoint (Tm) of the nanoparticles measured by differential scanning calorimetry is at least about 60 and a buffer having a pH of less than 7.0 is not used during preparation. A method. (Item 70) Recombinant trypsin-resistant HA nanoparticles prepared by the method of item 69. (Item 71) A vaccine composition comprising the nanoparticles of item 70 and a pharmaceutically acceptable carrier. (Item 72) A vaccine composition comprising the nanoparticles of item 71 and an adjuvant. (Item 73) The method of item 35, wherein the exchange of the first surfactant is substantially completed such that the amount of NP-9 detected by HPLC is less than about 0.1%, less than about 0.01%, less than about 0.001% or undetectable. (Item 74) Ebola virus glycoprotein (GP) nanoparticles comprising Ebola virus GP trimers associated with a nonionic surfactant core (Item 75) The nanoparticles of item 74, wherein the nonionic surfactant in the core comprises polysorbate-80 (PS80). (Item 76) The nanoparticles of item 74 having an average diameter of about 20 nm to about 40 nm. (Item 77) ​​The nanoparticle according to item 74, containing multiple copies of the Ebola virus GP trimer. (Item 78) The nanoparticle according to item 77, containing up to 15 trimers. (Item 79) The nanoparticle according to item 74, produced in Sf9 cells. (Item 80) The nanoparticle according to item 74, wherein the amino acid sequence of the Ebola virus GP is about 85%, about 90%, about 95%, about 97% or about 98% identical to SEQ ID NO: 29. (Item 81) The nanoparticle according to item 74, wherein the amino acid sequence of the Ebola virus GP contains SEQ ID NO: 29. (Item 82) The nanoparticle according to item 74, wherein the amino acid sequence of the Ebola virus GP consists of SEQ ID NO: 29. (Item 83) A vaccine composition comprising the nanoparticle according to any one of items 74 to 82 and a saponin adjuvant, wherein the saponin adjuvant consists of Matrix A and Matrix C. (Item 84) The vaccine composition according to item 83, wherein the Matrix A and the Matrix C are present in a ratio of 85:15. (Item 85) A method for inducing an immune response against the Ebola virus in a human, comprising administering to the human a composition comprising an Ebola virus glycoprotein (GP) nanoparticle and a saponin adjuvant, wherein the saponin adjuvant consists of Matrix A and Matrix C, and the nanoparticle comprises an Ebola virus GP trimer attached to a non-ionic surfactant core. Method. (Item 86) The method according to item 85, wherein the Matrix A saponin adjuvant and the Matrix C saponin adjuvant are present in a ratio of 85:15. (Item 87) The method according to item 85, wherein the amino acid sequence of said Ebola virus GP is about 85%, about 90%, about 95%, about 97% or about 98% identical to SEQ ID NO: 29. (Item 88) The method according to item 85, wherein the composition is administered intramuscularly. (Item 89) The method according to item 85, wherein the immune response comprises one or more of: induction of IgG1 antibody, induction of IgG2a antibody, formation of long-lived plasma cells, follicular helper T cell (TFH) response, CD4+ T cell response and CD8+ T cell response. (Item 90) The method according to item 85, wherein the composition comprises a heterogeneous population of GP nanoparticles having 2 to 6 trimers per nanoparticle. (Item 91) A method for preventing Ebola virus infection or disease in a human, comprising the step of intramuscularly administering the vaccine composition according to item 83 to the human. (Item 92) A nucleic acid encoding an Ebola protein comprising or consisting of SEQ ID NO: 29. (Item 93) An Sf9 host cell comprising the nucleic acid according to item 92.

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DETAILED DESCRIPTION OF THE INVENTION

[0072] Nanoparticles for inducing an immune response, methods for producing and administering them, and vaccine compositions containing them are disclosed herein. The nanoparticles surround a surfactant core that results in a structure that provides enhanced stability by a number of means and provides associated antigens. The surfactant core and the antigen associate through physicochemical interactions mediated by the antigen and surfactant properties. In addition, the nanoparticles, without being bound by theory, are thought to give the immune system particularly good antigen presentation resulting from the orientation of the antigen around the surfactant core.

[0073] In one aspect, the present disclosure provides a composition containing recombinant virus glycoprotein nanoparticles. In a specific aspect, the glycoprotein is recombinantly expressed in a suitable host cell. In one embodiment, the host cell is an insect cell. In an exemplary embodiment, the insect cell is an Sf9 cell.

[0074] In certain embodiments, the present disclosure provides an immunogenic composition comprising one or more viral glycoprotein molecular species within a nanoparticle structure, wherein the glycoprotein is in trimeric form and each nanoparticle contains at least one trimer associated with a nonionic surfactant core. In certain embodiments, the nanoparticle consists of an antigen, such as a viral glycoprotein, from only one pathogen.

[0075] The nanoparticles can be used for the prevention and / or treatment of viral infections. Thus, in another embodiment, the present disclosure provides a method for inducing an immune response against a virus. The method includes administering to a subject an immunologically effective amount of a composition containing the nanoparticles.

[0076] The present disclosure provides a vaccine composition comprising nanoparticles. The composition can contain nanoparticles having antigens from multiple pathogens. In some embodiments, the vaccine composition can contain nanoparticles comprising antigens from more than one viral strain from the same virus species. In an embodiment, the vaccine composition can contain nanoparticles comprising antigens from different virus species. In another embodiment, the present disclosure provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the components of the vaccine composition.

[0077] In another embodiment, the present disclosure includes adding an effective dose of nanoparticles to a composition The present disclosure provides a method of formulating a vaccine composition that induces immunity in a mammal against an infection or at least one of its disease symptoms. The disclosed nanoparticles are useful for preparing a composition that stimulates an immune response that confers immunity or substantial immunity against an infectious agent. Thus, in one embodiment, the present disclosure provides a method of inducing immunity in a subject against an infection or at least one of its disease symptoms, the method including administering at least one effective dose of the nanoparticles.

[0078] In some embodiments, the nanoparticles are administered with an adjuvant. In other embodiments, the nanoparticles are administered without an adjuvant. In some embodiments, the adjuvant may bind to the nanoparticles, such as by non-covalent interactions. In other embodiments, the adjuvant is co-administered with the nanoparticles, but the adjuvant and the nanoparticles do not substantially interact with each other.

[0079] Similarly, methods of making the nanoparticles and vaccine compositions are provided herein. Advantageously, the methods provide nanoparticles that are substantially free of contamination by other proteins, such as proteins associated with the recombinant expression of proteins in the baculovirus / Sf9 system.

[0080] Definitions As used herein and in 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 protein" may refer to one protein or a mixture of such proteins, reference to "the method" includes reference to equivalent steps and / or methods known to those of ordinary skill in the art, and the like.

[0081] As used herein, the term "adjuvant" refers to a compound that, when used in combination with an immunogen, enhances, otherwise modifies, or alters the immune response induced against the immunogen. Modification of the immune response may include strengthening or broadening the specificity of either or both of the antibody and cellular immune responses.

[0082] As used herein, the term "about" or "approximately," when preceding a numerical value, indicates a range of plus or minus 10% of the value. For example, "about 100" includes 90 and 110.

[0083] As used herein, the terms "immunogen", "antigen", and "epitope" refer to substances such as proteins and peptides, including glycoproteins, that can elicit an immune response.

[0084] As used herein, an "immunogenic composition" is a composition that contains an antigen, and administration of the composition to a subject results in the development in the subject of a humoral and / or cellular immune response to the antigen.

[0085] As used herein, a "subunit" composition, such as a vaccine, contains one or more selected antigens, but not all antigens are derived from a pathogen. Such compositions typically do not substantially contain intact viruses or lysates of such cells or particles and are typically prepared from immunogenic polypeptides that are at least partially purified, often substantially purified, from a pathogen. The antigens in the subunit compositions disclosed herein are typically prepared recombinantly, often using a baculovirus system.

[0086] As used herein, "substantially" refers to the isolation of a substance such that the substance forms the majority percentage in the sample in which it is contained (e.g., a compound, polynucleotide, or polypeptide ). For example, in a sample, a substantially purified component constitutes 85%, preferably 85% - 90%, more preferably at least 95% - 99.5%, and most preferably at least 99% of the sample. When a component is substantially replaced, the remaining amount in the sample is from about 0.5% to less than or equal to about 10%, preferably from about 0.5% to less than about 1.0%.

[0087] As used herein, the terms "treat", "treatment" and "treating" refer to an approach for obtaining beneficial or desirable results, such as clinical results. For the purposes of the present disclosure, beneficial or desirable results may include inhibiting or suppressing the induction or progression of an infection or disease; ameliorating or reducing the occurrence of symptoms of an infection or disease; or combinations thereof.

[0088] As used herein, "prevention" is used interchangeably with "prophylaxis" and can mean complete prevention of an infection or disease, or prevention of the occurrence of symptoms of that infection or disease; delay in the onset of an infection or disease or its symptoms; or reduction in the severity of an infection or disease or its symptoms that occur later.

[0089] As used herein, the term "effective dose" or "effective amount" refers to an amount of an immunogen sufficient to induce an immune response that reduces at least one symptom of a pathogen infection. The effective dose or effective amount can be determined, for example, by measuring the amount that neutralizes secreted and / or serum antibodies, such as by plaque neutralization, complement fixation, enzyme-linked immunosorbent assay (ELISA) or micro-neutralization assay.

[0090] As used herein, the term "vaccine" refers to an immunogenic composition, such as an immunogen derived from a pathogen, used to induce an immune response against the pathogen that provides protective immunity (e.g., immunity that protects a subject against infection by a pathogen and / or reduces the severity of a disease or condition caused by infection by a pathogen). The protective immune response may include the formation of antibodies and / or a cell-mediated response. Depending on the context, the term "vaccine" may also refer to a suspension or solution of an immunogen administered to a vertebrate to produce protective immunity.

[0091] As used herein, the term "subject" includes humans and other animals. Typically the subject is human. For example, the subject may be an adult, a teenager, a child (2 to 14 years old), an infant (1 to 24 months) or a neonate (up to 1 month). In some embodiments, an adult is an elderly person about 65 years of age or older or about 60 years of age or older. In some embodiments, the subject is a pregnant woman or a woman intending to become pregnant. In other embodiments, the subject is not human; for example, a non-human primate; for example, a baboon, chimpanzee, gorilla or macaque. In certain embodiments, the subject may be a pet such as a dog or a cat.

[0092] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the United States Federal or State government or listed in the U.S. Pharmacopeia, European Pharmacopeia or other generally recognized pharmacopeias for use in mammals, more particularly in humans. These compositions can be useful as vaccines and / or antigenic compositions for inducing a protective immune response in vertebrates.

[0093] As used herein, the term "about" means plus or minus 10% of the indicated numerical value.

[0094] Summary Pathogen-derived antigens are combined with non-ionic surfactants to provide nanoparticles that surround a surfactant core and have improved stability and excellent immunogenicity. The present disclosure also provides methods and compositions for vaccinating a subject against a pathogen. In certain embodiments, the pathogen is a virus. The antigen is typically a protein, often a glycoprotein. Similarly, compositions containing nanoparticles that have been found to be useful as vaccine compositions are disclosed. Methods for producing the nanoparticles and for producing the vaccine compositions are also disclosed.

[0095] Nanoparticle Structure and Morphology The nanoparticles of the present disclosure contain an antigen associated with a non-ionic surfactant core. The panel on FIG. 6 exemplifies an example of a plurality of RSV F antigens associated with the surfactant core. FIG. 35 shows Ebola nanoparticles. Advantageously, the nanoparticles have improved resistance to environmental stress such that they provide enhanced stability.

[0096] In certain embodiments, the nanoparticles are composed of a plurality of protein trimers surrounding a non-ionic surfactant core. For example, each nanoparticle may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 15 trimers. Typically, each nanoparticle contains from 2 to 9 trimers. In certain embodiments, each nanoparticle contains from 2 to 6 trimers. The compositions disclosed herein may contain nanoparticles having different numbers of trimers. For example, the composition may contain nanoparticles in which the number of trimers ranges from 2 to 9; in other embodiments, the nanoparticles in the composition may contain from 2 to 6 trimers. In certain embodiments, the composition contains a heterogeneous population of nanoparticles having from 2 to 6 trimers per nanoparticle, or from 2 to 9 trimers per nanoparticle. In other embodiments, the composition may contain a substantially homogeneous population of nanoparticles. For example, the population may contain nanoparticles about 95% of which have 5 trimers.

[0097] The antigen associates with the non-ionic surfactant-containing core of the nanoparticle. Typically, the surfactant is selected from polysorbate-20 (PS20), polysorbate-40 (PS40), polysorbate-60 (PS60), polysorbate-65 (PS65), and polysorbate-80 (PS80). The presence of the surfactant promotes the formation of the nanoparticles by forming a core that organizes and presents the antigen. Thereby, in certain embodiments, the nanoparticles may contain an antigen assembled into a multi-oligomeric glycoprotein-PS80 protein-surfactant nanoparticle that includes an outer protruding head region and a hydrophobic region, and in which the PS80 surfactant forms a central core surrounded by the antigen.

[0098] The nanoparticles disclosed herein are in the range of Z-average sizes from about 20 nm to about 60 nm, from about 20 nm to about 50 nm, from about 20 nm to about 45 nm or from about 25 nm to about 45 nm. The particle size (Z-average) is measured by dynamic light scattering (DLS) using a Malvern Zetasizer unless otherwise specified.

[0099] Several nanoparticle types may be included in the vaccine compositions disclosed herein. In some embodiments, the nanoparticle type is in the form of anisotropic rods, which may be dimers or monomers. In other embodiments, the nanoparticle type is a spherical oligomer. In yet other embodiments, the nanoparticles may be described as intermediate nanoparticles having sedimentation characteristics intermediate between the first two types. The formation of the nanoparticle type can be controlled by managing the concentration of surfactant and protein during the production process. The nanoparticle type can be determined by measuring the sedimentation coefficient. See, for example, FIGS. 9A and 9B showing RSV F nanoparticles. Similarly, see FIG. 8, which illustrates managing the nanoparticle size by adjusting the concentration of surfactant and protein.

[0100] Nanoparticle Production The nanoparticles of the present disclosure are non-naturally occurring products, and their components do not naturally occur together. Generally, the methods disclosed herein use a surfactant exchange approach in which a first surfactant is used to isolate a protein and then that first surfactant is exchanged for a second surfactant to form the nanoparticles.

[0101] The antigen contained in the nanoparticles is typically produced by recombinant expression in a host cell. Standard recombinant techniques may be used. Typically, the protein is expressed in insect host cells using a baculovirus system. In a preferred embodiment, the baculovirus is a cathepsin-L knockout baculovirus. In other preferred embodiments, the baculovirus is a chitinase knockout baculovirus. In still other preferred embodiments, the baculovirus is a double knockout for both cathepsin-L and chitinase. High-level expression can be obtained in an insect cell expression system. Non-limiting examples of insect cells are Spodoptera frugiperda (Sf) cells, such as Sf9, Sf21, Trichoplusia ni cells, such as High Five cells and Drosophila S2 cells.

[0102] Typical transfection and cell growth methods can be used to culture the cells. A vector, such as a vector containing a polynucleotide encoding a fusion protein, can be transfected into a host cell by methods well known in the art. For example, introducing nucleic acid into eukaryotic cells can be achieved by transfection using calcium phosphate coprecipitation, electroporation, microinjection, lipofection, and polyamine transfection reagents. In one embodiment, the vector is a recombinant baculovirus.

[0103] Methods for growing host cells include, but are not limited to, batch, fed-batch, continuous, and perfusion cell culture techniques. Cell culture refers to the growth and propagation of cells in a bioreactor (fermentation chamber) where the cells multiply and express proteins (e.g., recombinant proteins) for purification and isolation. Typically, cell culture is carried out in a bioreactor under sterile, controlled temperature, and atmospheric conditions. A bioreactor is a chamber used for culturing cells where environmental conditions such as temperature, atmosphere, agitation, and / or pH can be monitored. In one embodiment, the bioreactor is a stainless-steel chamber. In another embodiment, the bioreactor is a pre-sterilized plastic bag (e.g., Cellbag®, Wave Biotech, Bridgewater, N.J.). In other embodiments, the pre-sterilized plastic bag is a bag from about 50 L to 3500 L.

[0104] Surfactant Extraction and Purification of Nanoparticles After growth of the host cells, the protein may be collected from the host cells using a surfactant and a purification protocol. After the host cells have grown for 48 to 96 hours, the cells are isolated from the medium and a surfactant-containing solution is added to solubilize the cell membrane and release the protein into the surfactant extract. Triton X-100 and tergitol, also known as NP-9, are each preferred surfactants for extraction. The surfactant may be added at a final concentration of from about 0.1% to about 1.0%. For example, the concentration may be about 0.1%, about 0.2%, about 0.3%, about 0.5%, about 0.7%, about 0.8%, or about 1.0%. In certain embodiments, the range may be from about 0.1% to about 0.3%. Preferably the concentration is about 0.5%.

[0105] In other embodiments, different first surfactants may be used to isolate the protein from the host cell. For example, the first surfactant may be bis(polyethylene glycol bis[imidazolylcarbonyl]), nonoxynol-9, bis(polyethylene glycol bis[imidazolylcarbonyl]), Brij® 35, Brij® 56, Br ij (Registered Trademark) 72, Brij (Registered Trademark) 76, Brij (Registered Trademark) 92V, Brij (Registered Trademark) 97, Brij (Registered Trademark) 58P, Cremophor (Registered Trademark) EL, decaethylene glycol monododecyl ether, N-decanoyl-N-methylglucamine, n-decyl alpha-D-glucopyranoside, decyl beta-D-maltopyranoside, n-dodecanoyl-N-methylglucamide, n-dodecyl alpha-D-maltoside, n-dodecyl beta-D-maltoside, n-dodecyl beta-D-maltoside, heptaethylene glycol monodecyl ether, heptaethylene glycol monododecyl ether, heptaethylene glycol monotetradecyl ether, n-hexadecyl beta-D-maltoside, hexaethylene glycol monododecyl ether, hexaethylene glycol monohexadecyl ether, hexaethylene glycol monooctadecyl ether, hexaethylene glycol monotetradecyl ether,Igepal CA-630, Igepal CA-630, methyl-6-0-(N-heptylcarbamoyl)-alpha-D-glucopyranoside, nonaethylene glycol monododecyl ether, N-nonanoyl-N-methylglucamine, N-nonanoyl N-methylglucamine, octaethylene glycol monodecyl ether, octaethylene glycol monododecyl ether, octaethylene glycol monohexadecyl ether, octaethylene glycol monooctadecyl ether, octaethylene glycol monotetradecyl ether, octyl-beta-D-glucopyranoside, pentaethylene glycol monodecyl ether, pentaethylene glycol monododecyl ether, pentaethylene glycol monohexadecyl ether, pentaethylene glycol monohexyl ether, pentaethylene glycol monooctadecyl ether, pentaethylene glycol monooctyl ether, polyethylene glycol diglycidyl ether, polyethylene glycol ether W-1, polyoxyethylene 10 tridecyl ether, polyoxyethylene 100 stearate, polyoxyethylene 20 isohexadecyl ether, polyoxyethylene 20 oleyl ether, polyoxyethylene 40 stearate, polyoxyethylene 50 stearate,Polyoxyethylene 8 stearate, polyoxyethylene bis(imidazolylcarbonyl), polyoxyethylene 25 propylene glycol stearate, saponin derived from Quillaja bark, Span® 20, Span® 40, Span® 60, Span® 65, Span® 80, Span® 85, Tergitol 15 - S - 12 type, Tergitol 15 - S - 30 type, Tergitol 15 - S - 5 type, Tergitol 15 - S - 7 type, Tergitol 15 - S - 9 type, Tergitol NP - 10 type, Tergitol NP - 4 type, Tergitol NP - 40 type, Tergitol NP - 7 type, Tergitol NP - 9 type, Tergitol TMN - 10 type, Tergitol TMN - 6 type, Triton X - 100 or combinations thereof may be used.

[0106] Next, the nanoparticles may be isolated from cell debris using centrifugation. In some embodiments, gradient centrifugation using cesium chloride, sucrose, iodixanol, etc. may be used. Other techniques such as standard purification techniques including ion exchange, affinity, and gel filtration chromatography may be used alternatively or additionally.

[0107] For example, the first column may be an ion exchange chromatography resin such as Fractogel® EMD TMAE (EMD Millipore), the second column may be a lentil (Lens culinaris) lectin affinity resin, and the third column may be a cation exchange column such as Fractogel® EMD SO3 (EMD Millipore) resin. In other embodiments, the cation exchange column may be an MMC column or a Nuvia C Prime column (Bio - Rad Laboratories, Inc). Preferably, the methods disclosed herein do not use a surfactant extraction column; for example, a hydrophobic interaction column. Such Columns are often used to remove surfactants during purification, but may have an adverse effect on the methods disclosed herein.

[0108] Surfactant exchange To form nanoparticles, the first surfactant used to extract the protein from the host cell is substantially replaced with a second surfactant that reaches the nanoparticle structure. NP-9 is the preferred extraction surfactant. Typically, the nanoparticles do not contain detectable NP-9 as measured by HPLC. The second surfactant is typically selected from the group consisting of PS20, PS40, PS60, PS65, and PS80. Preferably, the second surfactant is PS80. To maintain the stability of the nanoparticle formulation, the ratio of the second surfactant to the protein is maintained within a certain range.

[0109] In certain embodiments, surfactant exchange is performed using affinity chromatography that binds glycoproteins via their carbohydrate components. For example, affinity chromatography may use a legume lectin column. Legume lectins are proteins originally identified in plants and have been found to interact specifically and reversibly with carbohydrate residues. See, for example, Sharon and Lis, "Legume lectins--a large family of homologous proteins", FASEB J. November 1990; 4(14): 3198-3208; Liener, "The Lectins: Properties, Functions, and Applications in Biology and Medicine", Elsevier, 2012. Suitable lectins include concanavalin A (con A), pea lectin, jack bean lectin (lect), and lentil lectin. Lentil lectin is a preferred column for surfactant exchange due to its binding properties. See, for example, Example 10, lectin columns are commercially available; for example, Captolensil lectin is available from GE Healthcare. In certain embodiments, a lentil lectin column may use a recombinant lectin. At the molecular level, the carbohydrate component binds to the lentil lectin, releasing the amino acids of the protein and allowing it to assemble around the surfactant, resulting in the formation of a surfactant core and providing nanoparticles having a glycoprotein oligomer that may be multiple copies of an antigen, such as a dimer, trimer, or tetramer tethered to the surfactant.

[0110] When the protein is incubated with the surfactant to form nanoparticles during surfactant exchange, the surfactant may be present up to about 0.1% (w / v) during the initial purification step, and this amount is reduced to obtain the final nanoparticles with optimal stability. For example, a non-ionic surfactant (e.g., PS80) may be from about 0.03% to about 0.1%. Preferably, for improved stability, the nanoparticles contain from about 0.03% to about 0.05% of PS80. An amount of PS80 less than about 0.03% in the formulation does not show good stability. Further, when PS80 is present in excess of about 0.05%, aggregates are formed. Thus, from about 0.03% to about 0.05% of PS80 provides structural and stability benefits that enable the long-term stability of nanoparticles with reduced degradation.

[0111] Surfactant exchange may be performed on the protein that has been purified, purified, frozen for storage, and then thawed for surfactant exchange as discussed above.

[0112] Enhanced stability and enhanced immunogenicity of nanoparticles Without being bound by theory, associating the antigen with a non-ionic surfactant core is thought to provide excellent stability and antigen presentation. The nanoparticles disclosed herein provide surprisingly good stability and immunogenicity. The advantageous stability is particularly useful for vaccines used in countries where proper storage is not possible; for example, in certain locations in Africa, refrigeration may not be available, and thus vaccines against diseases prevalent in regions facing difficulties in storage conditions, such as the Ebola virus and RSV, may particularly benefit from improved stability. Further, HA influenza nanoparticles produced using a neutral pH approach exhibit better folding than known recombinant influenza vaccines. in various places; for example, in certain locations in Africa, refrigeration may not be possible, and thus vaccines against diseases prevalent in regions facing difficulties in storage conditions, such as the Ebola virus and RSV, may particularly benefit from improved stability. Further, HA influenza nanoparticles produced using a neutral pH approach exhibit better folding than known recombinant influenza vaccines.

[0113] Notably, prior approaches that use surfactants to produce RSV vaccines, including split vaccines such as those described in US2004 / 0028698 to Colau et al., were unable to produce an effective structure. Instead of nanoparticles having proteins surrounding a surfactant core as disclosed herein, the Colau et al. compositions contain amorphous materials lacking a discernible viral structure and are presumed to have been unable to effectively present epitopes to the immune system. Additionally, the disclosed nanoparticles have enhanced stability because the orientation of antigens, often glycoproteins, around the surfactant core sterically hinders contact with proteolytic enzymes and other chemicals that would cause proteolysis.

[0114] Nanoparticles are enhanced in stability as determined by their ability to maintain immunogenicity after exposure to various stresses. Stability can be measured in a variety of ways. In one approach, peptide maps may be generated to determine the integrity of the antigen protein after various treatments designed to stress the nanoparticles by mimicking harsh storage conditions. Thereby, a measure of stability is the relative abundance of antigen peptides in the stressed sample compared to a control sample. Figure 12 shows that a strong immune response is achieved even after various different stresses to the RSV F nanoparticle composition. Figure 13 illustrates the improvement in protease resistance provided by nanoparticles using PS80 at levels above 0.015%. Notably, at 18 months, 0.03% PS80 shows a 50% reduction in the formation of truncated molecular species compared to 0.015% PS80. The nanoparticles disclosed herein are stable at 2 - 8°C. Advantageously, however, they are stable at 25°C for at least 2 months. In some embodiments, the composition is stable at 25°C for at least 3 months, at least 6 months, at least 12 months, at least 18 months or at least 24 months. For RSV-F nanoparticles, stability can be determined by measuring the formation of truncated F1 protein as shown in Figure 13. Advantageously, the RSV-F nanoparticles disclosed herein, as shown in Figure 12, favorably retain intact antigenic site II at an abundance measured by peptide mapping from 90 to 100% compared to the control RSV-F protein in response to various stresses including low pH (pH 3.7), high pH (pH 10), temperature increase (50°C, 2 weeks) and further oxidation by peroxide.

[0115] The position of the glycoprotein tethered to the surfactant core is thought to provide enhanced stability by reducing unwanted interactions. For example, improved protection against protease-based degradation can be achieved through a shielding effect where tethering the glycoprotein to the core at the molar ratios disclosed herein creates a steric hindrance that blocks protease access.

[0116] Thus, in certain embodiments, RSV-F nanoparticles, and compositions containing the same, are disclosed herein that retain from 90% to 100% of intact Site II peptides in response to one or more treatments selected from the group consisting of incubation at 50° C. for 2 weeks, incubation at 25° C., pH 3.7 for 1 week, incubation at pH 10 for 1 week, agitation at 25° C. for 1 week, and incubation with an oxidizing agent such as hydrogen peroxide at 25° C. for 1 week. Additionally, after such treatment, the functionality of the composition is retained. See FIGS. 12A-12D. For example, neutralizing antibody, anti-RSV IgG, and PCA titers are preserved compared to controls.

[0117] Enhanced immunogenicity is exemplified by cross-neutralization achieved by influenza nanoparticles. The orientation of the influenza antigen protruding from the core is thought to provide more efficient presentation of epitopes to the immune system.

[0118] Nanoparticle antigen In typical embodiments, the antigen used to produce the nanoparticles is a viral protein. In some embodiments, the protein may be modified, but retains the ability to stimulate an immune response against the native peptide. In some embodiments, the protein essentially contains, or is adapted to contain, a transmembrane domain that facilitates association of the protein with the surfactant core. Often the protein is a native glycoprotein.

[0119] RSV antigen In one aspect, the virus is respiratory syncytial virus (RSV), and the viral antigen is the fusion (F) glycoprotein. The structure and function of the RSV F protein are well characterized. See FIG. 1 as an example of the wild-type structure. Suitable RSV-F proteins for use in the compositions described herein may be derived from RSV strains such as A2, Long, ATCC VR-26, 19, 6265, E49, E65, B65, RSB89-6256, RSB89-5857, RSB89-6190, and RSB89-6614. In certain embodiments, the RSV F protein is mutated compared to their native variants. These mutations confer desirable features such as improved protein expression, enhanced immunogenicity, etc. Additional information describing the RSV-F protein structure can be found in Swanson et al., A Monomeric Uncleaved Respiratory Syncytial Virus F Antigen Retains Prefusion-Specific Neutralizing Epitopes. Journal of Virology, 2014, 88, 11802-11810, Jason S. McLellan et al., Structure of RSV Fusion Glycoprotein Trimer Bound to a Prefusion-Specific Neutralizing Antibody. Science, 2013, 340, 1113-1117.

[0120] The major fusion cleavage is located at residues 131 to 136 corresponding to SEQ ID NO: 2. Inactivation of the major fusion cleavage site can be achieved by mutating the residues in the site, such that furin can no longer recognize the consensus site. For example, inactivation of the major furin cleavage site can be achieved by introducing at least one amino acid substitution at positions corresponding to arginine 133, arginine 135, and arginine 136 of the wild-type RSV F protein (SEQ ID NO: 2). In certain embodiments, all one, two, or three arginines are mutated to glutamine. In other embodiments, inactivation is achieved by mutating the wild-type site to one of the following sequences: KKQKQQ (SEQ ID NO: 14), QKQKQQ (SEQ ID NO: 15), KKQKRQ (SEQ ID NO: 16), and GRRQQR (SEQ ID NO: 17).

[0121] In certain embodiments, 1 to 10 amino acids corresponding to acids 137 to 145 of SEQ ID NO: 2 may be deleted, including the specific examples of suitable RSV F proteins shown below. Each of SEQ ID NOS: 3 to 13 may optionally be prepared to include the active major fusion cleavage site KKRKRR (SEQ ID NO: 18). The wild-type strain in SEQ ID NO: 2 has sequencing errors (A to P, V to I, and V to M) that are corrected in SEQ ID NOS: 3 to 13. Following expression of the RSV-F protein in host cells, the N-terminal signal peptide is cleaved to provide the final sequence. Typically, the signal peptide is cleaved by a host cell protease. However, in other embodiments, the full-length protein may be isolated from the host cell and subsequently the signal peptide is cleaved. The N-terminal RSV F signal peptide is SEQ ID NO: 26 (MELLILKA It consists of the amino acids of (NAITTILTAVTFCFASG). Thus, for example, following cleavage of the signal peptide from SEQ ID NO: 8 during expression and purification, a mature protein having the sequence of SEQ ID NO: 19 is obtained and is used to produce the RSV F nanoparticle vaccine. See Figure 1B. Optionally, one or up to all of the RSV F signal peptide amino acids may be deleted, mutated, the entire signal peptide may be deleted, or may be replaced with a different signal peptide to enhance expression. The start methionine residue is maintained to initiate expression. [Table 1]

[0122] In some embodiments, the RSV F protein disclosed herein is modified from the wild-type strain only by a deletion in the fusion domain, optionally with inactivation of the major cleavage site. In other embodiments, additional modifications to the RSV F protein may be made. Typically, cysteine residues are mutated. Typically, N-linked glycosylation sites are not mutated. See Figure 1B. In addition, antigenic site II, also referred to herein as the palivizumab site due to the ability of the palivizumab antibody to bind to this site, is conserved. The motavizumab antibody also binds to site II. Additional suitable RSV-F proteins incorporated by reference are found in U.S. Publication US2011 / 0305727 and specifically include RSV-F proteins containing the sequence spanning residues 100 to 150 disclosed in its Figure 1C.

[0123] In certain other embodiments, the RSV F1 or F2 domain may have modifications compared to the wild-type strain shown in SEQ ID NO: 2. For example, the F1 domain may have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 changes that may be mutations or deletions. Similarly, the F2 domain may have 1, 2, 3, 4, 5 , may have 6, 7, 8, 9, or 10 modifications. The F1 and F2 domains may each independently retain at least 90%, at least 94%, at least 95%, at least 96%, at least 98%, at least 99%, or 100% identity to the wild-type sequence.

[0124] In certain examples, the RSV nanoparticle drug product may contain from about 0.025% to about 0.03% of PS80 with RSV F in the range from about 270 μg / mL to about 300 μg / mL, or from about 60 μg / mL to about 300 μg / mL. In other embodiments, the nanoparticle drug product may contain from about 0.035% to about 0.04% of PS80 with RSV F from 300 μg / mL to about 500 μg / mL in the composition. In yet other embodiments, the nanoparticle drug product may contain from about 0.035% to about 0.04% of PS80 with RSV F from 350 - 500 μg / mL in the composition.

[0125] Since the concentrations of the antigen and surfactant may vary, each amount may be referenced as the molar ratio of non-ionic surfactant:protein. For example, the molar ratio of PS80 to the protein is calculated using the PS80 concentration and the protein concentration of the antigen measured by ELISA / A280 as well as their respective molecular weights. The molecular weight of PS80 used for the calculation is 1310, and using RSV F as an example, the molecular weight of RSV F is 65 kD. The molar ratio is calculated as follows: (PS80 concentration × 10 × 65000) ÷ (1310 × RSV F concentration (mg / mL)). Thereby, for example, as shown in FIG. 13, the nanoparticle concentration measured by the protein is 270 μg / mL, and the PS80 concentrations are 0.015% and 0.03%. These have molar ratios of PS80 to the RSV F protein of 27:1 (i.e., 0.015 × 10 × 65000 / (1310 × 0.27)) and 55:1, respectively.

[0126] In certain embodiments, the molar ratio ranges from about 30:1 to about 80:1, from about 30:1 to about 70:1, from about 30:1 to about 60:1, from about 40:1 to about 70:1, or from about 40:1 to about 50:1. Often the nonionic surfactant is PS80 and the molar ratio is PS80:protein, from about 30:1 to about 50:1. For RSV-F glycoprotein, nanoparticles having a molar ratio in the range of 35:1 to about 65:1, specifically a ratio of about 45:1, are particularly stable.

[0127] Influenza antigen The nanoparticle platform is particularly useful for presenting influenza antigens to the immune system of a subject. Previous approaches for producing influenza nanoparticle vaccines used hydrophobic interaction columns to remove surfactants or contained only minimal amounts of surfactants to reduce non-specific interactions that occur during product purification. However, it has been discovered here that by performing a surfactant exchange step, nanoparticles having a nonionic surfactant core with excellent properties can be produced. The nanoparticles exhibit excellent stability demonstrated by resistance to degradation by environmental stress, allowing for a long shelf life as a particularly useful property for vaccines. In addition, the nanoparticle structure presents the antigen in a particularly advantageous manner.

[0128] Influenza nanoparticles are particularly useful as vaccines because the antibodies they induce contain a broad range of neutralizing antibodies. Thus, antibodies induced by the nanoparticles administered in a given year can neutralize influenza virus strains resulting from the "drift" process in subsequent years. These epitopes that induce these broad neutralizing antibodies are thought to have not been exposed at all, or effectively exposed, or the epitopes were not sufficiently stable in previous formulations, in previous influenza vaccines. The nanoparticles disclosed herein solve these problems by presenting cross-protective epitopes tethered around a nonionic surfactant core with enhanced stability.

[0129] Finally, the methods disclosed herein provide influenza nanoparticles with good purity in particularly high yields, which are generally economically advantageous and particularly important for viruses that require large-scale and rapid production, such as pandemic influenza viruses.

[0130] In certain embodiments, the nanoparticles may contain HA or NA proteins. For example, the nanoparticles may contain an HA protein selected from subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, and H16. The nanoparticles may contain an NA protein selected from subtypes N1, N2, N3, N4, N5, N6, N7, N8, and N9. Phylogenetically, the HA and NA proteins are grouped. For HA, group 1 contains H1, H2, H5, H6, H8, H9, H11, H12, H13, and H16, and group 2 contains H3, H4, H7, H10, H14, and H15. NA also forms two groups: group 1 contains N1, N4, N5, and N8, and group 2 contains N2, N3, N6, N7, and N9. In certain aspects, the antigen may have at least 90% identity, at least 95% identity, at least 97% identity, or at least 99% identity to a native influenza HA or NA protein.

[0131] The HA and NA proteins used for the nanoparticles are typically full-length sequences. In certain aspects, a portion of the C-terminus may be removed.

[0132] Advantageously, the influenza compositions can induce a response against heterologous strains of influenza even when co-administered with the additional pathogen nanoparticles disclosed herein. By inducing a response against heterologous influenza strains, broad protection is achieved. Thereby in certain embodiments, the homologous HAI titers induced by the matrix M-adjuvanted compositions are from about 800 to about 2000. In a specific embodiment, the heterologous HAI titer is about 1300. In certain embodiments, the heterologous HAI titers induced by the matrix M-adjuvanted compositions are from about 200 to about 400; for example, the heterologous HAI titer may be about 300.

[0133] In certain embodiments, the influenza nanoparticles are trypsin-resistant nanoparticles produced using neutral pH purification. Trypsin resistance is achieved by a neutral pH range above 6.8 and up to 8.5 during purification and formulation of the HA nanoparticles. In certain embodiments, the pH range during purification and formulation of the HA nanoparticles is from 7.0 to 8.5, from 7.0 to 7.5 or from 7.2 to 7.5. HA nanoparticle stability can be measured by differential scanning calorimetry (DSC). DSC measures the thermodynamic profile of a macromolecule in solution by specifically measuring the difference in heat energy uptake between a sample solution and an appropriate reference (buffer / solvent) by varying the temperature in a controlled manner. DSC provides data as a transition midpoint (Tm) defined as the temperature at which half of the protein is denatured / unfolded and half is in the non-denatured / folded state. In certain embodiments, the trypsin-resistant HA nanoparticles herein have a Tm peak within the range of about 60 °C to 75 °C; for example, the Tm may be 60 °C, 65 °C, 70 °C or 75 °C.

[0134] Trypsin resistance indicates that the HA protein is properly folded, thereby providing a vaccine product with better stability and immunogenicity. The sensitivity of the HA protein varies by strain, and thus the neutral pH production disclosed herein provides a process for maximizing immunogenicity for all strains, particularly pH-sensitive strains. Without being bound by theory, it is believed that the combination of surfactant exchange and neutral pH levels maintains the HA protein in a structure that is resistant to proteases, particularly trypsin. Thereby, neutral pH purification and in combination, by associating the HA protein around a nonionic surfactant core, particularly good stability and immunogenicity of the HA protein are achieved. In addition, the method of producing nanoparticles provides an excellent level of protein for use in vaccines. In certain embodiments, HA nanoparticles are produced that are measured by A280 to be from about 10 mg to about 30 mg, or higher, from about 20 mg to about 30 mg per liter of cell culture.

[0135] Trypsin-resistant HA nanoparticles may be prepared as described in FIG. 24. Briefly, the various steps, including surfactant exchange, are carried out at a pH above 7.0; often in a buffer in the range of about pH 7.2 to about pH 7.4. FIG. 25D provides an example of the better thermal stability achieved with trypsin-resistant nanoparticles. TFF and MMC production lots are obtained using neutral pH, while misfolded low pH lots are substantially degraded and / or misfolded.

[0136] Ebola antigen The present disclosure also provides methods and compositions for treating, ameliorating, or preventing Ebola virus infection and / or disease. Specifically, the composition is a vaccine composition. Advantageously, the vaccine compositions disclosed herein provide 100% survival to a lethal challenge in an animal model. The composition also maintains a viral load at or near the limit of detection when using RT-PCR to detect viral nucleic acids.

[0137] In one aspect, the present disclosure provides a composition comprising recombinant Ebola virus glycoprotein (GP) nanoparticles in combination with a saponin-based adjuvant.

[0138] In certain aspects, the present disclosure provides an immunogenic composition comprising one or more Ebola virus GP proteins in a nanoparticle structure where the GP protein is in trimeric form and each nanoparticle contains at least one trimer attached to a non-ionic surfactant core.

[0139] The Ebola GP nanoparticles may be used for the prevention and / or treatment of Ebola infection. In another aspect, the present disclosure provides a pharmaceutically acceptable vaccine composition comprising Ebola GP nanoparticles. In some aspects, there are nanoparticles from more than one strain in the vaccine. In another embodiment, the present disclosure provides a pharmaceutical pack or kit comprising one or more containers filled with one or more vaccine formulation ingredients.

[0140] Ebola glycoprotein The Ebola antigen used to prepare the nanoparticles is typically an Ebola glycoprotein (GP) antigen. The antigen may be from various strains. The compositions disclosed herein may contain nanoparticles from 1, 2, 3, 4, 5, or 6 separate Ebola strains. For example, the strain may be Makona, Sudan, Zaire, Reston. In other aspects, the Ebola GP may share amino acid or nucleic acid identity with one or more of these strains. For example, the GP may be about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to one or more GPs from Makona, Sudan, Zaire, or Reston virus, where the identity is measured over the full length of the protein or nucleic acid. In some aspects, the Ebola GP may comprise or consist of SEQ ID NO: 27 or 28 or a protein having identity thereto.

[0141] The representative Zaire strain sequence is provided at GenBank accession number AAB81004 (SEQ ID NO: 27). The first underlined portion indicates the N-terminus of the GP1 protein. The preceding signal peptide is cleaved and removed during processing in cells prior to purification and formulation into a vaccine. The furin cleavage site is indicated in bold. Following the bold string, the N-terminus of the GP2 protein is shown. Figure 7A shows a schematic diagram of the protein structure. It is removed by cleavage during subsequent processing that follows expression in cells prior to purification and formulation into a vaccine. The furin cleavage site is indicated in bold. Following the bold string, the N-terminus of the GP2 protein is shown. Figure 7A shows a schematic diagram of the protein structure. [Chemical Formula]

[0142] The Makona isolate sequence is provided at GenBank accession number AJG44192 (SEQ ID NO: 28). Similar to the above, the first underlined portion indicates the N-terminus of the GP1 protein. The preceding signal peptide is cleaved and removed during processing. The furin cleavage site is indicated in bold. Following the bold string, the N-terminus of the GP2 protein is shown. See also Figure 7B. [Chemical Formula]

[0143] The ability of the vaccine composition to stimulate an immune response was confirmed in three animal models. First, the mouse model was used. Recombinant EBOV / Mak full-length GP nanoparticle vaccines formulated with Matrix M, AlPO 4 or saline were evaluated. Non-adjuvanted or AlPO 4Immunization of mice with adjuvanted EBOV / Mak GP induced moderate antibody and cellular responses; however, when adjuvanted with matrix M, purified EBOV / Mak GP nanoparticles were highly immunogenic and protective in the mouse challenge model. Immunization of mice with matrix M-adjuvanted EBOV / Mak GP resulted in a significant increase in anti-EBOV / Mak GP IgG and Ebola virus neutralizing antibodies. Immunization with matrix M-adjuvanted EBOV / Mak GP conferred 100% protection from lethal Ebola virus challenge, while non-adjuvanted E BOV / Mak GP was only 10% protective, and no protection was observed in mice immunized with EBOV / Mak GP containing AlPO 4 . Thus, in certain embodiments, the compositions disclosed herein prevent Ebola infection.

[0144] Co-administration of EBOV / Mak GP and matrix M induced the production of a balanced IgG1 and IgG2a subclass response. In the absence of adjuvant or in the presence of AlPO 4 , minimal IgG2a antibodies were detected. Blaney et al., Antibody quality and protection from lethal Ebola virus challenge in nonhuman primates immunized with rabies virus based bivalent vaccine. PLoS Pathog. 2013;9(5): showed that antibody isotypes play a role in virus neutralization and protection against Ebola virus challenge in a non-human primate (NHP) rabies / EBOV chimera vaccine model. Mouse IgG2a antibodies are equivalent to human IgG1 antibodies that efficiently bind to IgG-Fc receptors (FcγR) and complement (C1q) (Bruhns, P. Properties of mouse and human IgG receptors and their contribution to disease models Blood. 2012;119:5640 - 5649; Vidarsson G, Dekkers G, Rispens T. IgG subclasses and allotypes: from structure to effector functions. Front. Immunol. 2014;5:520), and can help dissipate virus infection, for example, through antibody-dependent cell-mediated cytotoxicity. All antibodies that were fully protective in vivo were of the IgG2a subclass; that is, the same as human IgG1. Thus, the compositions disclosed herein stimulate the production of IgG1 antibodies as part of the protective immune response.

[0145] The use of Matrix M adjuvant provided a dose-dependent increase in the frequency of CD4+ and CD8+ cytokine-secreting T cells and the number of multifunctional T cells producing more than one cytokine. The observation that protection from lethal Ebola virus challenge was only observed in the Matrix M-adjuvanted EBOV / Mak GP group was associated with enhanced production of multifunctional T cells.

[0146] The use of Matrix M increased the frequency of GC B cells in the spleen and long-lived plasma cells in the bone marrow. GC is a microanatomical location for B cell differentiation, somatic hypermutation, antibody class switching, and the formation of memory B cells. Co-administration of EBOV / Mak GP with the saponin adjuvant Matrix M promotes T cell-mediated GC B cell differentiation and development. FHAn increase in the number of cells also occurred. GC and T cells induced by matrix M adjuvantation FH The increased frequency of cells is associated with enhanced magnitude of the antibody response and induction of a greater number of long-lived plasma cells, suggesting that matrix M adjuvanted EBOV / Mak GP vaccine can induce a particularly persistent antibody response.

[0147] Each dose of Ebola GP may be combined with an adjuvant. Administration of purified EBOV / Mak GP nanoparticles together with matrix M adjuvant provides a strong stimulation of an anti-EBOV / Mak GP immune response that results in 100% protective efficacy in a mouse model. The compositions and methods disclosed herein provide for a more rapid generation of anti-EBOV / Mak GP IgG and Ebola virus neutralizing antibodies, an increase in the concentration of IgG2a, as well as multifunctional CD4+ and CD8+ T cells, T FH cells, an increase in the frequency of germinal center B cells, and persistence of EBOV / Mak GP-specific plasma cells in the bone marrow.

[0148] Thus, analysis of the mouse study demonstrated that the compositions disclosed herein provided complete protection It has been confirmed. To further establish the protective effect, the study was conducted in two separate non-human primate models: baboons and macaques. See Perry et al., "The Baboon (Papio spp.) as a model of human Ebola virus infection", Viruses. October 23, 2012; 4(10):2400-16; Geisbert et al., "Pathogenesis of Ebola hemorrhagic fever in cynomolgus macaques: evidence that dendritic cells are early and sustained targets of infection", Am J Pathol. December 2003; 163(6):2347-70. Thus, in some aspects of the present disclosure, the protective effect includes a reduction in the amount of virus that is lower than the ability to detect by RT-PCR at about 7 days, about 10 days, about 14 days or about 21 days after virus exposure.

[0149] The non-human primate studies further confirmed that the compositions disclosed herein are protective. The Ebola GP nanoparticles were evaluated without an adjuvant and with either alum or Matrix M adjuvant. See Example 23. The immune response in baboons was very strong and sustained. Notably, the inclusion of Matrix M resulted in a stronger immune response than alum. The results in the macaque model were particularly unexpected. See Examples 24 and 24. The compositions were not only protective against challenge with live Ebola vaccine, but the amount of Ebola RNA was undetectable on day 10 after challenge with live virus. See Figure 48. Notably, in one macaque subject, a small signal was present at approximately day 7; however, by day 10, the level had returned below the limit of detection. In contrast, exposure of untreated animals to live Ebola virus resulted in infection and disease such that the subjects were euthanized on day 9.

[0150] Modified antigen The antigens disclosed herein include variants and mutants of these antigens. In certain embodiments, the antigen may share identity with the disclosed antigens. Generally, unless specifically defined in the context of the specifically identified antigen, the percent identity may be at least 80%, at least 90%, at least 95%, at least 97% or at least 98%. The percent identity can be calculated using the ClustalW2 alignment program available at www.ebi.ac.uk / Tools / msa / clustalw2 / . The following initial parameter settings may be used for pairwise alignment: protein weight matrix = Gonnet; gap open = 10; gap extension = 0.1.

[0151] In certain embodiments, the protein contained in the nanoparticle consists of that protein. In other embodiments, the protein contained in the nanoparticle comprises that protein. The addition to the protein itself may be for various purposes. In some embodiments, the antigen may be extended at the N-terminus, C-terminus or both. In some embodiments, the extension is a tag useful for functions such as purification or detection. In some embodiments, the tag contains an epitope. For example, the tag may be a polyglutamic acid tag, FLAG-tag, HA-tag, polyHis-tag (having about 5 - 10 histidines), Myc-tag, glutathione-S-transferase-tag, green fluorescent protein-tag, maltose binding protein-tag, thioredoxin-tag or Fc-tag. In other embodiments, the extension may be an N-terminal signal peptide fused to the protein to enhance expression. Such signal peptides are often cleaved upon expression in cells, while some nanoparticles may contain antigens with intact signal peptides. Thereby, when the nanoparticle contains the antigen, the antigen may contain an extension and thereby may be a fusion protein when incorporated into the nanoparticle. For the purpose of calculating identity with a sequence, the ex tension length is not included.

[0152] In some embodiments, the antigen may be truncated. For example, the N-terminus may be truncated by about 10 amino acids, about 30 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids or about 200 amino acids. The C-terminus may be truncated instead of or in addition to the N-terminus. For example, the C-terminus may be truncated by about 10 amino acids, about 30 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids or about 200 amino acids. For the purpose of calculating the identity with a truncated protein, the identity is measured over the remaining portion of the protein.

[0153] Combination nanoparticles As used herein, combination nanoparticles refer to nanoparticles that induce an immune response against two or more different pathogens. Depending on the particular combination, the pathogens may be different strains or subtypes of the same species, or the pathogens may be different species. Glycoproteins from multiple pathogens may be combined into a single nanoparticle by binding them at a surfactant exchange step. Surfactant exchange following binding of the glycoproteins to the column allows multiple glycoprotein types to form around the surfactant core, enabling the provision of combination nanoparticles.

[0154] The present disclosure also provides a vaccine composition that induces an immune response against two or more different pathogens by combining two or more nanoparticles that each induce a response against a different pathogen. Optionally, the vaccine composition may contain one or more combination nanoparticles alone or in combination with additional nanoparticles for the purpose of maximizing the immune response against multiple pathogens while reducing the number of vaccine compositions administered to a subject.

[0155] Such compositions are particularly desirable when a pathogen is involved in some way. In one example, the composition may contain nanoparticles against strains identified annually by the authorities as forming the seasonal influenza of a particular year. Typically, for seasonal influenza vaccines, the vaccine composition contains HA and / or NA nanoparticles that induce an immune response against three, four, or five strains of influenza subtypes. Thereby, different strains of influenza may be combined in the vaccine composition. In some embodiments, the combined nanoparticles may contain an HA protein from a first strain and an NA protein from a second strain. In other embodiments, the nanoparticles may contain one or more HAs and one or more NA proteins from the same or different subtypes. For example, the nanoparticles may contain one or more HA nanoparticles selected from subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, and H16 and / or one or more NA nanoparticles selected from subtypes N1, N2, N3, N4, N5, N6, N7, N8, and N9. Phylogenetically, the HA and NA proteins are divided into groups. For HA, group 1 contains H1, H2, H5, H6, H8, H9, H11, H12, H13, and H16, and group 2 contains H3, H4, H7, H10, H14, and H15. The NA proteins also form two groups: group 1 contains N1, N4, N5, and N8, and group 2 contains N2, N3, N6, N7, and N9. In certain embodiments, the antigen may have at least 90% identity, at least 95% identity, at least 97% identity, or at least 99% identity to the native influenza HA protein and / or NA protein.

[0156] In another example, both influenza and RSV cause respiratory diseases, and HA, NA, and / or RSV F may thus be mixed in the combined nanoparticles, or multiple nanoparticles may be combined in the vaccine composition to induce a response against RSV and one or more influenza strains thereby.

[0157] Vaccine composition The compositions disclosed herein may be used either prophylactically or therapeutically, but are typically prophylactic. Thus, the present disclosure includes methods for treating or preventing infection. The method includes administering to a subject a therapeutic or prophylactic amount of an immunogenic composition of the present disclosure. Preferably, the pharmaceutical composition is a vaccine composition that provides a protective effect. In other embodiments, the protective effect may include improvement of symptoms associated with infection in a percentage of the exposed population. For example, depending on the pathogen, the composition can prevent or reduce one or more viral disease symptoms selected from: fever, fatigue, muscle pain, headache, pharyngitis, vomiting, diarrhea, rash, symptoms of kidney and liver dysfunction, internal bleeding, and external bleeding compared to untreated subjects.

[0158] The nanoparticles may be formulated for administration as a vaccine in the presence of various excipients, buffers, etc. For example, the vaccine composition may contain sodium phosphate, sodium chloride, and / or histidine. Sodium phosphate may be present at about 10 mM to about 50 mM, about 15 mM to about 25 mM, or about 25 mM; in certain cases, about 22 mM sodium phosphate is present. Histidine may be present at about 0.1% (w / v), about 0.5% (w / v), about 0.7% (w / v), about 1% (w / v), about 1.5% (w / v), about 2% (w / v), or about 2.5% (w / v). When present, sodium chloride may be about 150 mM. In certain compositions, such as influenza vaccines, sodium chloride may be present in even greater amounts, including about 200 mM, about 300 mM, or about 350 mM.

[0159] Certain nanoparticles, specifically RSV F nanoparticles, have improved stability at slightly acidic pH levels. For example, the pH range for a composition containing the nanoparticles may be from about pH 5.8 to about pH 7.0, from about pH 5.9 to about pH 6.8, from about pH 6.0 to about pH 6.5, from about pH 6.1 to about pH 6.4, from about pH 6.1 to about pH 6.3 or about pH 6.2. Typically, the composition for RSV F protein nanoparticles is about pH 6.2. For other nanoparticles, the composition may tend to be neutral; for example, influenza nanoparticles may be from about pH 7.0 to pH 7.4; often about pH 7.2.

[0160] Adjuvant In certain embodiments, the compositions disclosed herein may be combined with one or more adjuvants to enhance the immune response. In other embodiments, the compositions are prepared without an adjuvant and can thus be utilized to be administered as an adjuvant-free composition. Advantageously, the adjuvant-free compositions disclosed herein can provide a protective immune response when administered as a single dose. The alum-free compositions that induce a strong immune response are particularly useful in adults 60 years of age and older.

[0161] Aluminum-based adjuvant In some embodiments, the adjuvant is alum (e.g., AlPO 4 or Al(OH) 3) may be. Typically, the nanoparticles are substantially bound to alum. For example, the nanoparticles may be bound to alum at least 80%, at least 85%, at least 90% or at least 95%. Often the nanoparticles are 92% to 97% bound to alum in the composition. The amount of alum present per dose is typically in the range of about 400 μg to about 1250 μg. For example, alum may be present in an amount of about 300 μg to about 900 μg, about 400 μg to about 800 μg, about 500 μg to about 700 μg, about 400 μg to about 600 μg or about 400 μg to about 500 μg per dose. Typically, alum is present at about 400 μg per 120 μg dose of protein nanoparticles.

[0162] Saponin adjuvant Adjuvants containing saponin may be combined with the immunogens disclosed herein. Saponin is a glycoside derived from the bark of the tree Quillaja saponaria Molina. Typically, saponin is prepared using a multi-step purification process that yields multiple fractions. The term "Quillaja saponaria Molina-derived saponin fraction" as used herein is used generically to describe a semi-purified or defined saponin fraction of Quillaja saponaria or a substantially pure fraction thereof.

[0163] Saponin fraction Several approaches for producing saponin fractions are suitable. Fractions A, B, and C are described in U.S. Patent No. 6,352,697 and can be prepared as follows. The lipophilic fraction derived from Quil A, the crude aqueous Quillaja saponaria Molina extract, is separated by chromatography and eluted with 70% acetonitrile in water to recover the lipophilic fraction. This lipophilic fraction is then separated by preparative HPLC using an elution with a gradient from 25% to 60% acetonitrile in acidic water. The fraction referred to herein as "Fraction A" or "QH-A" is the fraction eluted at approximately 39% acetonitrile, or corresponding thereto. The fraction referred to herein as "Fraction B" or "QH-B" is the fraction eluted at approximately 47% acetonitrile, or corresponding thereto. The fraction referred to herein as "Fraction C" or "QH-C" is the fraction eluted at approximately 49% acetonitrile, or corresponding thereto. Additional information regarding the purification of the fractions can be found in U.S. Patent No. 5,057,540. When prepared as described herein, Fractions A, B, and C of Quillaja saponaria Molina each represent a group or family of molecules that are chemically closely related and have definable properties. The chromatographic conditions under which they are obtained are highly reproducible from batch to batch with respect to the elution profile and biological activity.

[0164] Other saponin fractions are described. Fractions B3, B4, and B4b are described in EP0436620. Fractions QA1 - QA22 are described in EP03632279B2, Q-VAC (Nor-Feed, AS Denmark), Quillaja saponaria It is described in Molina Spikoside (lsconova AB, Ultunaallen 2B, 756 51 Uppsala, Sweden). Fractions QA-1, QA-2, QA-3, QA-4, QA-5, QA-6, QA-7, QA-8, QA-9, QA-10, QA-11, QA-12, QA-13, QA-14, QA-15, QA-16, QA-17, QA-18, QA-19, QA-20, QA-21 and QA-22 of EP03632279B2, in particular QA-7, QA-17, QA-18 and QA-21 can be used. They can be obtained as described in EP03632279B2, in particular on page 6 and in Example 1 on pages 8 and 9.

[0165] The saponin fraction described herein and used to form an adjuvant is often a substantially pure fraction; that is, the fraction is substantially free of contaminants from other materials. In certain embodiments, the substantially pure saponin fraction may contain up to 40% by weight, up to 30% by weight, up to 25% by weight, up to 20% by weight, up to 15% by weight, up to 10% by weight, up to 7% by weight, up to 5% by weight, up to 2% by weight, up to 1% by weight, up to 0.5% by weight, or up to 0.1% by weight of other compounds such as other saponins or other adjuvant materials.

[0166] ISCOM structure The saponin fraction may be administered in the form of cage-like particles called ISCOMs (Immune Stimulatin g COMplex). ISCOMs can be prepared as described in EP0109942B1, EP0242380B1 and EP0180546B1. In certain embodiments, a transport and / or passenger antigen as described in EP9600647-3 (PCT / SE97 / 00289) may be used.

[0167] Matrix adjuvant In one aspect, the ISCOM is an ISCOM matrix complex. The ISCOM matrix complex comprises at least one saponin fraction and a lipid. The lipid is at least one sterol such as cholesterol. In certain aspects, the ISCOM matrix complex also contains phospholipids. The ISCOM matrix complex may also contain one or more other immunomodulatory (adjuvant active) substances that are not necessarily glycosides and can be produced as described in EP0436620B1.

[0168] In other aspects, the ISCOM is an ISCOM complex. The ISCOM complex contains at least one saponin, at least one lipid, and at least one antigen or epitope. The ISCOM complex contains antigens associated by surfactant treatment, with a portion of the antigen incorporated into the particles. In contrast, the ISCOM matrix is formulated as a mixture with the antigen, and the association between the ISCOM matrix particles and the antigen is mediated by electrostatic and / or hydrophobic interactions.

[0169] According to one embodiment, the saponin fraction incorporated into the ISCOM matrix complex or ISCOM complex, or similarly at least one additional adjuvant incorporated into or mixed with the ISCOM or ISCOM matrix complex, is selected from fraction A, fraction B or fraction C of Quillaja saponaria, a semi-purified preparation of Quillaja saponaria, a purified preparation of Quillaja saponaria or any purified sub-fraction, such as QA1 - 21.

[0170] In certain embodiments, each ISCOM particle may contain at least two saponin fractions. Any combination of weight percentages of different saponin fractions may be used. Any combination of weight percentages of any two fractions may be used. For example, the particles may each contain fraction A at any weight percentage and another saponin fraction such as a crude saponin fraction or fraction C at any weight percentage. Thus, in certain embodiments, each ISCOM matrix particle or each ISCOM complex particle contains one saponin fraction, for example fraction A, at 0.1 to 99.9% by weight, 5 to 95% by weight, 10 to 90% by weight, 15 to 85% by weight, 20 to 80% by weight, 25 to 75% by weight, 30 to 70% by weight, 35 to 65% by weight, 40 to 60% by weight, 45 to 55% by weight, 40 to 60% by weight, or 50% by weight and another saponin in each case, for example any crude fraction or any other fraction, for example fraction C, up to the remaining 100%. The weight is calculated as the total weight of the saponin fractions. Examples of ISCOM matrix complexes and ISCOM complex adjuvants are disclosed in US Patent Application Publication No. 2013 / 0129770.

[0171] In certain embodiments, the ISCOM matrix or ISCOM complex contains one fraction, for example fraction A, at 5 to 99% by weight and another fraction, for example a crude saponin fraction or fraction C, up to the remaining 100% by weight. The weight is calculated as the total weight of the saponin fractions.

[0172] In another embodiment, the ISCOM matrix or ISCOM complex contains one fraction, for example fraction A, at 40% to 99% by weight and another fraction, for example a crude saponin fraction or fraction C, at 1% to 60% by weight. The weight is calculated as the total weight of the saponin fractions.

[0173] In yet another embodiment, the ISCOM matrix or ISCOM complex comprises, by weight, 70% to 95% of one fraction, such as fraction A, and 30% to 5% of another fraction, such as the crude saponin fraction or fraction C. The weight is calculated as the total weight of the saponin fraction. In other embodiments, the saponin fraction from Quillaja saponaria Molina is selected from any one of QA1-21.

[0174] In addition to particles containing a mixture of saponin fractions, ISCOM matrix particles and ISCOM complex particles may each be formed using only one saponin fraction. The compositions disclosed herein may contain a plurality of particles, where each particle contains only one saponin fraction. That is, a particular composition may contain one or more different types of ISCOM-matrix complex particles and / or one or more different types of ISCOM complex particles, where each individual particle contains one saponin fraction from Quillaja saponaria Molina, and the saponin fraction in one complex is different from the saponin fraction in other complex particles.

[0175] In certain embodiments, one type of saponin fraction or crude saponin fraction may be incorporated into one ISCOM matrix complex or particle, and another type of substantially pure saponin fraction or crude saponin fraction may be incorporated into another ISCOM matrix complex or particle. The composition or vaccine may comprise at least two types of complexes or particles, each type having one type of saponin incorporated into physically distinct particles.

[0176] In the composition, a mixture of ISCOM matrix complex particles and / or ISCOM complex particles may be used, where one saponin fraction from Quillaja saponaria Molina and another saponin fraction from Quillaja saponaria Molina are separately incorporated into different ISCOM matrix complex particles and / or ISCOM complex particles.

[0177] ISCOM matrices or ISCOM complex particles each having one saponin fraction may be present in the composition in any combination by weight %. In certain embodiments, the composition comprises an ISCOM matrix or complex containing a first saponin fraction from 0.1% to 99.9% by weight, from 5% to 95% by weight, from 10% to 90% by weight, from 15% to 85% by weight, from 20% to 80% by weight, from 25% to 75% by weight, from 30% to 70% by weight, from 35% to 65% by weight, from 40% to 60% by weight, from 45% to 55% by weight, from 40 to 60% by weight or 50% by weight, and the balance may consist of an ISCOM matrix or complex containing a different saponin fraction. In some embodiments, the balance is one or more ISCOM matrices or complexes, and each matrix or complex particle contains only one saponin fraction. In other embodiments, the ISCOM matrix or complex particles may contain more than one saponin fraction.

[0178] In certain compositions, the saponin fraction in the first ISCOM matrix or ISCOM complex particles is fraction A, and the saponin fraction in the second ISCOM matrix or ISCOM complex particles is fraction C.

[0179] Preferred compositions include a first ISCOM matrix containing fraction A and a second ISCOM matrix containing fraction C, wherein the fraction A ISCOM matrix constitutes about 70% by weight of the total saponin adjuvant and the fraction C ISCOM matrix constitutes about 30% by weight of the total saponin adjuvant. In another preferred composition, the fraction A ISCOM matrix constitutes about 85% by weight of the total saponin adjuvant and the fraction C ISCOM matrix constitutes about 15% by weight of the total saponin adjuvant. Thereby a certain special In certain compositions, the Fraction A ISCOM matrix is present in the range of from about 70% to about 85% of the total weight of the saponin adjuvant in the composition, and the Fraction C ISCOM matrix is present in the range of from about 15% to about 30%. Exemplary QS-7 and QS-21 fractions, their production, and their use are described in U.S. Patent Nos. 5,057,540; 6,231,859; 6,352,697; 6,524,584; 6,846,489; 7,776,343 and 8,173,141, which are incorporated herein by reference for their disclosures.

[0180] Other adjuvants In some compositions, other adjuvants may be used additionally or alternatively. The inclusion of any adjuvant described in Vogel et al., "A Compendium of Vaccine Adjuvants and Excipients (2nd Edition)", which is incorporated herein by reference in its entirety for all purposes, is contemplated to be within the scope of the present disclosure. Other adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing heat-killed Mycobacterium tuberculosis), incomplete Freund's adjuvant, and aluminum hydroxide adjuvant. Other adjuvants include MDP compounds such as GMCSP, BCG, thur-MDP, and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL), MF-59, RIBI containing three components extracted from bacteria, MPL, trehalose dimycolate (TDM), and cell wall skeleton (CWS) in a 2% squalene / Tween® 80 emulsion. In some embodiments, the adjuvant may be a paucilamellar lipid vesicle; for example, Novasome®. Novasome® is a paucilamellar non-liposomal vesicle in the range of from about 100 nm to about 500 nm. They contain Brij72, cholesterol, oleic acid, and squalene. Novasome has been shown to be an effective adjuvant (see U.S. Patent Nos. 5,629,021, 6,387,373, and 4,911,928).

[0181] Administration and Dosage The compositions disclosed herein may be administered via a systemic route, a mucosal route, a transdermal route, or directly to a particular tissue. As used herein, the term "systemic administration" includes parenteral administration routes. Specifically, parenteral administration includes subcutaneous, intraperitoneal, intravenous, intraarterial, intramuscular or intrasternal injection, intravenous or renal dialysis infusion techniques. Typically, systemic, parenteral administration is intramuscular injection. As used herein, the term "mucosal administration" includes oral, intranasal, intravaginal, rectal, intratracheal, intestinal, and ocular administration. Preferably, administration is intramuscular.

[0182] The compositions may be administered on a single-dose schedule or a multiple-dose schedule. Multiple doses may be used in a prime immunization schedule or a booster immunization schedule. In a multiple-dose schedule, the various doses may be given by the same or different routes, such as parenteral prime and mucosal boost, mucosal prime and parenteral boost, etc. In some embodiments, subsequent booster doses are administered about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or about 6 weeks after the preceding dose. Typically, however, the compositions disclosed herein are administered only once and nevertheless provide a protective immune response.

[0183] In some embodiments, the dose measured in μg may be the total weight of the dose containing the solute, or the weight of the RSV F nanoparticles, or the weight of the RSV F protein. The dose is measured using either an A280 or ELISA protein concentration assay.

[0184] The dose of antigen included for administration to pediatric patients is from about 30 μg to about 300 μg, about 90 μ It may be in the range of about 270 μg from g, about 100 μg to about 160 μg, about 110 μg to about 150 μg, about 120 μg to about 140 μg or about 140 μg to about 160 μg. In certain embodiments, the dosage is about 120 μg and is administered with alum. In some aspects, the pediatric dosage may be in the range of about 30 μg to about 90 μg. A particular population may be administered with or without an adjuvant. For example, when administered to the elderly, preferably alum is not included. In certain embodiments, the composition may not contain additional adjuvants. In such circumstances, the dosage may be increased by about 10%.

[0185] In some embodiments, the dosage may be administered in a volume of about 0.1 mL to about 1.5 mL, about 0.3 mL to about 1.0 mL, about 0.4 mL to about 0.6 mL or a typical amount of about 0.5 mL.

[0186] In certain embodiments, for the RSV vaccine, the dosage may contain RSV F protein at a concentration of about 175 μg / mL to about 325 μg / mL, about 200 μg / mL to about 300 μg / mL, about 220 μg / mL to about 280 μg / mL or about 240 μg / mL to about 260 μg / mL.

[0187] All patents, patent applications, references and academic papers cited in this disclosure are hereby expressly incorporated by reference in their entirety for all purposes.

Examples

[0188] (Example 1) Expression and purification of RSV F protein The RSV F protein having SEQ ID NO:8 was expressed in a baculovirus expression system, and recombinant plaques expressing the RSV F protein were selected and confirmed. Subsequently, the recombinant virus was amplified by infecting Sf9 insect cells. The culture of insect cells was infected with the baculovirus at approximately 3 MOI (multiplicity of infection = virus ffu or pfu / cell). The culture and supernatant were collected 48 - 72 hours after infection. The crude cell harvest, approximately 30 mL, was clarified by centrifugation at approximately 800×g for 15 minutes. The resulting crude cell harvest containing the RSV F protein was purified as follows.

[0189] The nonionic surfactant Tergitol® NP-9 (nonylphenol ethoxylate) was used in the membrane protein extraction protocol. The crude extract was further purified by passing through anion exchange chromatography, lentil lectin affinity / HIC, and cation exchange chromatography. The washed cells were lysed by treatment with a surfactant and then subjected to a low pH treatment that precipitated the DNA and proteins of BV as well as the Sf9 host cells. The neutralized low pH treatment lysate was clarified and further purified by anion exchange and affinity chromatography before performing a second low pH treatment.

[0190] Affinity chromatography was used to remove Sf9 / BV proteins, DNA, and NP-9 and concentrate the RSV F protein. Briefly, lentil lectin is a metal protein containing calcium and manganese, and lentil lectin reversibly binds to glycosylated proteins containing polysaccharides and glucose or mannose. The RSV F-containing anion exchange flow-through fraction was loaded onto a lentil lectin affinity chromatography resin (Capto Lentil Lectin, GE Healthcare). Nonglycosylated proteins and DNA were removed in the column flow-through, while the glycosylated RSV F protein selectively bound to the resin. Weakly bound glycoproteins were removed with a buffer containing high salt and low molarity methyl alpha-D-mannopyranoside (MMP).

[0191] In addition, column washing was also used to surfactant-exchange the NP-9 surfactant with the surfactant polysorbate 80 (PS80). To perform the surfactant exchange, after binding of the RSV F glycoprotein to the lentil lectin column, the column was incubated with 0.1% PS80. The RSV F protein was eluted from the lentil lectin column with high concentration MMP. After elution, the RSV F protein trimer was assembled into micelle nanoparticles composed of the RSV F protein trimer and the PS80 contained in the surfactant core. After surfactant exchange, there was a low pH inactivation step followed by incubation on a sulfate column in the presence of a buffer containing 0.1% PS80.

[0192] The dissolution material was diluted in a solution containing PS80 suitable for obtaining the bulk drug substance (DS) with a PS80:RSV F protein molar ratio of approximately 50. The appropriate composition of the DS was obtained by combining RSV F nanoparticles in a solution containing 22 mM sodium phosphate, 0.03% PS80 phosphate buffer at pH 6.2. During and after each step of the surfactant exchange, the antigen ratio to PS80 in the composition was maintained from a molar ratio of 35 to 60. The molar ratio was calculated using the PS80 concentration and RSV F concentration measured by ELISA / A280 and their respective molecular weights. The molecular weight of PS80 is 1310 and that of RSV is 65 kD.

[0193] (Example 2) Preparation of the vaccine composition To obtain nanoparticles for the vaccine product to be administered, the drug substance was diluted in a pharmaceutical product having a PS80:RSV protein molar ratio of approximately 50. The drug substance was thawed, diluted, and filled into glass vials or pre-filled syringes for storage at 2 - 8 °C before administration. The nanoparticles were conjugated to the alum adjuvant. The alum adjuvant was added and mixed to ensure that approximately 95% of the nanoparticles were conjugated to the alum, which means approximately 0.4 mg per 120 μg dose of RSV F nanoparticles in a 0.5 mL volume.

[0194] (Example 3) Characterization of the RSV F glycoprotein in nanoparticles The protein structure in the nanoparticles was analyzed by various analytical techniques. Figure 3 shows that the highest peak of the produced RSV F protein contains palmitoleic acid (peak 2A). The second largest peak contains palmitic acid (peak 2B). The remaining peaks were obtained without any fatty acids (peak 5) and in the soluble form (peak 1). Analysis by SDS-PAGE gel separated additional variants including the F1+2 protein, F1, F1A, F1B, and F1C moieties as well as F2. See Figure 4. Analysis of the peptide structure was performed using peptide mapping. See Figure 5. RSV To evaluate the glycan structure on the F glycoprotein, HPLC-FLD was performed. The results demonstrated that the major glycan structure was fucosylated.

[0195] (Example 4) Examination of RSV F nanoparticles by electron microscopy The nanoparticles prepared in Example 1 were visualized by electron microscopy. From the results, the formation of nanoparticles containing RSV F glycoprotein surrounding the surfactant core was confirmed. The exact composition of the surfactant core remained unknown. Figure 6 illustrates the types of nanoparticles obtained. The RSV F protein maintained its trimeric structure even after surfactant exchange. Several types of nanoparticles with varying numbers and morphologies of trimers / nanoparticles were obtained. Figure 6 shows that multiple trimers can be seen around the surfactant core. In the highlighted portion, seven trimers are shown surrounding the surfactant. The main panel of Figure 6 illustrates the range of trimers around the surfactant core produced. The schematic structure of the RSV F protein trimer in the lower left panel exemplifies the orientation of the trimer with a bottom that associates with the surfactant core, which is facilitated by the fatty acids attached to each RSV F glycoprotein.

[0196] (Example 5) Particle characterization of RSV F nanoparticles Dynamic light scattering (DLS) was utilized to determine the size distribution profile of the nanoparticles by measuring the change in the light scattering pattern of the particles in Brownian motion. The nanoparticle size could be determined as a linear function of the concentration of the ionic surfactant with respect to the concentration of the RSV F nanoparticles (see Figures 7 and 8).

[0197] Using analytical ultracentrifugation (AUC), the evolution of the sample concentration with respect to the axis of the rotational profile was measured as a result of the applied centrifugal field. From Figure 8, it becomes clear that mainly two shapes of nanoparticles appear based on the concentration of the existing nanoparticles. The obtained nanoparticle types include monomer and dimer anisotropic rods, as well as spherical oligomers. The structural intermediates between these two nanoparticle types are formed between the concentrations that result in anisotropic rods and spherical oligomers. Figure 9 shows that the nanoparticle type can be controlled by modulating the concentration of the RSV F protein, with higher concentrations (1 mg / mL) resulting in a predominantly spherical oligomer population, while lower concentrations (0.22 mg / mL) result in a predominantly monomer / dimer anisotropic rod population. These data illustrate that by controlling the amount of surfactant and the RSV F concentration, the nanoparticles can reach a specific diameter (z-average) between 20 nm and 60 nm.

[0198] (Example 6) Enhanced Stability of Nanoparticles: Molecular Characterization The five stressors utilized were heat stress at 50 °C for 48 hours, 1 week, and 2 weeks; low pH (3.7 at 25 °C) for 48 hours, 4 days, and 1 week; high pH (10 at 25 °C) for 24 hours, 48 hours, and 1 week; hydrogen peroxide oxidation at 25 °C for 12 hours, 48 hours, and 1 week; and physical agitation at 25 °C for 4 hours, 24 hours, and 1 week.

[0199] After various stress treatments, the differences in primary structure were evaluated. Figure 10 shows a comparison of how a specific region withstood stress compared to the control. The data indicate that the nanoparticles had excellent stability throughout the protein, and only the particularly stringent oxidation test using hydrogen peroxide was able to degrade the protein to any significant extent. However, even that treatment did not reduce the structural integrity of antigenic site II, which is the target of palivizumab. In fact, even under severe oxidation, the RSV-F protein only structurally deteriorated at positions 63 - 82, 237 - 258, and 360 - 364. Therefore, the nanoparticles remained substantially intact even after being subjected to severe stress.

[0200] Figure 11 further quantifies the nanoparticle stability with respect to antigenic site II. The data show that antigenic site II remained approximately 90% intact in each of the samples in response to each of heat stress, low pH, high pH, and agitation.

[0201] (Example 7) Enhanced Stability of Nanoparticles: Maintained Immunogenic Properties The stressed vaccine compositions described in Example 6 were evaluated for immunogenicity in a mouse model. The vaccine was administered to mice by two intramuscular injections over a range of RSV F doses consisting of 0.15, 0.45, 1.3, 4, and 12 μg / mL of RSV F protein. RSV F compositions stressed under the following conditions: 2 weeks at 50°C, 1 week at pH 10 at 25°C, 1 week at 0.5% hydrogen peroxide at 25°C, were administered with a control (thawed from storage at -70°C).

[0202] The immune response was evaluated in terms of the presence of anti-RSV F IgG, PCA titer, and the presence of RSV A neutralizing antibodies. Physical and chemical stressors did not significantly affect RSV F protein immunogenicity in vivo. Stressed samples induced anti-RSV F IgG antibody titers similar to those of the unstressed RSV F nanoparticle vaccine composition control, as well as comparable functional PCA and RSV A neutralizing titers. (See FIGS. 12A-12D). Collectively, these forced degradation studies indicate that nanoparticles induce a strong immune response even when exposed to intense environmental stress.

[0203] (Example 8) Protease resistance of nanoparticles The formation of nanoparticles with improved stability depends on the amount of PS80 used to produce the nanoparticles. FIG. 13 illustrates a dramatic improvement in stability up to 18 months when nanoparticles were formed with 0.03% PS80 (i.e., molar ratio 55) compared to 0.015% (i.e., molar ratio 27). The left panel shows the SDS-PAGE of nanoparticles produced at two concentrations at time 0. The data indicate that results similar to the reference preparation were obtained with both nanoparticle preparations. In particular, both show strong signals for F1 and F1+2, exemplifying essentially no degradation. An aliquot of each preparation was then incubated at 4° C. for 18 months and then SDS-PAGE was performed again. The data in the right panel and table exemplify that nanoparticles containing only 0.015% in the particles yielded truncated F1. In contrast, nanoparticles prepared at 0.03% in the particles exemplified excellent resistance to protease. The inventors believe that by maintaining the correct ratio of surfactant and protein, nanoparticles with an orientation of glycoprotein with protease-sensitive portions protected, perhaps by some steric hindrance mechanism, can be obtained. The inventors further observed that concentrations of PS80 of 0.06% or more increased aggregate formation. Collectively, the data indicate that the optimal PS80 level for nanoparticle stability is from about 0.03% to about 0.05%.

[0204] (Example 9) Purification of HA Nanoparticles The TMAE column was pre-equilibrated with buffer A1 (25 mM Tris pH 7.5, 70 mM NaCl, 0.02% NP9) at a flow rate of 91.7 cm 30 mL / min for 0.5 CV. The sample was loaded at 20 mL / min (retention time 25 minutes) and then washed with EQ buffer A1 (25 mM Tris pH 7.5, 70 mM NaCl, 0.02% NP-9). The purified sample was then eluted using 1.25 CV of 15% buffer B (25 mM Tris pH 8, 1 M NaCl, 0.02% NP9), followed by 1.1 cv of 100% B. A representative chromatogram is shown in Figure 18B. The product from the TMAE column was added to a lentil lectin affinity chromatography column pre-equilibrated with buffer A11: 25 mM sodium phosphate pH 6.0, 10 mM NaCl, 0.05% PS80 3 CV (flow rate: 147 cm / hour 13 mL / min). The sample was loaded at a retention time of 9.4 minutes, 6.5 mL / min, 73.5 cm / hour. After loading, a high salt wash was performed with 3 CV of buffer A12 (25 mM sodium phosphate pH 6.0, 500 mM NaCl, 0.5% NP-9). After the first wash, surfactant exchange was performed by washing the column with 6 CV of buffer A11 (25 mM sodium phosphate pH 6.0, 10 mM NaCl, 0.05% PS80). The nanoparticles containing PS80 were then eluted with 3 CV of 100% B, B1: buffer B: 25 mM sodium phosphate pH 6.0, 10 mM NaCl, 0.05% PS80, 500 mM methyl-alpha-D-mannopyranoside. A representative chromatogram trace is shown in Figure 18C. The product from the lentil lectin column was added to a sulfate column with 3 CV of buffer A1 (25 mM sodium phosphate pH 6.0, 10 mM NaCl, 0.05% PS80) and with 2 CV of buffer A1 Washed and then eluted with 100% buffer B1 (25 mM sodium phosphate pH 7.5, 500 mM NaCl, 0.05% PS80). The eluate was then combined 1:1 with 50 mM sodium phosphate pH 9 and filter sterilized. The final product had a pH of 7.2. The chromatogram is shown in Figure 18D. Figure 18E provides gels and western blots of various products obtained during the purification steps from the TMAE and LL columns. Figure 18F shows the eluate from the S03-column.

[0205] (Example 10) Analysis of HA nanoparticle purity HA nanoparticles were prepared as outlined in Figures 17 and 18. The purity of multiple HA nanoparticle preparations was measured using HA sequences obtained from various strains (A / New Hampshire / 1 / 2015, A / Switzerland / 9715293 / 2013, A / Hong Kong / 4801 / 2014, B / Puget / 3073 / 2013, and B / Brisbane 60 / 2008). The data showed that highly pure preparations were obtained in all cases. Analysis by gel densitometry showed a purity in the range of greater than 93% to 97%. See Figures 19A - 19H. The purity of three A-subtype strains was also analyzed by RP-HPLC and found to be 83% to 85%. See Figure 19I. In addition, the nanoparticle size was measured. The nanoparticles showed a diameter of 22.0 nm to 29.9 nm. See Figure 19.

[0206] (Example 11) Analysis of HA ultrastructure Electron microscopy was performed to evaluate the structure of HA nanoparticles. Like other glycoproteins, the HA glycoprotein was found to form trimers associated with the PS80 surfactant core. Each surfactant core contained multiple trimers. See Figure 20. Cryo-EM 2D class averaging was used to dock HA trimers onto the nanoparticles. Figure 21A shows the results of these in silico docking experiments. The upper panel shows the fitting of HA trimers onto the first nanoparticle. The lower panel shows the fitting onto another axis (stalk) of the nanoparticle.

[0207] For comparison, docking in VLP was performed. The VLP contains a lipid bilayer to which the HA protein is tethered. See Figure 21B. The central panel shows the HA protein structure superimposed on an axis extending radially from the bilayer. The upper right and lower panels show EM micrographs of free HA pointing straight down onto the HA trimer, alone (upper panel) and with the corresponding HA structure superimposed on the EM image (lower panel).

[0208] (Example 12) Immunogenicity analysis of HA nanoparticles co-administered with RSV F nanoparticles The immunogenicity of nanoparticles in the vaccine was evaluated in a mouse model. Combinations of nanoparticles containing two antigens (influenza HA protein derived from A / Switzerland H3 subtype and RSV F protein) were administered. Also, each nanoparticle was administered separately. The vaccine was administered alone or with the adjuvant AlPO 4 or together with the matrix M saponin adjuvant. Figure 22 shows the treatments administered to groups 1 - 10. Group 10, the control, was not treated. Treatments were administered on days 0 and 21. HAI against heterologous and homologous challenges was measured. See Figures 23A and 23B. Figure 23A shows that matrix-adjuvanted HA nanoparticles stimulated a particularly strong response against homologous challenge. Figure 23B shows that a strong HAI response was obtained against the heterologous influenza strain A / Texas / 50 / 2012 when administered with matrix M, and this response was not affected by co-administration with RSV F nanoparticles.

[0209] The ability of the RSV F nanoparticle component to induce the formation of antibodies that compete with palivizumab was also measured. Figure 23C. The data show that RSV F nanoparticles administered alone and RSV F nanoparticles administered with either AlPO4 or matrix M induced substantial antibody titers from approximately 80 μg / mL to 700 μg / mL. When induced by both RSV F and influenza nanoparticles, in the absence of an adjuvant or AlPO4 A low response of approximately 20 μg / mL to 40 μg / mL was obtained in the presence of 4 . However, when administered alone or in combination with HA nanoparticles, the RSV F response was strong in the presence of matrix M. Measurement of RSV neutralizing antibodies showed a pattern similar to that of PCA antibodies. See Figure 23D.

[0210] In addition to antibody responses, T cell responses induced by vaccines against RSV and influenza A / Switzerland / 9715293 / 2013 were measured. Figures 23E and 23F. When matrix M was used as an adjuvant, the data showed strong induction of IFNγ against both targets.

[0211] (Example 13) Trypsin-resistant nanoparticle production By a specific approach for producing influenza, the trypsin sensitivity of the HA protein can be obtained, and its sensitivity modifies the folding that results in a decrease in immunogenicity and the stability of the vaccine formulation. To produce trypsin-resistant HA nanoparticles, a surfactant exchange approach using a neutral pH buffer was used. See Figures 24A - C.

[0212] Sf9 cells were infected with the HA nanoparticle BV vector at an MOI of 0.1; 3E6 cells / ml. On day 3, the cells were harvested and lysed in buffered 0.5% NP9; pH 7.5. Anion exchange chromatography (Fractogel TMAE; EMD) was then performed. Figure 24B shows an exemplary chromatogram. The flow-through contains HA. After the anion exchange column, surfactant exchange was performed using a high salt / surfactant wash containing 0.01% PS80; pH 7.2 and a Capto lentil lectin column (GE), and elution was also in 0.01% PS80. Figure 24C shows an exemplary chromatogram of the surfactant exchange step. Finally, tangential flow filtration (TFF): 50kD MWCO filter; pH 7.2 was used to produce the bulk drug substance (BDS) used to store the product. During the TFF step and thereafter, PS80 was maintained at 0.05% in a pH 7.2 buffer.

[0213] (Example 14) Trypsin-resistant nanoparticle analysis HA nanoparticles from various strains using the processes and production described in Example 13 were evaluated. Figure 25A shows that the yield of strain A / New Hampshire / 1 / 2015 (H1N1) was approximately 20 mg / L. B strain influenza nanoparticles also had excellent productivity and purity. Figure 25B shows the yield and purity of B / Brisbane / 60 / 08 HA evaluated as the bulk drug substance. The yield was approximately 30 mg / L. Figure 25C shows the yield of the H3N2 strain, indicating that the process gave a purity of approximately 96% and a yield of approximately 20 mg / L.

[0214] Thermodynamic stability analysis Figure 25D provides a comparison of the thermodynamic profiles of HA nanoparticles produced using the trypsin-resistant neutral pH approach in Example 13 and an approach using a low pH purification step as shown in Figure 18. Using a differential scanning calorimetry (DSC) method, in particular, by varying the temperature in a controlled manner, the sample solution and an appropriate reference (buffer / solvent) The thermodynamic profile of the polymer in solution was determined by measuring the difference in heat energy uptake between it and []. For the Flu HA sample, differential scanning calorimetry (DSC) was able to visualize the transition midpoint (Tm), defined as the temperature at which half of the protein is denatured / unfolded and half is in the non-denatured / folded state.

[0215] In addition, DSC provides information on protein conformation and can approximately extrapolate the estimated stability profile for each HA strain that has undergone different process conditions. As an example, the difference between HA purified by a process step that exposes HA to pH 6.0 during purification and HA purified by another step (e.g., MMC resin or TFF membrane) is shown using B / Brisbane HA. The data show that the Tm value is higher in intensity, indicating a shift to a higher Tm for the main and sharper peaks, suggesting proper folding of the HA when purified by another step. However, the data for HA exposed to pH 6.0 show a significant drop in intensity as well, indicating an earlier appearance for the Tm of the main peak, a significantly broadened peak indicating slow, asymmetric unfolding, and / or a Tm value containing misfolded protein and an aggregation peak at a higher temperature. Similar profiles were observed for other strains (A / Cal, A / Hong Kong, A / New Hampshire). Based on these three observations in the DSC data, it is concluded that low pH produces nanoparticles as described above, which may have specific applications, but that another process step / condition using neutral pH produces HA proteins with significantly better thermodynamic and potentially improved stability profiles.

[0216] Trypsin resistance Figure 26 illustrates the improvement in trypsin resistance obtained when the nanoparticles were produced as described in Example 13. To test trypsin sensitivity, the HA samples were diluted to 0.24 mg / mL and incubated with decreasing trypsin at 37 °C for 60 minutes. A trypsin inhibitor was added to stop the digestion and then SDS-PAGE analysis was performed. Comparison of the left and right panels in Figure 26 illustrates the enhanced trypsin resistance. The purified HA nanoparticles produced in Sf9 insect cells are HA0. When exposed to trypsin, HA0 is cleaved into HA1 and HA2 at Arg AA344 in H1. The correctly folded HA trimer will withstand further cleavage when incubated with increasing concentrations of trypsin. B / Brisbane / 60 / 08 purified at neutral pH is resistant to trypsin and is correctly folded (left panel). B / Brisbane / 60 / 08 HA1 purified at acidic pH is trypsin-sensitive and misfolded (right panel). Figures 26B and 26C illustrate that trypsin resistance is achieved for various strains. Figure 26B shows strain A / Hong Kong / 4801 / 2014. A / Hong Kong / 4801 / 2014 (H3N2) purified at neutral pH is resistant to trypsin and is thus correctly folded (left panel). A / Hong Kong / 4801 / 2014 (H3N2) HA1 purified at acidic pH is trypsin-sensitive and not correctly folded (right panel).

[0217] Similar data were obtained with A / New Hampshire / 1 / 2015 and the H1N1 subtype. Like the other strains, acid-purified H1N1 was misfolded (data not shown), while the protein purified at neutral pH was trypsin-resistant and correctly folded.

[0218] Comparison with commercially available influenza vaccines Previous approaches for producing recombinant influenza vaccines have not met with broad success. To investigate whether egg-produced or recombinant-produced influenza vaccines exhibited trypsin resistance, the same protocol as above was used for egg-produced and recombinant The trypsin sensitivity was compared in egg-produced vaccines (Fluzone® and Flublok®, respectively). In particular, undiluted vaccines were incubated with various amounts of trypsin at 37 °C for 60 minutes, then a trypsin inhibitor was added, 2x sample buffer was added, and heated at 70 °C for 10 minutes prior to SDS-PAGE.

[0219] The egg-produced variants were found to be trypsin-resistant. In particular, the commercially available trivalent egg-derived high-dose Fluzone vaccine that is cleaved into HA1 and HA2 and HA1 is resistant to trypsin digestion. In contrast, the commercially available trivalent recombinant HA Flublok vaccine is converted to HA1 and HA2 and HA1 polypeptides and is sensitive to trypsin when exposed to trypsin. Figure 27 (right panel). These results demonstrate that at least one of the strains is likely denatured by purification at pH 5.89 (see, e.g., Wang et al., Vaccine 24 (2006); 2176).

[0220] Thus, commercially available recombinant influenza vaccines suffer from misfolding that may result from production under low pH conditions, which is likely, at least in part, the reason for their insufficient immunogenicity and lack of widespread adoption to date.

[0221] In contrast, by the method disclosed herein, purification of HA nanoparticles using a buffer of at least pH 7.0 confirms that misfolding of the HA protein that occurs when the HA protein is exposed to acidic conditions during purification is reduced or eliminated.

[0222] (Example 15) Construction of Ebola virus glycoprotein nanoparticles The wild-type, full-length, unmodified EBOV glycoprotein (GP) gene of the 2014 Makona Ebola virus was cloned into a recombinant baculovirus and expressed in Spodoptera frugiperda Sf9 insect cells. After expression, the N-terminal signal peptide was cleaved, and the mature protein was purified to form nanoparticles. The purified Ebola virus GP (EBOV / Mak GP) nanoparticles consisted of multiple GP trimers assembled into spherical particles 36 ± 4 nm (measured by dynamic light scattering). The recombinant GP nanoparticles had a core region containing the glycoprotein 2 (GP2) "fusion subunit" along with two to nine or up to 15 outer "cup-like" glycoprotein 1 (GP1) trimer "attachment subunits".

[0223] When co-administering matrix M, a saponin-based adjuvant consisting of two populations of individually formed matrix particles of size 40 nm was used. The matrix M used was 85% matrix A and 15% matrix C. The matrix particles were formed by formulating cholesterol and phospholipids with purified saponin from Quillaja saponaria Molina.

[0224] (Example 16) Immunization and protocol Balb / c mice (6 - 8 weeks old; Harlan Laboratories Inc., Frederick, MD) were housed in groups of 10 and immunized by subcutaneous (SC) or intramuscular (IM) administration. Phosphate-buffered saline (PBS) was used as a placebo. Blood samples for serum were collected via the retro-orbital route. Prior to blood collection, the animals were anesthetized with isoflurane.

[0225] On days 0 and 21, mice (n = 10 per group) were given EBOV / Mak GP alone or AlPO 4 (50 μg) or matrix M adjuvant (2.5 μg or 5 μ g) and immunized by IM administration (50 μl injection volume). Blood samples were collected on days 0, 14, 21, 28 and 60. Spleen and bone marrow samples were collected on days 28 and 60. Spleen and bone marrow samples were suspended in PBS containing 2% fetal bovine serum (FBS) for further preparation.

[0226] Serum samples on day 28 were evaluated for anti-EBOV / Mak neutralizing antibody response using a pseudovirus neutralization reporter assay at the U.S. Army Medical Research Institute of Infectious Diseases, Fredrick, MD. A hantavirus pulmonary syndrome (HPS) DNA vaccine delivered using a spring-loaded jet injector elicits a strong neutralizing antibody response in rabbits and non-human primates. Curr Gene Ther., 2014;14:200-210. For this assay, the vesicular stomatitis virus G protein was removed and replaced with a luciferase reporter. This VSV luciferase expression core was pseudotyped using the plasmid pWRG / EBOV-Z76(opt) expressing Zaire ebolavirus 1976 (Mayinga) GP. The plasmid used to obtain the pseudotyped ebola GP was pWRG / EBOV / Mak-Z76(opt) expressing Zaire ebolavirus 1976 (Mayinga) GP. PsVs were prepared in 293T cells. Mouse sera were heat-inactivated at 56 °C for 30 min and then a 1:20 dilution was prepared first, followed by 5-fold serial dilution in Eagle's minimum essential medium (EMEM) (Life Technologies) supplemented with 10% (volume / volume) heat-inactivated FBS, 100 IU / mL penicillin and 100 μg / mL streptomycin (cEMEM). Ebola GP PsVs were diluted in cEMEM. Equal volumes of 4×10 3A PsVs solution containing a focus-forming unit and 10% molar guinea pig complement (Cedarlane) were added to the serum dilutions at a final starting dilution of 1:40 and then incubated overnight at 4°C. Monolayers of Vero cells seeded in clear-bottom black 96-well plates (Corning) were infected with 50 μl of each PsVs-serum mixture and then incubated for an additional 18 - 24 hours at 37°C. The medium was discarded, the cells were lysed, and a luciferase substrate was added according to the protocol of the Renilla Luciferase Assay System (Promega #E2820). The flash luciferase signal was measured using a Tecan M200 microplate reader. The raw values were transferred to GraphPad Prism version 6.04 and the data were baseline corrected against the untreated PsVs signal. The data were fitted to a four-parameter logistic non-linear regression model using GraphPad Prism and then the PsVNA 50% (PsVNA50) neutralization titers were interpolated from the curve for each sample. Each sample was analyzed in triplicate. The assay positive control was serum from rabbits vaccinated three times with pWRG / EBOV-Z76 (opt), a Zaire ebolavirus 1976 Mayinga GP DNA vaccine.

[0227] EBOV / Mak GP-specific serum antibodies were quantified by enzyme-linked immunosorbent assay (ELISA). Briefly, NUNC MaxiSorp microtiter plates were coated overnight at 2 - 8 °C with 2 μg / mL of EBOV / Mak GP (Novavax). Unreacted surfaces were blocked for 1 hour at room temperature (RT) using StartingBlock Blocking Buffer (Pierce). The plates were sequentially reacted at RT with 5-fold serial dilutions of serum samples starting at 1:100 (2 hours), goat anti-mouse IgG (or IgG1 and IgG2a) conjugated to horseradish peroxidase (HRP) (Southern Biotech) (1 hour), peroxidase substrate 3,3,5,5-tetramethylbenzidine (TMB) (Sigma) (10 minutes), and TMB Stop Buffer (Scy Tek Laboratories). The plates were washed three times with PBS / Tween (Quality Biologicals) before addition of the HRP conjugate and TMB reagent.

[0228] The plates were read at 450 nm using a SpectraMax plus plate reader (Molecular Devices). SoftMax Pro software (Molecular Devices) was used to fit the concentration response to a four-parameter sigmoidal curve. Antibody titers were defined as the reciprocal of the highest dilution with a 50% maximum antibody binding (EC 50 ) response. If the serum IgG titer was outside the lower limit of detection, a titer (starting dilution) < 100 was recorded and a value of 50 was assigned to the sample, and the geometric mean titer (GMT) of the group was calculated. A mouse anti-EBOV / Mak GP monoclonal antibody (mAb) (4F3) from IBT Bioservices (Gaithersburg, MD) was used as a positive control.

[0229] ELISPOT assay to evaluate IFNγ and EBOV / Mak GP-specific IgG-secreting cells Single cell suspensions were prepared from individual spleens by gently grinding the tissue using the plunger of a syringe. Single bone marrow cell suspensions were prepared by flushing bone with PBS containing 2% FBS. Cells were washed twice with PBS containing 2% FBS and counted. The IFNγ ELISPOT assay was performed using a mouse IFNγ ELISPOT kit (eBioscience, San Diego, CA) according to the manufacturer's procedure. Briefly, ELISPOT plates (Millipore, Darmstadt, Germany) were coated overnight at 4°C with anti-IFNγ antibody (15 μg / ml in PBS) at 100 μl / well. Plates were washed four times with PBS and blocked for 1 - 2 hours at room temperature with RPMI1640 medium + 5% FBS. A total of 3×10 5 spleen cells in a volume of 200 μl were stimulated with a 15-mer EBOV GP peptide pool (2.5 μg / ml) containing 11 overlapping amino acids that span the full-length EBOV GP sequence. Phorbol myristate acetate (PMA) (50 ng / ml) plus ionomycin (200 ng / ml) were used as a positive control and medium as a negative control. Each stimulation condition was performed in triplicate. Assay plates were incubated overnight at 37°C in a 5% CO 2 incubator, and signals were developed according to the manufacturer's instructions. Spots were counted and analyzed using an ELISPOT reader and Immunospot software (Cellular Technology, Ltd., Shaker Heights, OH). The number of Ebola GP-specific spots was obtained by subtracting the background number of medium controls from the GP peptide-stimulated wells. Data shown in graphs are the mean of triplicate wells. To measure GP-specific IgG-secreting cells, ELISPOT plates were coated with EBOV / Mak GP (2.5 μg / ml in PBS) and incubated overnight at 4°C. Plates were washed and blocked as described above. 3 - 5×10 5Triplicates of individual splenocytes or bone marrow cells were cultured in a flat plate, and the plate was incubated overnight at 37°C. On the second day, the plate was washed, goat anti-mouse IgG-HRP was added, and incubated for 1.5 hours. The spots were developed and counted as described above. The average number of spots was calculated from the triplicate wells and shown.

[0230] Surface staining for cell phenotype and intracellular staining for cytokines For surface staining, cells were first incubated with anti-CD16 / 32 antibody (clone 2.4G2) to block Fc receptors. To characterize germinal center cells, 1×10 6 fresh splenocytes were incubated with a mixture of the following antibodies: B220-PerCP, CD19-APC, GL7-BV421, CD95-PE-Cy7 (BD Biosciences, CA) and yellow LIVE / DEAD® dye (Life Technologies, NY) for 30 minutes at 4°C. The cells were washed twice and resuspended in PBS containing 2% FBS for analysis. To stain follicular helper T cells, 1×10 6 fresh splenocytes were incubated with CXCR5-biotin, washed twice, and then incubated with a mixture of antibodies including CD3-BV650, B220-PerCP, CD4-PE-Cy7, streptavidin-BV421, PD-1-APC, CD69-FITC and CD49b-PE (BD Biosciences, CA) and yellow LIVE / DEAD® dye (Life Technologies). The cells were washed twice and resuspended in PBS containing 2% FBS for analysis.

[0231] For intracellular staining of cytokines, splenocytes were seeded at 1×10 per well 6cells were cultured in a 96-well U-bottom plate. Peptide stimulation was performed as described for ELISPOT culture. The plates were incubated at 37 °C for 6 h in the presence of BD GolgiPlug™ and BD GolgiStop™ (BD Biosciences). Cells were washed twice and incubated with a mixture of antibodies against cell surface markers including CD3-BV 650, CD4-PerCP, CD8-FITC, CD44-APC-Cy7, and CD62L-PE-Cy7 (BD Pharmingen, CA) and the yellow LIVE / DEAD® dye (Life Technologies, NY) for 20 min at 4 °C. After two washes, cells were fixed with Cytofix / Cytoperm (BD Biosciences) for 30 min at 4 °C, followed by two washes with BD Perm / Wash™ (BD Biosciences). Cells were incubated overnight at 4 °C with antibodies against IFNγ-APC, IL-2-BV 421, and TNFα-PE (BD Biosciences). Cells were washed and resuspended in 1× BD Perm / Wash buffer for data acquisition. All stained samples were acquired using an LSR-Fortessa flow cytometer (Becton Dickinson, San Jose, CA), and the data were analyzed with Flowjo software version Xv10 (Tree Star Inc., Ashland, OR).

[0232] Statistical analysis was performed using SAS software version 9.4. Pairwise comparisons with Tukey adjustment from ANOVA were used to determine significance between groups using the group as the independent variable and the log-transformed titer results as the dependent variable.

[0233] (Example 17) EBOV / Mak GP-Induced Antibody Responses and Protective Efficacy The immunogenicity of the EBOV / Mak GP nanoparticle vaccine was evaluated in a mouse model, with and without an adjuvant. On days 0, 14, and 28, mice were vaccinated by SC injection with 5 μg of EBOV / Mak GP formulated alone or in Matrix M or AlPO 4 adjuvant. Analysis of sera obtained on day 28 (14 days after the second immunization) showed that Matrix M-adjuvanted EBOV / Mak GP induced high levels of antigen-specific IgG antibodies against Mayinga GP with a geometric mean titer (GMT) of 26,991. The response obtained after immunization with EBOV / Mak GP containing Matrix M was significantly higher than that induced by EBOV / Mak GP alone (GMT = 266, p = 0.001) or EBOV / Mak GP adjuvanted with AlPO 4 (GMT = 436, p = 0.0001) (Figure 28A). AlPO 4 adjuvant presented only a slight increase in anti-EBOV / Mak GP IgG compared to EBOV / Mak GP alone.

[0234] The neutralizing activity of sera on day 28 was analyzed using Ebola GP pseudovirions (PsVs) (Figure 28B). In the absence of an adjuvant, the neutralizing GMT titer in sera from mice immunized with EBOV / Mak GP alone was 197, and lower titers were observed when EBOV / Mak GP was adjuvanted with AlPO 4 (GMT = 49, p = 0.1). The neutralizing titers observed in sera from mice immunized with EBOV / Mak GP containing Matrix M had a GMT of 6,463, which was 32-fold higher than that obtained with EBOV / Mak GP alone. In this assay, PsVs expressing the EBOV / Mak 2014 strain GP were not available, so PsVs expressing the EBOV 1976 Mayinga strain GP were used. Thus, the assay measures the cross-neutralizing activity of anti-EBOV / Mak GP against Mayinga GP. Since PsVs expressing the EBOV / Mak 2014 strain GP were not available, PsVs expressing the EBOV 1976 Mayinga strain GP were used. Thus, the assay measures the cross-neutralizing activity of anti-EBOV / Mak GP against Mayinga GP.

[0235] On day 42, 2 weeks after the third vaccination on day 28, mice were challenged by intraperitoneal inoculation with 1,000 pfu of the mouse-adapted Zaire ebolavirus strain 1976 Mayinga. Control mice began to die of infection 3 days later, while mice vaccinated with EBOV / Mak GP alone or EBOV / Mak GP adjuvanted with AlPO 4 died on days 5 or 6, respectively. Twenty-one days after challenge infection, all mice vaccinated with matrix M-adjuvanted EBOV / Mak GP and one mouse vaccinated with EBOV / Mak GP alone survived and were healthy. In contrast, all other mice died of ebolavirus infection or were euthanized by day 8 (Figure 28C).

[0236] (Example 18) Kinetics of Ebola GP IgG, IgG1, and IgG2a responses To further characterize in more detail the immune response to matrix M-adjuvanted EBOV / Mak GP, two groups of Balb / c mice (10 mice / group) were injected with 5 μg of EBOV / Mak GP adjuvanted with either 2.5 or 5 μg of matrix M . Groups of mice injected with PBS, EBOV / Mak GP alone, or EBOV / Mak GP containing AlPO 4 were used as controls. On days 14, 21, 28, and 60, EBOV / Mak GP-specific IgG and IgG subclasses (IgG1 and IgG2a) were measured by ELISA.

[0237] Fourteen days after the first injection, all mice injected with EBOV / Mak GP containing matrix M (2.5 or 5 μg) responded with EBOV / Mak GP-specific IgG (GMT = 755 and 1,499, respectively, data not shown). The EBOV / Mak GP group and AlPO 4None of the 10 mice in the EBOV / Mak GP group containing [substance] produced EBOV / Mak GP-specific IgG (data not shown). By day 21, the IgG response to EBOV / Mak GP further increased in the matrix M-adjuvanted group (Figure 29A). In the groups given EBOV / Mak GP alone or containing AlPO 4 there was still no response. On day 21, all mice received a second injection. On day 28, in mice receiving matrix M (2.5 or 5 μg), there were strong increases in the IgG response with ELISA GMT titers of 3.0×10 5 and 4.9×10 5 respectively (Figure 29A). On days 28 and 60, specific IgG responses were detected in several mice in the groups given EBOV / Mak GP alone and containing AlPO 4 but were significantly lower than in mice immunized with EBOV / Mak GP containing matrix M (Figure 29A). By day 60, the anti-GP IgG titers induced by EBOV / Mak GP containing 2.5 or 5 μg of matrix M did not decrease significantly compared to day 28 and were 67-fold and 139-fold higher respectively than the EBOV / Mak GP group containing AlPO 4 (Figure 29A).

[0238] EBOV / Mak GP-specific IgG1 and IgG2a responses were also determined. Similar to total IgG, the matrix M-adjuvanted EBOV / Mak GP vaccine induced high anti-GP IgG1 and IgG2a levels on days 28 and 60 (Figures 29B and 29C). In contrast, only 1 out of 10 mice given EBOV / Mak GP alone and 4 out of 10 mice given EBOV / Mak GP containing AlPO 4 produced low levels of IgG1 on day 28 (Figure 29B). On day 60, antigen-specific IgG1 was detected in the sera of all 5 remaining mice in the group given EBOV / Mak GP containing AlPO 4 but the mean titer was lower than that of the matrix M 2.5 or 5 It was 1 / 51 and 1 / 41, respectively, of those in the group given EBOV / Mak GP containing 0.0 μg (Figure 29B). Furthermore, on days 28 and 60, EBOV / Mak GP alone did not induce detectable IgG2a antibodies.

[0239] (Example 19) CD4+, CD8+ and multifunctional T cell responses Next, T cell responses to different EBOV / Mak GP formulations were evaluated by measuring the number of IFNγ-secreting T cells after ex vivo stimulation of spleen cells with EBOV / Mak GP peptides in an ELISPOT assay. On day 28, IFNγ-secreting cells increased in a matrix M dose-dependent manner in the spleens of mice immunized with EBOV / Mak GP containing matrix M (Figures 30A, 30B). The average numbers of IFNγ-secreting cells in the groups receiving EBOV / Mak GP containing matrix M 5.0 and 2.5 μg were 17-fold and 10-fold higher, respectively, than those in the group receiving EBOV / Mak GP alone, and 4 8-fold and 5-fold higher, respectively, than those in the group receiving EBOV / Mak GP containing AlPO (Figure 30A).

[0240] By day 60, the number of IFNγ-secreting cells in the spleens of mice immunized with EBOV / Mak GP containing matrix M 5 μg was still 12-fold higher than that in the spleens of mice immunized with EBOV / Mak GP alone, and 4 3-fold higher than that in the spleens of mice immunized with EBOV / Mak GP containing AlPO (Figure 30B). The increase in the number of IFNγ-secreting cells in the spleens of mice immunized with EBOV / Mak GP containing matrix M 2.5 μg was also maintained on day 60 but at a lower level than that containing matrix M 5 μg.

[0241] Intracellular staining of cytokines in combination with cell surface markers was used to further evaluate the matrix M-induced CD4+ and CD8+ T cell responses. On day 28, analysis of splenocytes by flow cytometry staining showed that both CD4+ and CD8+ T cells from the EBOV / Mak GP group containing matrix M secreted IFNγ, TNFα, and IL-2 (Figures 30C and 30D). The frequencies of cytokine-secreting CD4+ and CD8+ T cells were very high in the spleens from the EBOV / Mak GP group containing matrix M compared to the baseline or minimal responses observed in control mice, mice receiving EBOV / Mak GP alone, or EBOV / Mak GP containing AlPO 4 (Figures 30C and 30D). The frequencies of T cells producing two or more cytokines (IFNγ, TNFα, and IL-2) were also evaluated on day 28. Both CD4+ and CD8+ T cells producing either two or three cytokines were detected at significant levels only in the spleens from mice immunized with EBOV / Mak GP containing matrix M.

[0242] (Example 20) Germinal center and follicular helper T cell responses The frequencies and absolute numbers of GC B cells in the spleen were analyzed by flow cytometry staining (Figure 31A). The analysis showed that on day 28 (7 days after the second vaccine injection), EBOV / Mak GP adjuvanted with 2.5 and 5 μg of matrix M induced responses with GC frequencies of 1.22 and 2.12%, respectively, compared to placebo, EBOV / Mak GP alone, or EBOV / Mak GP containing AlPO 4 (0.38, 0.41, and 0.44%, respectively) (Figure 31B). Thus, the absolute numbers of GC cells in the spleen also increased in the groups receiving matrix M (Figure 31C). By day 60, the frequencies and absolute numbers had returned to background levels (Figures 31D and 31E).

[0243] T on day 28 FHAnalysis of cell frequency was performed with Matrix M at 2.5 or 5 μg included EBOV / Mak GP induced higher T 4 cell frequencies than those with EBOV / Mak GP alone or AlPO FH (Figures 32A and 32B). Thus, the absolute number of T FH cells was also enhanced by EBOV / Mak GP containing Matrix M compared to those with EBOV / Mak GP alone or AlPO 4 included (Figure 32C). By day 60, the frequency and absolute number of T FH cells regressed to levels close to background (Figures 32D and 32E).

[0244] (Example 21) EBOV / Mak GP-specific plasma cells To evaluate the effect of Matrix M on EBOV / Mak GP-specific plasma cells, the number of IgG-producing cells in the spleen and bone marrow was analyzed on day 60 after immunization. Analysis on day 60 demonstrated very few EBOV / Mak GP-specific IgG-secreting cells (<6 / 10 6 spleen cells) in the spleens of mice immunized with Matrix M-adjuvanted EBOV / Mak GP vaccine (Figure 33A). IgG-secreting cells were not detected in the spleens of mice immunized with EBOV / Mak GP alone and EBOV / Mak GP containing AlPO 4 (Figure 33A). In contrast, a large number of EBOV / Mak GP-specific IgG-secreting cells were found in the bone marrow of mice that received Matrix M-adjuvanted EBOV / Mak GP (Figure 33B), demonstrating the formation of long-lived plasma B cells.

[0245] (Example 22) Characterization of antibody binding to nanoparticles The ability of several anti-Ebola antibodies that bind to nanoparticles was tested. The antibodies are 13C6, 13F6, 6D8 and KZ52. The EC50 curves and values are shown in Figure 36, and additional binding kinetic data are shown in Figure 37. Figure 38 shows the efficacy data using 13C6 as a reference. Three of the four antibodies exhibited excellent binding to GP.

[0246] (Example 23) Non-human primate study: baboon To confirm the results obtained in mice in a non-human primate model, a baboon study was conducted. The study was designed as shown in Figure 39. Four groups were formed. Group 1 was the control. Group 2 received an antigen containing AlPO 4 Groups 2 and 3 received 60 μg and 5 μg of the antigen together with 50 μg of Matrix M, respectively. The baboons were immunized on days 0 and 21. A strong response was obtained against both Makona GP and Mayinga GP. See Figure 40. Additional analysis confirmed that the response persisted. Figure 41 shows the EC50 values of IgG against Makona at a later time point. The data establish that the response is persistent.

[0247] Additional studies confirmed that IFNγ levels increased substantially after immunization. Figure 42 shows that Matrix M combined with GP increased more than when combined with alum adjuvant. Interestingly, a low dose of GP 5 μg resulted in a more significant increase in IFNγ levels. The TNFα and IFNγ responses in T cells are shown in Figure 43, and the cytokine responses are shown in Figure 44. Also, the response is more prominent with GP and Matrix M than with alum in each case. These data highlight the strong immune response of the disclosed formulations in the baboon model.

[0248] (Example 24) Non-human primate study: macaque study 1 To further confirm the protective effect by nanoparticles, a macaque study was conducted as shown in Figure 45. As shown, macaques were immunized intramuscularly with the vaccine on days 0 and 21 and challenged on day 42. The anti-GP response was measured on days 0 and 28. As shown in Figure 46 immunized macaques showed a dramatic induction of anti-IgG antibodies. The immune response was characterized as shown in Figure 47. IFNγ-secreting cells responding to various peptide pools were measured at weeks 0, 3, and 5. The results demonstrate that immunized macaques induced IFNγ-secreting cells in immunized macaques.

[0249] The survival of the animals is notable. Figure 48. By day 7, the amount of Ebola virus in placebo-treated macaques was 10 7 . By day 9, placebo animals were euthanized. In contrast, 100% of the treated animals survived. Notably, the immune response was able to make the virus amount undetectable by RT-PCR in almost all animals at almost all time points. Animal 33362 showed a virus amount exceeding the detection limit by about 10% on day 7. However, by day 10, the level had dropped to a level below the ability of the assay to detect it.

[0250] (Example 25) Non-human primate study: Macaque study 2 A second study was conducted in macaques. At week 0, animals were dosed with 5 μg of GP + 50 μg of Matrix M, and an additional boost was given either at week 3 or week 6. The immunized animals were then challenged with wild-type Ebola virus at weeks 9 and 12, respectively. Figure 49.

[0251] ELISA data for IgG are shown in Figure 50. The left panel shows that high titers appeared and were persistent 3 weeks after the first injection. The right panel illustrates the results in animals with a 6-week gap between administrations. Those animals showed a substantial increase 2 weeks after the second booster administration, exemplifying the beneficial effect of the prime-boost approach.

[0252] The vaccine composition was completely protective in macaques. By 18 days after challenge with live virus, all saline control-treated mice had died. In contrast, 100% of the macaques immunized with the vaccine composition survived the challenge. Collectively, these data confirm that the immune response elicited by the composition is protective regardless of whether the boost dose is within 3 weeks or within 6 weeks.

Claims

**Claim 1** An immunogenic composition comprising: (i) a first nanoparticle comprising a non-ionic surfactant core and a viral glycoprotein, wherein the viral glycoprotein is an influenza hemagglutinin (HA) glycoprotein, the head region of the influenza HA glycoprotein protrudes outward from the non-ionic surfactant core, and the transmembrane domain of the influenza HA glycoprotein associates with the non-ionic surfactant core, the first nanoparticle, wherein the non-ionic surfactant is polysorbate-80 (PS80); (ii) a second nanoparticle comprising a non-ionic surfactant core and a viral glycoprotein, wherein the viral glycoprotein is not an influenza HA glycoprotein, the head region of the glycoprotein protrudes outward from the non-ionic surfactant core, and the transmembrane domain of the glycoprotein associates with the non-ionic surfactant core, the second nanoparticle, wherein the non-ionic surfactant is PS80; and (iii) a pharmaceutically acceptable buffer. An immunogenic composition. **Claim 2** (iv) a third nanoparticle comprising a non-ionic surfactant core and a viral glycoprotein, wherein the viral glycoprotein is an influenza hemagglutinin (HA) glycoprotein, the head region of the influenza HA glycoprotein protrudes outward from the non-ionic surfactant core, and the transmembrane domain of the influenza HA glycoprotein associates with the non-ionic surfactant core, the non-ionic surfactant is polysorbate-80 (PS80), and the influenza HA glycoprotein of the third nanoparticle is derived from an influenza strain different from the influenza HA glycoprotein of the first nanoparticle. The immunogenic composition according to claim 1, further comprising the third nanoparticle. **Claim 3** (v) a fourth nanoparticle comprising a non-ionic surfactant core and a viral glycoprotein, wherein the viral glycoprotein is an influenza hemagglutinin (HA) glycoprotein, the head region of the influenza HA glycoprotein protrudes outward from the non-ionic surfactant core, and the transmembrane domain of the influenza HA glycoprotein associates with the non-ionic surfactant core, The nonionic surfactant is polysorbate-80 (PS80), The immunogenic composition according to claim 2, further comprising a fourth nanoparticle, wherein the influenza HA glycoprotein of the fourth nanoparticle is derived from an influenza strain different from the influenza HA glycoprotein of the first nanoparticle or the third nanoparticle.

4. (vi) A fifth nanoparticle comprising a nonionic surfactant core and a viral glycoprotein, wherein the viral glycoprotein is an influenza hemagglutinin (HA) glycoprotein, the head region of the influenza HA glycoprotein protrudes outward from the nonionic surfactant core, and the transmembrane domain of the influenza HA glycoprotein associates with the nonionic surfactant core, The nonionic surfactant is polysorbate-80 (PS80), The immunogenic composition according to claim 3, further comprising a fifth nanoparticle, wherein the influenza HA glycoprotein of the fourth nanoparticle is derived from an influenza strain different from the influenza HA glycoproteins of the first nanoparticle, the third nanoparticle, and the fourth nanoparticle.

5. The immunogenic composition according to any one of claims 1 to 4, wherein the immunogenic composition comprises an adjuvant.

6. The immunogenic composition according to claim 5, wherein the adjuvant comprises at least two ISCOM particles, the first ISCOM particle comprises fraction A of Quillaja saponaria Molina and does not comprise fraction C of Quillaja saponaria Molina, and the second ISCOM particle comprises fraction C of Quillaja saponaria Molina and does not comprise fraction A of Quillaja saponaria Molina.

7. The immunogenic composition according to claim 6, wherein the two ISCOM matrix particles are present in a composition of about 70% (w / w) of the first ISCOM particle and about 30% (w / w) of the second ISCOM particle.

8. The immunogenic composition according to claim 5, wherein the two ISCOM matrix particles are present in a composition of about 85% (w / w) of the first ISCOM particle and about 15% (w / w) of the second ISCOM particle.

9. The immunogenic composition according to claim 5, wherein the adjuvant comprises ISCOM matrix particles comprising a mixture of fraction A of Quillaja saponaria Molina and fraction C of Quillaja saponaria Molina.

10. The immunogenic composition according to claim 9, wherein the mixture comprises about 70% (w / w) fraction A of Quillaja saponaria Molina and about 30% (w / w) fraction C of Quillaja saponaria Molina.

11. The immunogenic composition according to claim 5, wherein the mixture comprises about 85% (w / w) fraction A of Quillaja saponaria Molina and about 15% (w / w) fraction C of Quillaja saponaria Molina.

12. The immunogenic composition according to claim 5, wherein the adjuvant is an alum adjuvant.

13. The immunogenic composition according to any one of claims 1 to 12, wherein the immunogenic composition comprises about 30 μg to about 300 μg of viral glycoprotein.

14. The immunogenic composition according to any one of claims 1 to 13, wherein the pharmaceutically acceptable buffer comprises about 300 mM sodium chloride.

15. The immunogenic composition according to any one of claims 1 to 13, wherein the pharmaceutically acceptable buffer comprises about 25 mM sodium phosphate.

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