Vaccine compositions 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-REFERENCE TO RELATED APPLICATIONS This application incorporates in their entirety for all purposes the disclosures of U.S. Provisional Application Nos. 62 / 213,947, filed September 3, 2015; 62 / 255,786, filed November 16, 2015; 62 / 309,216, filed March 16, 2016; and 62 / 350,973, filed June 16, 2016.

[0002] Description of electronically submitted text files The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: Computer-readable copy of the Sequence Listing (Filename: NOVV_060_03US_SeqList_ST25.txt, Recorded: 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 technology]

[0003] Infectious diseases remain a challenge worldwide. While progress has been made in developing vaccines against some pathogens, many remain a threat to human health. HIV, the most notorious, remains a vaccine-free disease. Attempts to produce vaccines against certain pathogens have failed, resulting in additional pathology. Other pathogens remain a challenge, including Ebola, an epidemic that occurs sporadically, particularly in Africa, resulting in loss of life and global economic impact. Influenza virus is yet another virus for which existing vaccines offer some protection, but technical obstacles in virus production mean that seasonal influenza vaccines may offer insufficient protection.

[0004] Deploying an effective vaccine depends on a combination of accomplishments: 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 for use in challenging environments where refrigeration may not be available. Summary of the Invention [Problem to be solved by the invention]

[0005] Thus, there is a continuing interest in producing vaccines against viruses that represent public health challenges worldwide, and there is a continuing need to produce effective vaccines with good stability. [Means for solving the problem]

[0006] The present disclosure provides nanoparticles suitable for inducing an immune response against pathogens. The nanoparticles offer improved stability and effective immunogenicity. In certain aspects, 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 viral proteins with enhanced stability. In some embodiments, the present disclosure includes vaccine compositions comprising nanoparticles comprising a non-ionic surfactant, a viral glycoprotein, and a pharmaceutical buffer. In typical embodiments, the non-ionic 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 non-ionic surfactant. One or more glycoprotein antigen molecules surround a surfactant core containing a non-ionic 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 a further embodiment, 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 of between 15 mM and 25 mM; NaCl at a concentration of between 125 mM and 175 mM; histidine at a concentration of between 0.25% and 2% w / v; and the pH of the composition is between 5.8 and 7.2.

[0011] In some embodiments, an 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; histidine at 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, the method for preventing infection includes administering one or more doses of a vaccine composition. In some method embodiments, a single dose of the composition is administered to induce a protective immune response. In some method embodiments, each dose consists of between about 100 μg and about 150 μg of protein antigen. In further method embodiments, the one or more doses are administered subcutaneously. In some method embodiments, the composition includes an adjuvant. In further method embodiments, the adjuvant is alum. In some method embodiments, the composition does not include an adjuvant.

[0014] In some embodiments of the method, one or more doses of the composition are administered to an adult. In further embodiments of the method, the adult is a woman, and the woman may be pregnant. In further embodiments of the method, the adult is over 65 years of age or over 60 years of age. In some embodiments of the method, one or more doses of the composition are administered to a child. In further embodiments of the method, the child is a newborn or an infant.

[0015] For RSV vaccines, 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 surrounding a surfactant-containing core comprising PS80, and wherein a first RSV F nanoparticle type comprises an anisotropic rod and a second RSV F nanoparticle type comprises a spherical oligomer. and a third RSV F nanoparticle type includes intermediates between anisotropic rods and spherical oligomers.

[0016] In some embodiments, a method for producing RSV F protein nanoparticles includes preparing a 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. In a further embodiment 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 agitation resistance. In some embodiments of the method, the molar ratio of PS80:RSV F protein is 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 about 20 nm to about 60 nm as measured by dynamic light scattering. In some embodiments of the F nanoparticles, each RSV F protein trimer contains a 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 contains a 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 a deletion of 10 amino acids corresponding to residues 137-146, or SEQ ID NO: 2, and inactivation of the major furin cleavage site by mutation of the 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 the mature peptide SEQ ID NO: 19. In certain embodiments of the RSV F nanoparticles, the RSV F protein comprises or consists of SEQ ID NO: 8. RSV Vaccine formulations containing F nanoparticles consist essentially of the mature peptide (SEQ ID NO: 8) with any full-length peptide. Over time, small amounts of truncated RSV F peptides may be generated due to proteolysis. Advantageously, however, the RSV F nanoparticles disclosed herein minimize such degradation and provide long-term stability.

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

[0020] For Ebola, Ebola virus nanoparticles include vaccine compositions containing Ebola virus glycoprotein (GP) trimers and nanoparticles attached to a non-ionic surfactant core, optionally in combination with a matrix M saponin adjuvant. Additionally, the present disclosure provides methods for inducing an immune response to Ebola virus in humans by administering compositions containing Ebola virus nanoparticles and a saponin adjuvant. Methods for protecting against Ebola infection are also provided.

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

[0022] [Figure 1A] Figures 1A and 1B show the primary protein structure of the RSV F protein along with the polypeptide sequence. Figure 1A shows the primary protein structure of the modified RSV F protein relative to that of the wild-type RSV A2 strain. The furin cleavage site is indicated by a triangle. Figure 1B shows the amino acid sequence of the modified RSV F protein (SEQ ID NO: 19), including the F1 domain (residues 1-84) in light-shaded text, the F2 domain (residues 85-539) in dark-shaded text, and the black line connecting the cysteines that form disulfide bonds; the underlined asparagine indicates the N-linked glycosylation site, the light-shaded vertical dashed line indicates the furin cleavage site, and the dark-shaded vertical dashed line indicates the primary cleavage site. [Figure 1B]Figures 1A and 1B show the primary protein structure of the RSV F protein along with the polypeptide sequence. Figure 1A shows the primary protein structure of the modified RSV F protein relative to that of the wild-type RSV A2 strain. The furin cleavage site is indicated by a triangle. Figure 1B shows the amino acid sequence of the modified RSV F protein (SEQ ID NO: 19), including the F1 domain (residues 1-84) in light-shaded text, the F2 domain (residues 85-539) in dark-shaded text, and the black line connecting the cysteines that form disulfide bonds; the underlined asparagine indicates the N-linked glycosylation site, the light-shaded vertical dashed line indicates the furin cleavage site, and the dark-shaded vertical dashed line indicates the primary cleavage site.

[0023] [Figure 2] Figure 2 shows the separation peak of RSV F protein by reverse-phase HPLC, and four major species were identified, corresponding to four major peaks. The peak containing the lowest molecular weight species (approximately 51.2 kDa to approximately 51.3 kDa) is a soluble trimer; the next peak contains a full-length trimer lacking fatty acids (approximately 64.5 kDa). The final two major peaks are full-length trimers containing palmitoleic acid (approximately 64.7 kDa) and palmitic acid (approximately 64.788 kDa), respectively.

[0024] [Figure 3] Figure 3 shows the separation of RSV F protein in reduced SDS-PAGE. The highest molecular weight protein contains high molecular weight species, followed by variants containing the F1 and F2 domains, then the F1 domain alone and its variants, followed by the F2 domain alone.

[0025] [Figure 4] Figure 4 shows the chromatogram of LC-UV peptide mapping covering 90% of the amino acids comprising the primary protein structure of the RSV F protein. The coverage of the combined sequence, including the early eluting peptides, was found to be 98%, thereby confirming the amino acid sequence of the RSV F protein.

[0026] [Figure 5] Figure 5 shows the glycosylation analysis of purified RSV F protein using HPLC coupled with fluorescence detection (FLD). The major glycan structure detected is fucosylated Man3 glycan.

[0027] [Figure 6] Figure 6 shows electron micrographs of RSV F protein trimers and RSV F nanoparticles associated with the core of PS80. The figure further illustrates the orientation of the F1 and F2 domains, antigenic site II recognized by the palivizumab antibody, and the C- and N-termini of the F1 domain, which further contain fatty acids such as palmitic acid and palmitoleic acid.

[0028] [Figure 7] Figure 7 shows dynamic light scattering (DLS) measurements of the particle size of RSV F nanoparticles. DLS measurements show that the size of the nanoparticles is modulated by both the available PS80 and the RSV F concentration. At a constant RSV F concentration, an increase in PS80 results in a decrease in the average nanoparticle size (Z average).

[0029] [Figure 8] Figure 8 shows the molecular weight distribution of nanoparticles versus individual sample concentrations, where the concentration of RSV F and the percentage of PS80 were varied. The maximum signal intensity of the nanoparticles was achieved at 0.2% PS80 and 1 mg / mL RSV F, suggesting greater uniformity of the nanoparticles and confirming modulation of particle size as a function of the concentration of PS80 and RSV.

[0030] [Figure 9A]Figures 9A and 9B show the shape of RSV F nanoparticle types produced by various PS80 percentages and RSV F concentrations. Figure 9A shows that a composition using 0.2% PS80 and 0.22 mg / mL RSV F produces three primary types: monomer / dimer anisotropic rods, spherical oligomers, and intermediates. Figure 9B shows that a composition using 0.05% PS80 and 0.22 mg / mL RSV produces a population dominated by monomer / dimer anisotropic rods, and a composition using 0.05% PS80 and 1.0 mg / mL produces a population dominated by spherical oligomers. [Figure 9B] Figures 9A and 9B show the shape of RSV F nanoparticle types produced by various PS80 percentages and RSV F concentrations. Figure 9A shows that a composition using 0.2% PS80 and 0.22 mg / mL RSV F produces three primary types: monomer / dimer anisotropic rods, spherical oligomers, and intermediates. Figure 9B shows that a composition using 0.05% PS80 and 0.22 mg / mL RSV produces a population dominated by monomer / dimer anisotropic rods, and a composition using 0.05% PS80 and 1.0 mg / mL produces a population dominated by spherical oligomers.

[0031] [Figure 10] 10 shows the effect of stressors on specific subdivisions of the RSV F protein in nanoparticles, as shown by relative abundance compared to the control in a reduced Lys-C peptide map. The stressors were 50°C for 2 weeks, 25°C at pH 3.7 for 1 week, 25°C at pH 10 for 1 week, 25°C at hydrogen peroxide for 1 week, and 25°C with stirring for 1 week.

[0032] [Figure 11]Figure 11 illustrates the stability of antigenic site 2 (palivizumab site) exposed to various stress conditions. Percentages are shown as relative abundance compared to the control. The closer to or exceeding 100%, the greater the resilience in view of stress conditions. The data show that the nanoparticles maintain excellent antigenic site robustness and therefore generate a stable immune response. NSELLSLINDMPITNDQK / K; SEQ ID NO: 20 and LMSNN (SEQ ID NO: 21) are portions of antigenic site II.

[0033] [Figure 12A] Figures 12A, 12B, 12C, and 12D show the stability of RSV F nanoparticle compositions by demonstrating mouse immunogenicity after exposure of the nanoparticle compositions to environmental stress. Mice were sampled for anti-RSV F IgG on day 21, PCA titers on day 35, and RSV / A neutralization titers on day 35. Figure 12A shows the results of the -70°C control. Figure 12B shows the results of exposing the composition to 50°C for two weeks. Figure 12C shows the results of exposing the composition to pH 10 at 25°C for two weeks. Figure 12D shows the results of exposing the composition to 0.5% hydrogen peroxide at 25°C for one week. [Figure 12B] Figures 12A, 12B, 12C, and 12D show the stability of RSV F nanoparticle compositions by demonstrating mouse immunogenicity after exposure of the nanoparticle compositions to environmental stress. Mice were sampled for anti-RSV F IgG on day 21, PCA titers on day 35, and RSV / A neutralization titers on day 35. Figure 12A shows the results of the -70°C control. Figure 12B shows the results of exposing the composition to 50°C for two weeks. Figure 12C shows the results of exposing the composition to pH 10 at 25°C for two weeks. Figure 12D shows the results of exposing the composition to 0.5% hydrogen peroxide at 25°C for one week. [Figure 12C]Figures 12A, 12B, 12C, and 12D show the stability of RSV F nanoparticle compositions by demonstrating mouse immunogenicity after exposure of the nanoparticle compositions to environmental stress. Mice were sampled for anti-RSV F IgG on day 21, PCA titers on day 35, and RSV / A neutralization titers on day 35. Figure 12A shows the results of the -70°C control. Figure 12B shows the results of exposing the composition to 50°C for two weeks. Figure 12C shows the results of exposing the composition to pH 10 at 25°C for two weeks. Figure 12D shows the results of exposing the composition to 0.5% hydrogen peroxide at 25°C for one week. [Figure 12D] Figures 12A, 12B, 12C, and 12D show the stability of RSV F nanoparticle compositions by demonstrating mouse immunogenicity after exposure of the nanoparticle compositions to environmental stress. Mice were sampled for anti-RSV F IgG on day 21, PCA titers on day 35, and RSV / A neutralization titers on day 35. Figure 12A shows the results of the -70°C control. Figure 12B shows the results of exposing the composition to 50°C for two weeks. Figure 12C shows the results of exposing the composition to pH 10 at 25°C for two weeks. Figure 12D shows the results of exposing the composition to 0.5% hydrogen peroxide at 25°C for one week.

[0034] [Figure 13] Figure 13 shows the enhanced protease resistance of nanoparticles with higher PS80. Over 18 months, RSV F nanoparticles formulated in the presence of a higher PS80 percentage (0.03%) exhibited lower protease degradation than RSV F nanoparticles formulated in the presence of a lower PS80 percentage (0.015%), as assessed by SDS-PAGE. In addition, fewer high molecular weight (HMW) structures were observed at higher PS80 concentrations.

[0035] [Figure 14] Figure 14 shows a comparison of mAb binding to RSV F nanoparticles to RSV FA strain viral proteins, and the equilibrium dissociation constants for site I, II, and IV antibodies reveal that the mAb antibody binding at each site is comparable.

[0036] [Figure 15] Figure 15 shows the results of a competitive binding assay in which antibodies present in the serum of cotton rats exposed to placebo, RSV / A infection, formaldehyde-inactivated RSV, RSV F nanoparticles, and RSV F nanoparticles containing alum were compared to each other for binding to sites I, II, and IV.

[0037] [Figure 16] FIG. 16 illustrates a process flow chart of the method for fabricating nanoparticles disclosed herein.

[0038] [Figure 17] Figure 17 illustrates a flow chart of the method for fabricating HA nanoparticles disclosed herein. Sf9 cells containing baculovirus-expressed HA are grown, and the HA is then extracted using the non-ionic detergent NP9. The extract undergoes sequential purification and detergent exchange steps using a lectin affinity column, then is filtered and formulated into a bulk drug substance.

[0039] [Figure 18A] Figures 18A-18F illustrate steps for producing influenza nanoparticles using HA glycoprotein as an example and the results obtained using this method. Figure 18A shows the sequential purification steps from cell infection using three columns (TMAE (trimethylaminoethyl), followed by lentil lectin, followed by sulfuric acid (SO3-) column) used to obtain cell lysis and purified nanoparticles. Figure 18B illustrates a chromatogram obtained using the TMAE column. Figure 18C illustrates a chromatogram obtained using the lentil lectin column purification step. Figure 18D illustrates a chromatogram obtained using the sulfuric acid column purification step. Figure 18E shows a gel of the TMAE and LL column steps (upper right panel). The lower panel shows a Western blot of the gel. Lanes are as shown in the upper left panel. Figure 18F shows a gel containing the eluate from the SO3- column. [Figure 18B] Figures 18A-18F illustrate steps for producing influenza nanoparticles using HA glycoprotein as an example and the results obtained using this method. Figure 18A shows the sequential purification steps from cell infection using three columns (TMAE (trimethylaminoethyl), followed by lentil lectin, followed by sulfuric acid (SO3-) column) used to obtain cell lysis and purified nanoparticles. Figure 18B illustrates a chromatogram obtained using the TMAE column. Figure 18C illustrates a chromatogram obtained using the lentil lectin column purification step. Figure 18D illustrates a chromatogram obtained using the sulfuric acid column purification step. Figure 18E shows a gel of the TMAE and LL column steps (upper right panel). The lower panel shows a Western blot of the gel. Lanes are as shown in the upper left panel. Figure 18F shows a gel containing the eluate from the SO3- column. [Figure 18C] Figures 18A-18F illustrate steps for producing influenza nanoparticles using HA glycoprotein as an example and the results obtained using this method. Figure 18A shows the sequential purification steps from cell infection using three columns (TMAE (trimethylaminoethyl), followed by lentil lectin, followed by sulfuric acid (SO3-) column) used to obtain cell lysis and purified nanoparticles. Figure 18B illustrates a chromatogram obtained using the TMAE column. Figure 18C illustrates a chromatogram obtained using the lentil lectin column purification step. Figure 18D illustrates a chromatogram obtained using the sulfuric acid column purification step. Figure 18E shows a gel of the TMAE and LL column steps (upper right panel). The lower panel shows a Western blot of the gel. Lanes are as shown in the upper left panel. Figure 18F shows a gel containing the eluate from the SO3- column. [Figure 18D]Figures 18A-18F illustrate steps for producing influenza nanoparticles using HA glycoprotein as an example and the results obtained using this method. Figure 18A shows the sequential purification steps from cell infection using three columns (TMAE (trimethylaminoethyl), followed by lentil lectin, followed by sulfuric acid (SO3-) column) used to obtain cell lysis and purified nanoparticles. Figure 18B illustrates a chromatogram obtained using the TMAE column. Figure 18C illustrates a chromatogram obtained using the lentil lectin column purification step. Figure 18D illustrates a chromatogram obtained using the sulfuric acid column purification step. Figure 18E shows a gel of the TMAE and LL column steps (upper right panel). The lower panel shows a Western blot of the gel. Lanes are as shown in the upper left panel. Figure 18F shows a gel containing the eluate from the SO3- column. [Figure 18E] Figures 18A-18F illustrate steps for producing influenza nanoparticles using HA glycoprotein as an example and the results obtained using this method. Figure 18A shows the sequential purification steps from cell infection using three columns (TMAE (trimethylaminoethyl), followed by lentil lectin, followed by sulfuric acid (SO3-) column) used to obtain cell lysis and purified nanoparticles. Figure 18B illustrates a chromatogram obtained using the TMAE column. Figure 18C illustrates a chromatogram obtained using the lentil lectin column purification step. Figure 18D illustrates a chromatogram obtained using the sulfuric acid column purification step. Figure 18E shows a gel of the TMAE and LL column steps (upper right panel). The lower panel shows a Western blot of the gel. Lanes are as shown in the upper left panel. Figure 18F shows a gel containing the eluate from the SO3- column. [Figure 18F]Figures 18A-18F illustrate steps for producing influenza nanoparticles using HA glycoprotein as an example and the results obtained using this method. Figure 18A shows the sequential purification steps from cell infection using three columns (TMAE (trimethylaminoethyl), followed by lentil lectin, followed by sulfuric acid (SO3-) column) used to obtain cell lysis and purified nanoparticles. Figure 18B illustrates a chromatogram obtained using the TMAE column. Figure 18C illustrates a chromatogram obtained using the lentil lectin column purification step. Figure 18D illustrates a chromatogram obtained using the sulfuric acid column purification step. Figure 18E shows a gel of the TMAE and LL column steps (upper right panel). The lower panel shows a Western blot of the gel. Lanes are as shown in the upper left panel. Figure 18F shows a gel containing the eluate from the SO3- column.

[0040] [Figure 19A]Figures 19A-19J illustrate purity analyses of HA nanoparticles produced using different subtypes and different insect cell lines. Figure 19A shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing A / New Hampshire / 1 / 2015 HA. Figure 19B shows quantification of the HA bands, indicating that the HA is 99.1% pure by densitometry. Figure 19C shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing A / Switzerland / 9715293 / 2013 HA. Figure 19D shows quantification of the HA bands, indicating that the HA is 94.5% pure by densitometry. Figure 19E shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing A / Hong Kong / 4801 / 2014 HA. Figure 19F shows quantification of the HA bands, indicating that the HA is 93.3% pure by densitometry. Figure 19G shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing B / Phuket / 3073 / 2013 HA in Sf9 and Sf22a cells. The right panel shows quantification of the HA bands, indicating that the HA was 95.4% pure by densitometry. Figure 19H shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing B / Brisbane / 60 / 2008 HA in Sf9 cells. The right panel shows quantification of the HA bands, indicating that the HA was 96.7% pure by densitometry. Figure 19I measures HA purity using RP-HPLC. Figure 19J summarizes the data for HA nanoparticles using three influenza A subtypes and two influenza B subtypes. [Figure 19B]Figures 19A-19J illustrate purity analyses of HA nanoparticles produced using different subtypes and different insect cell lines. Figure 19A shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing A / New Hampshire / 1 / 2015 HA. Figure 19B shows quantification of the HA bands, indicating that the HA is 99.1% pure by densitometry. Figure 19C shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing A / Switzerland / 9715293 / 2013 HA. Figure 19D shows quantification of the HA bands, indicating that the HA is 94.5% pure by densitometry. Figure 19E shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing A / Hong Kong / 4801 / 2014 HA. Figure 19F shows quantification of the HA bands, indicating that the HA is 93.3% pure by densitometry. Figure 19G shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing B / Phuket / 3073 / 2013 HA in Sf9 and Sf22a cells. The right panel shows quantification of the HA bands, indicating that the HA was 95.4% pure by densitometry. Figure 19H shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing B / Brisbane / 60 / 2008 HA in Sf9 cells. The right panel shows quantification of the HA bands, indicating that the HA was 96.7% pure by densitometry. Figure 19I measures HA purity using RP-HPLC. Figure 19J summarizes the data for HA nanoparticles using three influenza A subtypes and two influenza B subtypes. [Figure 19C]Figures 19A-19J illustrate purity analyses of HA nanoparticles produced using different subtypes and different insect cell lines. Figure 19A shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing A / New Hampshire / 1 / 2015 HA. Figure 19B shows quantification of the HA bands, indicating that the HA is 99.1% pure by densitometry. Figure 19C shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing A / Switzerland / 9715293 / 2013 HA. Figure 19D shows quantification of the HA bands, indicating that the HA is 94.5% pure by densitometry. Figure 19E shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing A / Hong Kong / 4801 / 2014 HA. Figure 19F shows quantification of the HA bands, indicating that the HA is 93.3% pure by densitometry. Figure 19G shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing B / Phuket / 3073 / 2013 HA in Sf9 and Sf22a cells. The right panel shows quantification of the HA bands, indicating that the HA was 95.4% pure by densitometry. Figure 19H shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing B / Brisbane / 60 / 2008 HA in Sf9 cells. The right panel shows quantification of the HA bands, indicating that the HA was 96.7% pure by densitometry. Figure 19I measures HA purity using RP-HPLC. Figure 19J summarizes the data for HA nanoparticles using three influenza A subtypes and two influenza B subtypes. [Figure 19D]Figures 19A-19J illustrate purity analyses of HA nanoparticles produced using different subtypes and different insect cell lines. Figure 19A shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing A / New Hampshire / 1 / 2015 HA. Figure 19B shows quantification of the HA bands, indicating that the HA is 99.1% pure by densitometry. Figure 19C shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing A / Switzerland / 9715293 / 2013 HA. Figure 19D shows quantification of the HA bands, indicating that the HA is 94.5% pure by densitometry. Figure 19E shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing A / Hong Kong / 4801 / 2014 HA. Figure 19F shows quantification of the HA bands, indicating that the HA is 93.3% pure by densitometry. Figure 19G shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing B / Phuket / 3073 / 2013 HA in Sf9 and Sf22a cells. The right panel shows quantification of the HA bands, indicating that the HA was 95.4% pure by densitometry. Figure 19H shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing B / Brisbane / 60 / 2008 HA in Sf9 cells. The right panel shows quantification of the HA bands, indicating that the HA was 96.7% pure by densitometry. Figure 19I measures HA purity using RP-HPLC. Figure 19J summarizes the data for HA nanoparticles using three influenza A subtypes and two influenza B subtypes. [Figure 19E]Figures 19A-19J illustrate purity analyses of HA nanoparticles produced using different subtypes and different insect cell lines. Figure 19A shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing A / New Hampshire / 1 / 2015 HA. Figure 19B shows quantification of the HA bands, indicating that the HA is 99.1% pure by densitometry. Figure 19C shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing A / Switzerland / 9715293 / 2013 HA. Figure 19D shows quantification of the HA bands, indicating that the HA is 94.5% pure by densitometry. Figure 19E shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing A / Hong Kong / 4801 / 2014 HA. Figure 19F shows quantification of the HA bands, indicating that the HA is 93.3% pure by densitometry. Figure 19G shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing B / Phuket / 3073 / 2013 HA in Sf9 and Sf22a cells. The right panel shows quantification of the HA bands, indicating that the HA was 95.4% pure by densitometry. Figure 19H shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing B / Brisbane / 60 / 2008 HA in Sf9 cells. The right panel shows quantification of the HA bands, indicating that the HA was 96.7% pure by densitometry. Figure 19I measures HA purity using RP-HPLC. Figure 19J summarizes the data for HA nanoparticles using three influenza A subtypes and two influenza B subtypes. [Figure 19F]Figures 19A-19J illustrate purity analyses of HA nanoparticles produced using different subtypes and different insect cell lines. Figure 19A shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing A / New Hampshire / 1 / 2015 HA. Figure 19B shows quantification of the HA bands, indicating that the HA is 99.1% pure by densitometry. Figure 19C shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing A / Switzerland / 9715293 / 2013 HA. Figure 19D shows quantification of the HA bands, indicating that the HA is 94.5% pure by densitometry. Figure 19E shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing A / Hong Kong / 4801 / 2014 HA. Figure 19F shows quantification of the HA bands, indicating that the HA is 93.3% pure by densitometry. Figure 19G shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing B / Phuket / 3073 / 2013 HA in Sf9 and Sf22a cells. The right panel shows quantification of the HA bands, indicating that the HA was 95.4% pure by densitometry. Figure 19H shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing B / Brisbane / 60 / 2008 HA in Sf9 cells. The right panel shows quantification of the HA bands, indicating that the HA was 96.7% pure by densitometry. Figure 19I measures HA purity using RP-HPLC. Figure 19J summarizes the data for HA nanoparticles using three influenza A subtypes and two influenza B subtypes. [Figure 19G]Figures 19A-19J illustrate purity analyses of HA nanoparticles produced using different subtypes and different insect cell lines. Figure 19A shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing A / New Hampshire / 1 / 2015 HA. Figure 19B shows quantification of the HA bands, indicating that the HA is 99.1% pure by densitometry. Figure 19C shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing A / Switzerland / 9715293 / 2013 HA. Figure 19D shows quantification of the HA bands, indicating that the HA is 94.5% pure by densitometry. Figure 19E shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing A / Hong Kong / 4801 / 2014 HA. Figure 19F shows quantification of the HA bands, indicating that the HA is 93.3% pure by densitometry. Figure 19G shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing B / Phuket / 3073 / 2013 HA in Sf9 and Sf22a cells. The right panel shows quantification of the HA bands, indicating that the HA was 95.4% pure by densitometry. Figure 19H shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing B / Brisbane / 60 / 2008 HA in Sf9 cells. The right panel shows quantification of the HA bands, indicating that the HA was 96.7% pure by densitometry. Figure 19I measures HA purity using RP-HPLC. Figure 19J summarizes the data for HA nanoparticles using three influenza A subtypes and two influenza B subtypes. [Figure 19H]Figures 19A-19J illustrate purity analyses of HA nanoparticles produced using different subtypes and different insect cell lines. Figure 19A shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing A / New Hampshire / 1 / 2015 HA. Figure 19B shows quantification of the HA bands, indicating that the HA is 99.1% pure by densitometry. Figure 19C shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing A / Switzerland / 9715293 / 2013 HA. Figure 19D shows quantification of the HA bands, indicating that the HA is 94.5% pure by densitometry. Figure 19E shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing A / Hong Kong / 4801 / 2014 HA. Figure 19F shows quantification of the HA bands, indicating that the HA is 93.3% pure by densitometry. Figure 19G shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing B / Phuket / 3073 / 2013 HA in Sf9 and Sf22a cells. The right panel shows quantification of the HA bands, indicating that the HA was 95.4% pure by densitometry. Figure 19H shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing B / Brisbane / 60 / 2008 HA in Sf9 cells. The right panel shows quantification of the HA bands, indicating that the HA was 96.7% pure by densitometry. Figure 19I measures HA purity using RP-HPLC. Figure 19J summarizes the data for HA nanoparticles using three influenza A subtypes and two influenza B subtypes. [Figure 19I]Figures 19A-19J illustrate purity analyses of HA nanoparticles produced using different subtypes and different insect cell lines. Figure 19A shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing A / New Hampshire / 1 / 2015 HA. Figure 19B shows quantification of the HA bands, indicating that the HA is 99.1% pure by densitometry. Figure 19C shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing A / Switzerland / 9715293 / 2013 HA. Figure 19D shows quantification of the HA bands, indicating that the HA is 94.5% pure by densitometry. Figure 19E shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing A / Hong Kong / 4801 / 2014 HA. Figure 19F shows quantification of the HA bands, indicating that the HA is 93.3% pure by densitometry. Figure 19G shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing B / Phuket / 3073 / 2013 HA in Sf9 and Sf22a cells. The right panel shows quantification of the HA bands, indicating that the HA was 95.4% pure by densitometry. Figure 19H shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing B / Brisbane / 60 / 2008 HA in Sf9 cells. The right panel shows quantification of the HA bands, indicating that the HA was 96.7% pure by densitometry. Figure 19I measures HA purity using RP-HPLC. Figure 19J summarizes the data for HA nanoparticles using three influenza A subtypes and two influenza B subtypes. [Figure 19J]Figures 19A-19J illustrate purity analyses of HA nanoparticles produced using different subtypes and different insect cell lines. Figure 19A shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing A / New Hampshire / 1 / 2015 HA. Figure 19B shows quantification of the HA bands, indicating that the HA is 99.1% pure by densitometry. Figure 19C shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing A / Switzerland / 9715293 / 2013 HA. Figure 19D shows quantification of the HA bands, indicating that the HA is 94.5% pure by densitometry. Figure 19E shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing A / Hong Kong / 4801 / 2014 HA. Figure 19F shows quantification of the HA bands, indicating that the HA is 93.3% pure by densitometry. Figure 19G shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing B / Phuket / 3073 / 2013 HA in Sf9 and Sf22a cells. The right panel shows quantification of the HA bands, indicating that the HA was 95.4% pure by densitometry. Figure 19H shows gel and Western blot analysis of HA and gp64 for HA nanoparticles containing B / Brisbane / 60 / 2008 HA in Sf9 cells. The right panel shows quantification of the HA bands, indicating that the HA was 96.7% pure by densitometry. Figure 19I measures HA purity using RP-HPLC. Figure 19J summarizes the data for HA nanoparticles using three influenza A subtypes and two influenza B subtypes.

[0041] [Figure 20] FIG. 20 shows HA nanoparticles in an electron micrograph.

[0042] [Figure 21A]Figures 21A and 21B show a comparison of the docking of the HA trimer to an HA nanoparticle (Figure 21A) and to an influenza VLP containing both HA and NA proteins (Figure 21B) by cryoEM structure. [Figure 21B] Figures 21A and 21B show a comparison of the docking of the HA trimer to an HA nanoparticle (Figure 21A) and to an influenza VLP containing both HA and NA proteins (Figure 21B) by cryoEM structure.

[0043] [Figure 22] FIG. 22 illustrates a representative study of a combined nanoparticle composition containing RSV F nanoparticles and HA nanoparticles.

[0044] [Figure 23A] Figures 23A-23F illustrate the results obtained from the study in Figure 22. Figure 23A shows HAI titers against the homologous strain. Figure 23B shows heterologous HAI titers against the heterologous strain. Figure 23C shows palivizumab competitive antibodies. Figure 23D shows neutralizing antibodies against the RSV A strain. Figure 23E shows T cell responses to the RSV F protein. The responses obtained with matrix-adjuvanted nanoparticles are significant. Figure 23F shows T cell responses to influenza proteins. [Figure 23B] Figures 23A-23F illustrate the results obtained from the study in Figure 22. Figure 23A shows HAI titers against the homologous strain. Figure 23B shows heterologous HAI titers against the heterologous strain. Figure 23C shows palivizumab competitive antibodies. Figure 23D shows neutralizing antibodies against the RSV A strain. Figure 23E shows T cell responses to the RSV F protein. The responses obtained with matrix-adjuvanted nanoparticles are significant. Figure 23F shows T cell responses to influenza proteins. [Figure 23C]Figures 23A-23F illustrate the results obtained from the study in Figure 22. Figure 23A shows HAI titers against the homologous strain. Figure 23B shows heterologous HAI titers against the heterologous strain. Figure 23C shows palivizumab competitive antibodies. Figure 23D shows neutralizing antibodies against the RSV A strain. Figure 23E shows T cell responses to the RSV F protein. The responses obtained with matrix-adjuvanted nanoparticles are significant. Figure 23F shows T cell responses to influenza proteins. [Figure 23D] Figures 23A-23F illustrate the results obtained from the study in Figure 22. Figure 23A shows HAI titers against the homologous strain. Figure 23B shows heterologous HAI titers against the heterologous strain. Figure 23C shows palivizumab competitive antibodies. Figure 23D shows neutralizing antibodies against the RSV A strain. Figure 23E shows T cell responses to the RSV F protein. The responses obtained with matrix-adjuvanted nanoparticles are significant. Figure 23F shows T cell responses to influenza proteins. [Figure 23E] Figures 23A-23F illustrate the results obtained from the study in Figure 22. Figure 23A shows HAI titers against the homologous strain. Figure 23B shows heterologous HAI titers against the heterologous strain. Figure 23C shows palivizumab competitive antibodies. Figure 23D shows neutralizing antibodies against the RSV A strain. Figure 23E shows T cell responses to the RSV F protein. The responses obtained with matrix-adjuvanted nanoparticles are significant. Figure 23F shows T cell responses to influenza proteins. [Figure 23F] Figures 23A-23F illustrate the results obtained from the study in Figure 22. Figure 23A shows HAI titers against the homologous strain. Figure 23B shows heterologous HAI titers against the heterologous strain. Figure 23C shows palivizumab competitive antibodies. Figure 23D shows neutralizing antibodies against the RSV A strain. Figure 23E shows T cell responses to the RSV F protein. The responses obtained with matrix-adjuvanted nanoparticles are significant. Figure 23F shows T cell responses to influenza proteins.

[0045] [Figure 24A]Figures 24A-24C illustrate the steps and results for obtaining HA nanoparticles with enhanced stability. Specifically, the pH range during this purification is neutral, from pH 7.0 to pH 7.4. Figure 24A shows the purification steps from the use of thawed cells expressing HA protein to a bulk drug substance (BDS) product. Figure 24B shows a chromatogram trace of a representative nanoparticle using strain A / New Hampshire / 1 / 2015. The flow-through from the column is collected, leaving behind the unwanted product. Figure 24C shows a chromatogram trace of the detergent exchange step on a lentil lectin column. The flow-through from this column is discarded, as is the wash solution. Elution is performed with 0.01% PS80. The buffers are as follows: A1: 25 mM sodium phosphate, pH 7.2, 150 mM NaCl, 0.01% PS80, A2: 25 mM sodium phosphate, pH 7.2, 500 mM NaCl, 0.5% NP9, A3: 25 mM sodium phosphate, pH 7.2, 150 mM NaCl, 0.1% PS80, B1: 25 mM sodium phosphate, pH 7.2, 150 mM. The HA nanoparticles are then concentrated and stored in 0.05% PS80 buffer as shown in Figure 24A. [Figure 24B]Figures 24A-24C illustrate the steps and results for obtaining HA nanoparticles with enhanced stability. Specifically, the pH range during this purification is neutral, from pH 7.0 to pH 7.4. Figure 24A shows the purification steps from the use of thawed cells expressing HA protein to a bulk drug substance (BDS) product. Figure 24B shows a chromatogram trace of a representative nanoparticle using strain A / New Hampshire / 1 / 2015. The flow-through from the column is collected, leaving behind the unwanted product. Figure 24C shows a chromatogram trace of the detergent exchange step on a lentil lectin column. The flow-through from this column is discarded, as is the wash solution. Elution is performed with 0.01% PS80. The buffers are as follows: A1: 25 mM sodium phosphate, pH 7.2, 150 mM NaCl, 0.01% PS80, A2: 25 mM sodium phosphate, pH 7.2, 500 mM NaCl, 0.5% NP9, A3: 25 mM sodium phosphate, pH 7.2, 150 mM NaCl, 0.1% PS80, B1: 25 mM sodium phosphate, pH 7.2, 150 mM. The HA nanoparticles are then concentrated and stored in 0.05% PS80 buffer as shown in Figure 24A. [Figure 24C]Figures 24A-24C illustrate the steps and results for obtaining HA nanoparticles with enhanced stability. Specifically, the pH range during this purification is neutral, from pH 7.0 to pH 7.4. Figure 24A shows the purification steps from the use of thawed cells expressing HA protein to a bulk drug substance (BDS) product. Figure 24B shows a chromatogram trace of a representative nanoparticle using strain A / New Hampshire / 1 / 2015. The flow-through from the column is collected, leaving behind the unwanted product. Figure 24C shows a chromatogram trace of the detergent exchange step on a lentil lectin column. The flow-through from this column is discarded, as is the wash solution. Elution is performed with 0.01% PS80. The buffers are as follows: A1: 25 mM sodium phosphate, pH 7.2, 150 mM NaCl, 0.01% PS80, A2: 25 mM sodium phosphate, pH 7.2, 500 mM NaCl, 0.5% NP9, A3: 25 mM sodium phosphate, pH 7.2, 150 mM NaCl, 0.1% PS80, B1: 25 mM sodium phosphate, pH 7.2, 150 mM. The HA nanoparticles are then concentrated and stored in 0.05% PS80 buffer as shown in Figure 24A.

[0046] [Figure 25A] Figures 25A-25D show the results of purification of trypsin-resistant nanoparticles from several strains. Figure 25A shows a representative strain of the H1N1 subtype, A / New Hampshire / 1 / 2015. Figure 25B shows a representative strain of influenza B, B / Brisbane / 60 / 08 HA. Figure 25C shows a representative strain of the H1N1 subtype, A / New Hampshire / 1 / 2015. In each case, the data demonstrate high levels of production and excellent purity. Figure 25D provides a differential scanning calorimetry (DSC) comparison of trypsin-resistant nanoparticles to nanoparticles produced using a step involving exposure to low pH, approximately pH 6.0. The DSC data demonstrate greater thermal stability at the neutral pH step, establishing that the HA protein in the nanoparticles was properly folded. [Figure 25B]Figures 25A-25D show the results of purification of trypsin-resistant nanoparticles from several strains. Figure 25A shows a representative strain of the H1N1 subtype, A / New Hampshire / 1 / 2015. Figure 25B shows a representative strain of influenza B, B / Brisbane / 60 / 08 HA. Figure 25C shows a representative strain of the H1N1 subtype, A / New Hampshire / 1 / 2015. In each case, the data demonstrate high levels of production and excellent purity. Figure 25D provides a differential scanning calorimetry (DSC) comparison of trypsin-resistant nanoparticles to nanoparticles produced using a step involving exposure to low pH, approximately pH 6.0. The DSC data demonstrate greater thermal stability at the neutral pH step, establishing that the HA protein in the nanoparticles was properly folded. [Figure 25C] Figures 25A-25D show the results of purification of trypsin-resistant nanoparticles from several strains. Figure 25A shows a representative strain of the H1N1 subtype, A / New Hampshire / 1 / 2015. Figure 25B shows a representative strain of influenza B, B / Brisbane / 60 / 08 HA. Figure 25C shows a representative strain of the H1N1 subtype, A / New Hampshire / 1 / 2015. In each case, the data demonstrate high levels of production and excellent purity. Figure 25D provides a differential scanning calorimetry (DSC) comparison of trypsin-resistant nanoparticles to nanoparticles produced using a step involving exposure to low pH, approximately pH 6.0. The DSC data demonstrate greater thermal stability at the neutral pH step, establishing that the HA protein in the nanoparticles was properly folded. [Figure 25D]Figures 25A-25D show the results of purification of trypsin-resistant nanoparticles from several strains. Figure 25A shows a representative strain of the H1N1 subtype, A / New Hampshire / 1 / 2015. Figure 25B shows a representative strain of influenza B, B / Brisbane / 60 / 08 HA. Figure 25C shows a representative strain of the H1N1 subtype, A / New Hampshire / 1 / 2015. In each case, the data demonstrate high levels of production and excellent purity. Figure 25D provides a differential scanning calorimetry (DSC) comparison of trypsin-resistant nanoparticles to nanoparticles produced using a step involving exposure to low pH, approximately pH 6.0. The DSC data demonstrate greater thermal stability at the neutral pH step, establishing that the HA protein in the nanoparticles was properly folded.

[0047] [Figure 26A] Figures 26A-26C show the results of enhanced trypsin resistance of trypsin-resistant nanoparticles from several strains expressed in Sf9 cells. Purified HA nanoparticles produced in Sf9 insect cells are HA0. When exposed to trypsin, HA0 is cleaved at Arg AA344 in H1 to HA1 and HA2. When incubated with increasing concentrations of trypsin, correctly folded HA trimers resist further cleavage. Figure 26A shows that B / Brisbane / 60 / 08 purified at neutral pH is resistant to trypsin and therefore correctly folded (left panel), while B / Brisbane / 60 / 08 HA1 purified at acidic pH is trypsin-sensitive and therefore misfolded (right panel). Figure 26B shows that HA nanoparticles purified at acidic pH but not neutral pH from A / Hong Kong / 4801 / 2014 are misfolded. Figure 26C shows the trypsin resistance of neutral pH A / New Hampshire / 1 / 2015(H1N) HA nanoparticles. Nanoparticles purified at the corresponding acidic pH were trypsin sensitive (not shown). [Figure 26B]Figures 26A-26C show the results of enhanced trypsin resistance of trypsin-resistant nanoparticles from several strains expressed in Sf9 cells. Purified HA nanoparticles produced in Sf9 insect cells are HA0. When exposed to trypsin, HA0 is cleaved at Arg AA344 in H1 to HA1 and HA2. When incubated with increasing concentrations of trypsin, correctly folded HA trimers resist further cleavage. Figure 26A shows that B / Brisbane / 60 / 08 purified at neutral pH is resistant to trypsin and therefore correctly folded (left panel), while B / Brisbane / 60 / 08 HA1 purified at acidic pH is trypsin-sensitive and therefore misfolded (right panel). Figure 26B shows that HA nanoparticles purified at acidic pH but not neutral pH from A / Hong Kong / 4801 / 2014 are misfolded. Figure 26C shows the trypsin resistance of neutral pH A / New Hampshire / 1 / 2015(H1N) HA nanoparticles. Nanoparticles purified at the corresponding acidic pH were trypsin sensitive (not shown). [Figure 26C]Figures 26A-26C show the results of enhanced trypsin resistance of trypsin-resistant nanoparticles from several strains expressed in Sf9 cells. Purified HA nanoparticles produced in Sf9 insect cells are HA0. When exposed to trypsin, HA0 is cleaved at Arg AA344 in H1 to HA1 and HA2. When incubated with increasing concentrations of trypsin, correctly folded HA trimers resist further cleavage. Figure 26A shows that B / Brisbane / 60 / 08 purified at neutral pH is resistant to trypsin and therefore correctly folded (left panel), while B / Brisbane / 60 / 08 HA1 purified at acidic pH is trypsin-sensitive and therefore misfolded (right panel). Figure 26B shows that HA nanoparticles purified at acidic pH but not neutral pH from A / Hong Kong / 4801 / 2014 are misfolded. Figure 26C shows the trypsin resistance of neutral pH A / New Hampshire / 1 / 2015(H1N) HA nanoparticles. Nanoparticles purified at the corresponding acidic pH were trypsin sensitive (not shown).

[0048] [Figure 27] Figure 27 shows the trypsin sensitivity of a commercial egg-purified influenza vaccine (left panel) and a commercial recombinant influenza vaccine (right panel). HA0 is cleaved into HA1 and HA2 in the left panel. However, properly folded HA1 is resistant to further trypsin. In contrast, the commercial recombinant vaccine showed that HA1 was degraded by trypsin, indicating that misfolded protein was present in the vaccine.

[0049] [Figure 28]Figures 28A-28C show antibody induction and protection from infection. Mice were immunized SC on days 0, 14, and 28 with 5 μg of EBOV / Mak GP, 5 μg of EBOV GP adjuvanted with 50 μg of AlPO4, or 5 μg of EBOV / Mak GP adjuvanted with 5 μg of matrix M. Sera were obtained on day 28 and evaluated by ELISA for anti-EBOV / Mak GP IgG (Figure 28A) or anti-Ebola virus neutralizing antibodies (Figure 28B). Black bars represent group GMTs, and error bars indicate the 95% confidence intervals of the GMTs. On day 42, mice were infected with 1,000 pfu of mouse-adapted Zaire Ebola virus strain 1976 Mayinga. After challenge, mice were observed daily for 21 days for morbidity and mortality. Figure 28C shows Kaplan-Meier survival curves for infected mice.

[0050] [Figure 29] Figures 29A-29C show that Matrix M enhanced EBOV / Mak GP-specific IgG and IgG subclass responses. On days 0 and 21, mice were immunized IM with 5 μg of EBOV / Mak GP alone or in combination with either 2.5 or 5 μg of Matrix M or 50 μg of AlPO4. As a placebo control, mice received PBS. Serum samples were collected 21, 28, and 60 days after the first injection and tested for EBOV / Mak GP-IgG (Figure 29A), IgG1 (Figure 29B), and IgG2a (Figure 29C). Results are representative of two separate experiments. Black bars represent group GMTs, and error bars indicate 95% confidence intervals for GMTs.

[0051] [Figure 30-1]Figures 30A-30D show that Ebola nanoparticles containing Matrix M induced strong CD4+ and CD8+ T cell responses and polyfunctional T cells. Splenocytes were stimulated with an Ebola / Mak GP peptide pool covering the entire GP sequence. Culture medium or PMA (50 ng / ml) plus ionomycin (200 ng / ml) served as negative and positive controls. IFNγ-positive spots were counted on days 28 (Figure 30A) and 60 (Figure 30B) and analyzed using an ELISPOT reader and associated software. The background counts for the medium control were subtracted from the peptide-stimulated wells, and averages were obtained from triplicates. On day 28, cells from all five mice in the same group were pooled and incubated for 6 hours at 37°C with either medium alone, the GP peptide pool, or PMA plus ionomycin in the presence of BD Golgi-stop / Golgi-plug. Cells were then harvested and stained for cell surface markers and intracellular cytokines. Cytokine frequencies were analyzed using Flowjo software and Flowjo Boolean functions by gating on CD3+CD44+CD62-CD4+ live effector memory T cells or CD3+CD44+CD62-CD8+ live effector memory T cells (Figures 30C and 30D). Values ​​for single, dual, or triple cytokines represent the sum of the frequencies of cells expressing any one of the three cytokines (IFNγ, TNFα, and IL-2), any two of the three cytokines, or all three cytokines. Results are representative of two separate experiments. Black bars indicate group means, and error bars indicate standard deviations. [Figure 30-2]Figures 30A-30D show that Ebola nanoparticles containing Matrix M induced strong CD4+ and CD8+ T cell responses and polyfunctional T cells. Splenocytes were stimulated with an Ebola / Mak GP peptide pool covering the entire GP sequence. Culture medium or PMA (50 ng / ml) plus ionomycin (200 ng / ml) served as negative and positive controls. IFNγ-positive spots were counted on days 28 (Figure 30A) and 60 (Figure 30B) and analyzed using an ELISPOT reader and associated software. The background counts for the medium control were subtracted from the peptide-stimulated wells, and averages were obtained from triplicates. On day 28, cells from all five mice in the same group were pooled and incubated for 6 hours at 37°C with either medium alone, the GP peptide pool, or PMA plus ionomycin in the presence of BD Golgi-stop / Golgi-plug. Cells were then harvested and stained for cell surface markers and intracellular cytokines. Cytokine frequencies were analyzed using Flowjo software and Flowjo Boolean functions by gating on CD3+CD44+CD62-CD4+ live effector memory T cells or CD3+CD44+CD62-CD8+ live effector memory T cells (Figures 30C and 30D). Values ​​for single, dual, or triple cytokines represent the sum of the frequencies of cells expressing any one of the three cytokines (IFNγ, TNFα, and IL-2), any two of the three cytokines, or all three cytokines. Results are representative of two separate experiments. Black bars indicate group means, and error bars indicate standard deviations.

[0052] [Figure 31-1]Figures 31A-31E show that matrix M enhanced germinal center (GC) cell responses. Fresh splenocytes were stained for GC B cells, and data were acquired as described in Materials and Methods. Data were analyzed using Flowjo software. Dead cells were excluded from the analysis using Invitrogen LIVE / DEAD™ fixable yellow dye. (Figure 31A) GC cells were defined as CD95+GL-7+ cells in the B220+ B cell gate. Numbers in the dot plots for a representative mouse indicate the mean and standard deviation of GC frequencies for all five mice in the same group on day 28. GC cell frequencies for individual mice are shown for days 28 (Figure 31B) and 60 (Figure 31C). The absolute number of GC cells per spleen on days 28 (Figure 31D) and 60 (Figure 31E) was calculated by multiplying the total number of splenocytes in the spleen by the frequency of GC cells. Black bars indicate group means, and error bars represent standard deviations. [Figure 31-2] Figures 31A-31E show that matrix M enhanced germinal center (GC) cell responses. Fresh splenocytes were stained for GC B cells, and data were acquired as described in Materials and Methods. Data were analyzed using Flowjo software. Dead cells were excluded from the analysis using Invitrogen LIVE / DEAD™ fixable yellow dye. (Figure 31A) GC cells were defined as CD95+GL-7+ cells in the B220+ B cell gate. Numbers in the dot plots for a representative mouse indicate the mean and standard deviation of GC frequencies for all five mice in the same group on day 28. GC cell frequencies for individual mice are shown for days 28 (Figure 31B) and 60 (Figure 31C). The absolute number of GC cells per spleen on days 28 (Figure 31D) and 60 (Figure 31E) was calculated by multiplying the total number of splenocytes in the spleen by the frequency of GC cells. Black bars indicate group means, and error bars represent standard deviations.

[0053] [Figure 32-1]Figures 32A-32E show that matrix M enhanced the frequency and absolute number of THF cells in the spleen. THF cells, defined as CXCR5+PD-1+ T cells within the B220-CD49b-CD3+CD4+ T cell gate, were identified in the spleen on days 28 and 60. Representative dot plots of THF cell analysis for each group are shown (Figure 32A). Numbers in the dot plots represent the mean frequency and standard deviation on day 28. The frequency of THF cells within the CD4+ T cell population on days 28 (Figure 32B) and 60 (Figure 32D) is shown. The absolute number of THF cells per spleen on days 28 (Figure 32C) and 60 (Figure 32E) was calculated by multiplying the total number of splenocytes in the spleen by the frequency of THF cells. Black bars indicate group means, and error bars represent standard deviations. [Figure 32-2] Figures 32A-32E show that matrix M enhanced the frequency and absolute number of THF cells in the spleen. THF cells, defined as CXCR5+PD-1+ T cells within the B220-CD49b-CD3+CD4+ T cell gate, were identified in the spleen on days 28 and 60. Representative dot plots of THF cell analysis for each group are shown (Figure 32A). Numbers in the dot plots represent the mean frequency and standard deviation on day 28. The frequency of THF cells within the CD4+ T cell population on days 28 (Figure 32B) and 60 (Figure 32D) is shown. The absolute number of THF cells per spleen on days 28 (Figure 32C) and 60 (Figure 32E) was calculated by multiplying the total number of splenocytes in the spleen by the frequency of THF cells. Black bars indicate group means, and error bars represent standard deviations.

[0054] [Figure 33]Figures 33A-33B show that matrix M induced long-lived plasma cells in bone marrow. Spleen and bone marrow cells were incubated overnight in EBOV / Mak GP-coated ELISPOT plates. EBOV / Mak GP-specific IgG spots were detected by incubating with goat anti-mouse IgG-HRP followed by spot development. The number of spots was counted and analyzed using an ELISPOT reader. The number of antibody-secreting cells (ASCs) per million cells is shown. (Figure 33A) EBOV / Mak GP-IgG ASC count in the spleen at day 60; (Figure 33B) EBOV / Mak GP-IgG ASC count in the bone marrow at day 60. Black bars indicate group means, and error bars represent standard deviations.

[0055] [Figure 34A] Figures 34A-34B show the characteristics of the Ebola glycoprotein. Figure 34A shows the domain structure. Figure 34B shows the amino acid sequence of GP, including the cleaved signal peptide and the N- and C-termini of the mature protein, and the furin cleavage sequence (SEQ ID NO: 22). [Figure 34B] Figures 34A-34B show the characteristics of the Ebola glycoprotein. Figure 34A shows the domain structure. Figure 34B shows the amino acid sequence of GP, including the cleaved signal peptide and the N- and C-termini of the mature protein, and the furin cleavage sequence (SEQ ID NO: 22).

[0056] [Figure 35A] Figures 35A-35C show electron micrographs of nanoparticles of the present disclosure. Note that Figure 35B illustrates a nonionic surfactant core with up to five copies of the trimer attached to the core. In some cases, the additional trimers are outside the plane of the view. Figure 35C shows a docking study with a GP trimer overlaid on the nanoparticle in the micrograph. [Figure 35B]Figures 35A-35C show electron micrographs of nanoparticles of the present disclosure. Note that Figure 35B illustrates a nonionic surfactant core with up to five copies of the trimer attached to the core. In some cases, the additional trimers are outside the plane of the view. Figure 35C shows a docking study with a GP trimer overlaid on the nanoparticle in the micrograph. [Figure 35C] Figures 35A-35C show electron micrographs of nanoparticles of the present disclosure. Note that Figure 35B illustrates a nonionic surfactant core with up to five copies of the trimer attached to the core. In some cases, the additional trimers are outside the plane of the view. Figure 35C shows a docking study with a GP trimer overlaid on the nanoparticle in the micrograph.

[0057] [Figure 36] Figure 36 illustrates the ability of three monoclonal anti-Ebola antibodies to detect Ebola nanoparticles.

[0058] [Figure 37] FIG. 37 shows surface plasmon resonance (SPR) data for antibody binding to epitopes on Ebola GP nanoparticles (SEQ ID NOs: 23-25).

[0059] [Figure 38] FIG. 38 illustrates the high efficiency of binding of the 13C6 antibody to nanoparticles of the present disclosure.

[0060] [Figure 39] Figure 39 illustrates the baboon immunogenicity study design. Group 1 was 60 μg of GP nanoparticles without adjuvant. Group 2 was 60 μg of GP nanoparticles with 800 μg of AlPO4 adjuvant. Group 3 was 60 μg of GP nanoparticles with 50 μg of Matrix M adjuvant. Group 4 was 5 μg of GP nanoparticles with 50 μg of Matrix M adjuvant.

[0061] [Figure 40A]Figures 40A-40B illustrate the results of the baboon immunogenicity study in Figure 39. At day 21, EC90 titers increased in groups 2 and 3 (Figure 40A). Titers were approximately the same in both groups, as well as for nanoparticles containing glycoproteins of the Makona Ebola virus and the Mayinga strain (a prototype variant of the Zaire Ebola strain). By day 31, immune responses were significant in all cases, particularly for compositions containing GP and matrix M adjuvants. Notably, a low dose of GP (5 μg) performed as well as a high dose (60 μg), highlighting the dose-sparing effect of matrix M. [Figure 40B] Figures 40A-40B illustrate the results of the baboon immunogenicity study in Figure 39. At day 21, EC90 titers increased in groups 2 and 3 (Figure 40A). Titers were approximately the same in both groups, as well as for nanoparticles containing glycoproteins of the Makona Ebola virus and the Mayinga strain (a prototype variant of the Zaire Ebola strain). By day 31, immune responses were significant in all cases, particularly for compositions containing GP and matrix M adjuvants. Notably, a low dose of GP (5 μg) performed as well as a high dose (60 μg), highlighting the dose-sparing effect of matrix M.

[0062] [Figure 41] Figure 41 illustrates the durable immune response achieved by the nanoparticle compositions. The data show the EC50 GMT response to IgG after administration on days 0 and 21. Nanoparticles containing GP and matrix M show a superior response to alum adjuvant, and the response remains high over time.

[0063] [Figure 42] Figure 42 illustrates the stimulation of immune responses associated with IFNγ-releasing cells. Matrix M combined with 5 μg of GP nanoparticles gave the greatest response, followed by high-dose GP nanoparticles (60 μg). The use of alum resulted in a small but detectable increase in IFNγ-secreting peripheral blood mononuclear cells (PBMCs).

[0064] [Figure 43] FIG. 43 illustrates the IFNγ and TNFα release profiles of CD4+ and CD8+ T cells isolated from baboons administered a vaccine composition containing GP nanoparticles as disclosed herein.

[0065] [Figure 44] Figure 44 illustrates the cytokine release profile of T cells isolated from baboons administered a vaccine composition containing GP nanoparticles as disclosed herein. The data show that the Matrix M-adjuvanted GP nanoparticle composition stimulates an immune response with a broader cytokine release profile.

[0066] [Figure 45] Figure 45 shows the design of a vaccine trial conducted in Cynomolgus macaques. Animals were administered a vaccine composition of 5 μg GP + 50 μg Matrix M on days 0 and 21, and then challenged on day 42. Animals 33360, 33362, and 33355 were treated with the vaccine composition. Animal 33356 received a placebo.

[0067] [Figure 46] Figure 46 shows the IgG titers obtained in the Cynomolgus macaque test. By day 28, EC50 titers exceeded 10.

[0068] [Figure 47-1] Figure 47 shows the induction of IFNγ-secreting PBMC cells isolated from treated macaques. Peptides derived from Ebola Zaire GP were pooled and used in the assay. Consensus peptides derived from the Zaire and Sudan strains were also tested. The data shown illustrate cell responses to the peptides at weeks 0 (top panel), 3 (middle panel), and 5 (bottom panel). Control animals injected with placebo showed essentially no response. In contrast, vaccine-treated animals showed a strong increase in cell-released IFNγ in response to the various peptides tested. [Figure 47-2] Figure 47 shows the induction of IFNγ-secreting PBMC cells isolated from treated macaques. Peptides derived from Ebola Zaire GP were pooled and used in the assay. Consensus peptides derived from the Zaire and Sudan strains were also tested. The data shown illustrate cell responses to the peptides at weeks 0 (top panel), 3 (middle panel), and 5 (bottom panel). Control animals injected with placebo showed essentially no response. In contrast, vaccine-treated animals showed a strong increase in cell-released IFNγ in response to the various peptides tested. [Figure 47-3] Figure 47 shows the induction of IFNγ-secreting PBMC cells isolated from treated macaques. Peptides derived from Ebola Zaire GP were pooled and used in the assay. Consensus peptides derived from the Zaire and Sudan strains were also tested. The data shown illustrate cell responses to the peptides at weeks 0 (top panel), 3 (middle panel), and 5 (bottom panel). Control animals injected with placebo showed essentially no response. In contrast, vaccine-treated animals showed a strong increase in cell-released IFNγ in response to the various peptides tested.

[0069] [Figure 48] Figure 48 shows viral load and survival in macaques. By day 7 post-challenge, placebo animals exhibited a substantial increase in viral nucleic acid, indicating Ebola infection. By day 9, the animals were euthanized. All vaccinated animals survived. Only animal 33360 exhibited a detectable increase in viral nucleic acid, which was near the limit of detection. By day 10, viral RNA levels in that single animal also fell below the detectability of RT-PCR.

[0070] [Figure 49]Figure 49 shows the vaccine trial design for additional macaque studies. Animals were administered saline or 5 μg GP + 50 μg Matrix M. Group F received the vaccine at weeks 0 and 6. Group G received the vaccine at weeks 0 and 3. Both groups were challenged 6 weeks after administration of the boost vaccine.

[0071] [Figure 50] Figure 50 shows the results of the second study. Substantial increases in anti-Ebola GP were obtained in both groups. 18 days after challenge with live virus, the survival rate of saline control animals was 0%. In contrast, both animals in groups F and G, respectively, survived, establishing the vaccine composition as protective. DETAILED DESCRIPTION OF THE INVENTION

[0072] Disclosed herein are nanoparticles for inducing an immune response, methods for producing and administering them, and vaccine compositions containing them. The nanoparticles provide an antigen surrounding and associated with a surfactant core, resulting in a structure that provides enhanced stability by multiple means. The surfactant core and antigen associate through physicochemical interactions mediated by the properties of the antigen and the surfactant. Additionally, without being bound by theory, the nanoparticles provide particularly good antigen presentation to the immune system, which is believed to result from the orientation of the antigen around the surfactant core.

[0073] In one aspect, the present disclosure provides a composition containing a recombinant viral glycoprotein nanoparticle. 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 immunogenic compositions comprising one or more viral glycoprotein molecular species in a nanoparticle structure, wherein the glycoproteins are in the form of trimers and each nanoparticle contains at least one trimer associated with a non-ionic surfactant core. In certain embodiments, the nanoparticles consist of antigens, such as viral glycoproteins, from only one pathogen.

[0075] The nanoparticles can be used for the prevention and / or treatment of viral infections. Accordingly, in another aspect, 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 nanoparticles.

[0076] The present disclosure provides vaccine compositions comprising nanoparticles. The compositions can contain nanoparticles bearing antigens from multiple pathogens. In some aspects, the vaccine compositions can contain nanoparticles comprising antigens from more than one virus strain from the same virus species. In some aspects, the vaccine compositions can contain nanoparticles comprising antigens from different virus species. In another embodiment, the present disclosure provides pharmaceutical packs or kits comprising one or more containers filled with one or more of the components of the vaccine composition.

[0077] In another embodiment, the present disclosure provides a method for treating a pulmonary arthritis comprising the step of: Methods for formulating vaccine compositions that induce immunity in a mammal against an infection or at least one disease symptom thereof are provided. The disclosed nanoparticles are useful for preparing compositions that stimulate an immune response that confers immunity or substantial immunity against an infectious agent. Thus, in one embodiment, the present disclosure provides a method for inducing immunity in a subject against an infection or at least one disease symptom thereof, comprising administering at least one effective dose of nanoparticles.

[0078] In some embodiments, the nanoparticles are administered with an adjuvant. In other aspects, the nanoparticles are administered without an adjuvant. In some aspects, the adjuvant may be bound to the nanoparticles, such as by a non-covalent interaction. In other aspects, the adjuvant is co-administered with the nanoparticles, but does not substantially interact with the nanoparticles.

[0079] Also provided herein are methods for producing nanoparticles and vaccine compositions, which advantageously provide nanoparticles that are substantially free of contaminating other proteins, such as proteins associated with recombinant expression of proteins in the baculovirus / Sf9 system.

[0080] definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the content 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 skill in the art, and so forth.

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

[0082] As used herein, the terms "about" or "approximately," when preceding a numerical value, indicate 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 are capable of eliciting an immune response.

[0084] As used herein, an "immunogenic composition" is a composition that comprises an antigen, wherein 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, e.g., a vaccine, comprises one or more selected antigens, but not all of the antigens are derived from a pathogen. Such compositions are typically prepared from immunogenic polypeptides that are substantially free of intact viruses or lysates of such cells or particles and that have been at least partially purified, often substantially purified, from the 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 a substance (e.g., a compound, polynucleotide, or polypeptide) that forms the majority percentage of the sample in which it is contained. ) refers to the isolation of a component. For example, in a sample, a substantially purified component comprises 85% of the sample, preferably 85% to 90%, more preferably at least 95% to 99.5%, and most preferably at least 99%. When a component has been substantially replaced, the amount remaining in the sample is less than or equal to about 0.5% to about 10%, preferably about 0.5% to about 1.0%.

[0087] As used herein, the terms "treat," "treatment," and "treating" refer to an approach for obtaining a beneficial or desired result, e.g., a clinical result. For purposes of this disclosure, a beneficial or desired result may include inhibiting or suppressing the initiation or progression of an infection or disease; ameliorating or reducing the occurrence of symptoms of an infection or disease; or a combination thereof.

[0088] As used herein, "prevention" is used interchangeably with "prophylaxis" and can mean preventing an infection or disease altogether, or preventing the onset of symptoms of that infection or disease; delaying the onset of an infection or disease or its symptoms; or reducing the severity of a subsequently occurring infection or disease or its symptoms.

[0089] As used herein, an "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. An effective dose or amount can be determined, for example, by measuring the amount of neutralizing secreted and / or serum antibodies, for example, by plaque neutralization, complement fixation, enzyme-linked immunosorbent (ELISA), or microneutralization assays.

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

[0091] The term "subject" as used herein includes humans and other animals. Typically, the subject is a human. For example, the subject may be an adult, a teenager, a child (2 to 14 years old), an infant (1 to 24 months old), or a newborn (up to 1 month old). In some embodiments, the adult is about 65 years old or older, or about 60 years old or older. In some embodiments, the subject is a pregnant woman or a woman who is planning to become pregnant. In other embodiments, the subject is not a 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 cat.

[0092] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the U.S. federal or state government or listed in the U.S. Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias for use in mammals, more particularly humans. These compositions may 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] overview Pathogen-derived antigens are combined with non-ionic surfactants to provide nanoparticles surrounding a surfactant core with improved stability and superior immunogenicity. The present disclosure also provides methods and compositions for vaccinating a subject against a pathogen. In certain aspects, the pathogen is a virus. The antigen is typically a protein, often a glycoprotein. Also disclosed are compositions containing nanoparticles that find use as vaccine compositions. Methods of producing the nanoparticles and of producing the vaccine compositions are also disclosed.

[0095] Nanoparticle structure and morphology The nanoparticles of the present disclosure comprise an antigen associated with a non-ionic surfactant core. The upper panel of Figure 6 illustrates an example of multiple RSV F antigens associated with a surfactant core. Figure 35 shows Ebola nanoparticles. Advantageously, the nanoparticles have improved resistance to environmental stress, providing enhanced stability.

[0096] In certain embodiments, the nanoparticles are composed of multiple protein trimers surrounding a nonionic 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 2 to 9 trimers. In certain embodiments, each nanoparticle contains 2 to 6 trimers. The compositions disclosed herein may contain nanoparticles with different numbers of trimers. For example, the composition may contain nanoparticles with a trimer count ranging from 2 to 9; in other embodiments, the nanoparticles in the composition may contain 2 to 6 trimers. In certain embodiments, the composition contains a heterogeneous population of nanoparticles with 2 to 6 trimers per nanoparticle, or 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 about 95% nanoparticles with 5 trimers.

[0097] The antigen is associated 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 nanoparticle formation by forming a core that organizes and presents the antigen. Thus, in certain embodiments, the nanoparticle may contain an antigen assembled into a multi-oligomeric glycoprotein-PS80 protein-surfactant nanoparticle that includes an outwardly protruding head region and a hydrophobic region, with the PS80 surfactant forming a central core surrounded by the antigen.

[0098] The nanoparticles disclosed herein range in Z-average size 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. 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 types are in the form of anisotropic rods, which may be dimeric or monomeric. In other embodiments, the nanoparticle types are spherical oligomers. In still other embodiments, the nanoparticles may be described as intermediate nanoparticles with sedimentation properties intermediate between the first two types. The formation of nanoparticle types can be controlled by controlling the concentrations of surfactants and proteins during the production process. Nanoparticle types can be determined by measuring the sedimentation coefficient. See, for example, Figures 9A and 9B, which show RSV F nanoparticles. See also Figure 8, which illustrates controlling nanoparticle size by adjusting the concentrations of surfactants and proteins.

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

[0101] Antigens contained in nanoparticles are typically produced by recombinant expression in host cells. Standard recombinant techniques may be used. Typically, proteins are expressed in insect host cells using the baculovirus system. In a preferred embodiment, the baculovirus is a cathepsin-L knockout baculovirus. In another preferred embodiment, the baculovirus is a chitinase knockout baculovirus. In yet another preferred embodiment, the baculovirus is a double knockout for both cathepsin-L and chitinase. High-level expression can be obtained in insect cell expression systems. Non-limiting examples of insect cells include 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 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 acids into eukaryotic cells can be achieved by calcium phosphate co-precipitation, electroporation, microinjection, lipofection, and transfection using 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 cells propagate 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 to cultivate cells in which 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, NJ). In other embodiments, the pre-sterilized plastic bag is a bag of approximately 50 L to 3500 L.

[0104] Surfactant extraction and purification of nanoparticles After host cell growth, proteins may be harvested from the host cells using detergents and purification protocols. After host cell growth for 48 to 96 hours, the cells are isolated from the culture medium, and a detergent-containing solution is added to solubilize the cell membrane and release the protein into a detergent extract. Triton X-100 and tergitol, also known as NP-9, are preferred detergents for extraction. The detergent may be added at a final concentration of 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 about 0.1% to about 0.3%. Preferably, the concentration is about 0.5%.

[0105] In other embodiments, a different first detergent may be used to isolate proteins from host cells, for example, the first detergent may be bis(polyethylene glycol bis[imidazoylcarbonyl]), nonoxynol-9, bis(polyethylene glycol bis[imidazoylcarbonyl]), Brij® 35, Brij® 56, Br Brij® 72, Brij® 76, Brij® 92V, Brij® 97, Brij® 58P, Cremophor® 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 Glycoside Hexaethylene 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 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,The hydroxybenzoate may be polyoxyethylene 8 stearate, polyoxyethylene bis(imidazolylcarbonyl), polyoxyethylene 25 propylene glycol stearate, Quillaja bark-derived saponin, 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 a combination thereof.

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

[0107] For example, the first column can be an ion exchange chromatography resin such as Fractogel® EMD TMAE (EMD Millipore), the second column can be a Lens culinaris lectin affinity resin, and the third column can be a cation exchange column such as Fractogel® EMD SO3 (EMD Millipore) resin. In other embodiments, the cation exchange column can be an MMC column or a Nuvia C Prime column (Bio-Rad Laboratories, Inc.). Preferably, the methods disclosed herein do not use detergent extraction columns; e.g., hydrophobic interaction columns. Such columns can be used in combination with other column types. Columns are often used during purification to remove detergents, which can adversely affect the methods disclosed herein.

[0108] Surfactant Exchange To form nanoparticles, the first surfactant used to extract proteins from host cells is essentially replaced with a second surfactant to achieve a nanoparticle structure. NP-9 is a preferred extraction surfactant. Typically, the nanoparticles contain no 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, detergent exchange is performed using affinity chromatography, which binds glycoproteins via their carbohydrate moieties. 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, e.g., Sharon and Lis, "Legume lectins—a large family of homologous proteins," FASEB J. November 1990; Issue 4(14):3198-208; Liener, "The Lectins: Properties, Functions, and Applications in Biology and Medicine," Elsevier, 2012. Suitable lectins include concanavalin A (con A), legume lectin, sainfoin lectin, and lentil lectin. Lentil lectin is a preferred column for detergent exchange due to its binding properties. See, e.g., Example 10. Lectin columns are commercially available; for example, Caputo lentil lectin is available from GE Healthcare. In certain embodiments, the lentil lectin column may use recombinant lectins. At the molecular level, it is believed that carbohydrate moieties bind to the lentil lectin, liberating amino acids of the protein so that they can coalesce around the surfactant, resulting in the formation of a surfactant core, providing nanoparticles with multiple copies of the antigen, e.g., a glycoprotein oligomer that may be a dimer, trimer, or tetramer, tethered to the surfactant.

[0110] When incubated with proteins to form nanoparticles during detergent exchange, detergents can be present at up to about 0.1% (w / v) during the initial purification step, with this amount being reduced to obtain final nanoparticles with optimal stability. For example, nonionic detergents (e.g., PS80) can be present at about 0.03% to about 0.1%. Preferably, for improved stability, the nanoparticles contain about 0.03% to about 0.05% PS80. PS80 in amounts less than about 0.03% in the formulation does not exhibit good stability. Furthermore, if PS80 is present at more than about 0.05%, aggregates will form. Therefore, about 0.03% to about 0.05% PS80 provides structural and stability benefits that allow for long-term stability of the nanoparticles with reduced degradation.

[0111] Detergent exchange may be performed on proteins that have been purified as discussed above, purified, frozen for storage, and then thawed for detergent exchange.

[0112] Enhanced stability and immunogenicity of nanoparticles Without being bound by theory, it is believed that associating the antigen with a non-ionic surfactant core provides superior stability and antigen presentation. The nanoparticles disclosed herein provide surprisingly good stability and immunogenicity. The advantageous stability is due to the fact that they can be easily stored in countries where proper storage is not possible. For example, refrigeration may not be possible in certain parts of Africa, and thus vaccines against endemic diseases such as Ebola virus and RSV, which face difficult storage conditions, may particularly benefit from improved stability. Furthermore, the HA influenza nanoparticles produced using the neutral pH approach exhibit superior folding compared to known recombinant influenza vaccines.

[0113] Notably, previous approaches using surfactants to produce RSV vaccines, including split vaccines, such as those described in US 2004 / 0028698 to Colau et al., have failed to produce effective structures. Unlike the nanoparticles with proteins surrounding the surfactant core disclosed herein, the compositions of Colau et al. contain amorphous material lacking identifiable viral structures, presumably failing to effectively present epitopes to the immune system. In addition, the orientation of antigens, often glycoproteins, around the surfactant core sterically hinders contact with enzymes and other chemicals that cause proteolysis, resulting in particularly enhanced stability of the disclosed nanoparticles.

[0114] The nanoparticles have enhanced stability, as determined by their ability to maintain immunogenicity after exposure to various stresses. Stability can be measured in various ways. In one approach, peptide maps can be generated to determine the integrity of antigen proteins after various treatments designed to stress the nanoparticles by mimicking harsh storage conditions. The stability is then measured by the relative abundance of antigen peptides in stressed samples compared to control samples. Figure 12 shows that a strong immune response is achieved even after various different stresses on the RSV F nanoparticle composition. Figure 13 illustrates the improved protease resistance provided by nanoparticles using PS80 at levels greater than 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, but preferably are stable at 25°C for at least 2 months. In some embodiments, the compositions are 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 advantageously retain intact antigenic site II at an abundance of 90-100% compared to the control RSV-F protein, as measured by peptide mapping, in response to various stresses, including pH (pH 3.7), high pH (pH 10), elevated temperature (50° C. for 2 weeks), and even peroxide oxidation, as shown in Figure 12.

[0115] The position of the glycoprotein tethered to the detergent core is believed to provide enhanced stability by reducing undesired 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 steric hindrance that blocks protease access.

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

[0117] Enhanced immunogenicity is exemplified by the cross-neutralization achieved by influenza nanoparticles, and it is believed that the orientation of influenza antigens protruding from the core provides more effective presentation of epitopes to the immune system.

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

[0119] RSV antigen In one aspect, the virus is a respiratory syncytial virus (RSV), and the viral antigen is a fusion (F) glycoprotein. The structure and function of the RSV F protein have been well characterized. See Figure 1 for 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 its native variant. These mutations confer desirable characteristics, such as improved protein expression and enhanced immunogenicity. 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, Vol. 88, pp. 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 site 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 residues within the site, such that furin no longer recognizes 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, one, two, or all 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 amino acids 137 to 145 of SEQ ID NO: 2 may be deleted, including the specific example of a suitable RSV F protein shown below. Each of SEQ ID NOs: 3-13 may optionally be prepared with an 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: 3-13. Following expression of the RSV-F protein in a host cell, 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, followed by cleavage of the signal peptide. The N-terminal RSV F signal peptide is SEQ ID NO: 26 (MELLILKA). 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 used to produce RSV F nanoparticle vaccines. See Figure 1B. Optionally, one or more of the RSV F signal peptide amino acids can be deleted, mutated, or the entire signal peptide can be deleted and replaced with a different signal peptide to enhance expression. The initial methionine residue is maintained to initiate expression. [Table 1]

[0122] In some embodiments, the RSV F protein disclosed herein is altered from the wild-type strain only by deletions in the fusion domain, optionally with inactivation of the major cleavage site. In other embodiments, additional alterations 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. Motavizumab antibodies also bind to site II. Additional suitable RSV-F proteins incorporated by reference include RSV-F proteins containing the sequence spanning residues 100 to 150 found in US Publication US2011 / 0305727 and disclosed in Figure 1C thereof.

[0123] In certain other embodiments, the RSV F1 or F2 domain may have modifications compared to the wild-type strain set forth in SEQ ID NO: 2. For example, the F1 domain may have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 alterations, which may be mutations or deletions. Similarly, the F2 domain may have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 alterations, which may be mutations or deletions. 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 a specific example, the RSV nanoparticle drug product may contain about 0.025% to about 0.03% PS80 with about 270 μg / mL to about 300 μg / mL, or about 60 μg / mL to about 300 μg / mL of RSV F. In other embodiments, the nanoparticle drug product may contain about 0.035% to about 0.04% PS80 in the composition with 300 μg / mL to about 500 μg / mL of RSV F. In yet other embodiments, the nanoparticle drug product may contain about 0.035% to about 0.04% PS80 in the composition with 350-500 μg / mL of RSV F.

[0125] Because the concentrations of antigen and surfactant may vary, each amount may be referred to as the molar ratio of non-ionic surfactant to protein. For example, the molar ratio of PS80 to protein is calculated using the PS80 concentration and the protein concentration of the antigen measured by ELISA / A280 and their respective molecular weights. The molecular weight of PS80 used for calculation is 1310, and using RSV F as an example, RSV The molecular weight of F is 65 kD. The molar ratio is calculated as follows: (PS80 concentration × 10 × 65000) ÷ (1310 × RSV F concentration (mg / mL)). Thus, as shown in Figure 13, the nanoparticle concentration measured by protein is 270 μg / mL, and the PS80 concentrations are 0.015% and 0.03%, respectively. These are PS80 to RSV F protein molar ratios 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, about 30:1 to about 70:1, about 30:1 to about 60:1, about 40:1 to about 70:1, or about 40:1 to about 50:1. Often, the substituted nonionic surfactant is PS80, and the molar ratio of PS80:protein is about 30:1 to about 50:1. For RSV-F glycoprotein, nanoparticles having a molar ratio ranging from 35:1 to about 65:1, specifically a ratio of about 45:1, are particularly stable.

[0127] Influenza antigen Nanoparticle platforms are particularly useful for presenting influenza antigens to a subject's immune system. Previous approaches to producing influenza nanoparticle vaccines have used hydrophobic interaction columns to remove detergents or have included only minimal amounts of detergent to reduce nonspecific interactions that occur during product purification. However, it has now been discovered that by performing a detergent exchange step, nanoparticles with a nonionic surfactant core can be produced that have superior properties. The nanoparticles exhibit excellent stability, evidenced by resistance to degradation due to environmental stress, allowing for a long shelf life, a particularly useful property for vaccines. In addition, the nanoparticle structure presents antigens in a particularly advantageous manner.

[0128] Influenza nanoparticles are particularly useful as vaccines because the antibodies they induce contain broadly neutralizing antibodies. Thus, antibodies induced by nanoparticles administered in one year can neutralize influenza virus strains arising from the "drift" process in subsequent years. It is believed that the epitopes that induce these broadly neutralizing antibodies were not exposed at all or not effectively exposed in prior influenza vaccines, or that the epitopes were not sufficiently stable in prior formulations. The nanoparticles disclosed herein contain cross-protective nanoparticles tethered around a non-ionic surfactant core with enhanced stability. These problems are solved by displaying epitopes.

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

[0130] In certain embodiments, nanoparticles may contain HA or NA protein.For example, nanoparticles may contain HA protein selected from subtype H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 and H16.Nanoparticles may contain NA protein selected from subtype N1, N2, N3, N4, N5, N6, N7, N8 and N9.Phylogenetically, HA and NA protein 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 NAs 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 a native influenza HA or NA protein.

[0131] The HA and NA proteins used for the nanoparticles are typically full-length sequences, although in certain embodiments, portions of the C-terminus may be removed.

[0132] Advantageously, the influenza-containing composition can induce a response against heterologous strains of influenza, even when additional pathogen nanoparticles disclosed herein are co-administered. By inducing a response against heterologous influenza strains, broad protection is achieved. Thus, in certain embodiments, the matrix M-adjuvanted composition induces a homologous HAI titer of about 800 to about 2000. In a specific embodiment, the heterologous HAI titer is about 1300. In certain embodiments, the matrix M-adjuvanted composition induces a heterologous HAI titer of 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 of greater than 6.8 to 8.5 during the purification and formulation of HA nanoparticles. In certain embodiments, the pH range during the purification and formulation of HA nanoparticles is 7.0 to 8.5, 7.0 to 7.5, or 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 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 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. 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 can 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 HA proteins varies from strain to strain, and thus the neutral pH production disclosed herein provides a step to maximize immunogenicity for all strains, particularly pH-sensitive strains. Without being bound by theory, it is believed that the combination of detergent exchange and neutral pH levels keeps the HA protein in a conformation that makes it resistant to proteases, particularly trypsin. Therefore, neutral pH purification and In combination, by associating HA protein around the nonionic surfactant core, particularly good stability and immunogenicity of HA protein are achieved.In addition, the method for producing nanoparticles provides excellent levels of protein for use in vaccines.In certain embodiments, A280 produces HA nanoparticles that measure about 10 mg to about 30 mg per liter of cell culture, or higher, about 20 mg to about 30 mg per liter.

[0135] Trypsin-resistant HA nanoparticles may be prepared as described in Figure 24. Briefly, various steps, including detergent exchange, are performed in buffers above pH 7.0; often in the range of about pH 7.2 to about pH 7.4. Figure 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 compositions are vaccine compositions. Advantageously, the vaccine compositions disclosed herein provide 100% survival to lethal challenge in animal models. The compositions also maintain viral loads at or below the limit of detection when using RT-PCR to detect viral nucleic acid.

[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 immunogenic compositions comprising one or more Ebola virus GP proteins in a nanoparticle structure, wherein the GP proteins are in the form of trimers, and each nanoparticle contains at least one trimer attached to a non-ionic surfactant core.

[0139] 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, nanoparticles from more than one strain are present 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 components.

[0140] Ebola glycoprotein The Ebola antigen used to prepare the nanoparticles is typically an Ebola glycoprotein (GP) antigen. The antigen may be derived from various strains. The compositions disclosed herein may contain nanoparticles derived from one, two, three, four, five, or six different Ebola strains. For example, the strains may be Makona, Sudan, Zaire, or Reston. In other embodiments, 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% identical, about 85% identical, about 90% identical, about 95% identical, about 97% identical, about 98% identical, or about 99% identical to one or more GPs from Makona, Sudan, Zaire, or Reston viruses, where identity is measured over the entire length of the protein or nucleic acid. In some embodiments, the Ebola GP may comprise or consist of SEQ ID NO: 27 or 28, or a protein having identity thereto.

[0141] A representative Zaire strain sequence is provided in GenBank Accession No. AAB81004 (SEQ ID NO: 27). The first underlined portion indicates the N-terminus for the GP1 protein. The preceding signal peptide is used to regulate expression in cells prior to purification and formulation into a vaccine. It is subsequently cleaved off during subsequent processing. The furin cleavage site is shown in bold. The N-terminus for the GP2 protein is shown following the bold text. Figure 7A shows a schematic diagram of the protein structure. [ka]

[0142] The Makona isolate sequence is provided in GenBank accession number AJG44192 (SEQ ID NO: 28). As before, the first underlined portion indicates the N-terminus for the GP1 protein. The preceding signal peptide is cleaved off during processing. In bold is the furin cleavage site. Following the bold text is the N-terminus for the GP2 protein. See also Figure 7B. [ka]

[0143] The ability of the vaccine composition to stimulate an immune response was confirmed in three animal models. A mouse model was used first. Recombinant EBOV / Mak full-length GP nanoparticle vaccines formulated with matrix M, AlPO4, or saline were evaluated. Immunization of mice with unadjuvanted or AlPO4-adjuvanted EBOV / Mak GP induced modest antibody and cellular responses; however, when adjuvanted with matrix M, purified EBOV / Mak GP nanoparticles were highly immunogenic and protective in a 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 a lethal Ebola virus challenge, while unadjuvanted EBOV / Mak GP induced 100% protection. BOV / Mak GP provided only 10% protection, and no protection was observed in mice immunized with EBOV / Mak GP containing AlPO. Thus, in certain embodiments, the compositions disclosed herein prevent Ebola infection.

[0144] Co-administration of EBOV / Mak GP with Matrix M induced the production of balanced IgG1 and IgG2a subclass responses. Minimal IgG2a antibodies were detected in the absence of adjuvant or with AlPO4. Blaney et al., Antibody quality and protection from lethal Ebola virus challenge in nonhuman primates immunized with rabies virus based bivalent In a rabies / EBOV chimeric vaccine model in nonhuman primates (NHPs), a study in PLoS Pathog. 2013;9(5): demonstrated that antibody isotype plays a role in virus neutralization and protection against Ebola virus challenge. Mouse IgG2a antibodies are equivalent to human IgG1 antibodies, which efficiently bind IgG-Fc receptors (FcγRs) 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 resolve viral infections, for example, through antibody-dependent cell-mediated cytotoxicity. All antibodies that were fully protective in vivo were of the IgG2a subclass; i.e., identical to human IgG1. Thus, the compositions disclosed herein stimulate the production of IgG1 antibodies as part of a 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 polyfunctional T cells producing more than one cytokine. The observation that protection from lethal Ebola virus challenge was observed only in the Matrix M-adjuvanted EBOV / Mak GP group was associated with enhanced production of polyfunctional 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. GCs are microanatomical locations for B cell differentiation, somatic hypermutation, antibody class switching, and memory B cell formation. Coadministration of EBOV / Mak GP with the saponin adjuvant Matrix M enhanced T cell differentiation and development, promoting GC B cell differentiation and development. FHThere was also an increase in the number of GC and T cells induced by matrix M adjuvanting. FH The increased frequency of cells was associated with an enhanced magnitude of the antibody response and the induction of greater numbers of long-lived plasma cells, suggesting that the matrix M-adjuvanted EBOV / Mak GP vaccine is capable of inducing particularly durable antibody responses.

[0147] Each dose of Ebola GP may be combined with an adjuvant. Administration of purified EBOV / Mak GP nanoparticles with Matrix M adjuvant provides a strong stimulation of anti-EBOV / Mak GP immune responses resulting in 100% protective efficacy in mouse models. The compositions and methods disclosed herein result in a more rapid development of anti-EBOV / Mak GP IgG and Ebola virus neutralizing antibodies, increased levels of IgG2a, and polyfunctional CD4+ and CD8+ T cells, T FH cells, providing an increase in the frequency of germinal center B cells and the persistence of EBOV / Mak GP-specific plasma B cells in the bone marrow.

[0148] Thus, analysis of the mouse study demonstrated that the compositions disclosed herein provided complete protection. confirms. To further establish the protective effect, studies were 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. 2012 Oct. 23; 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. 2003 Dec. 163(6):2347-70. Thus, in some embodiments of the present disclosure, the protective effect includes a reduction in viral load below the ability of RT-PCR to detect it at about 7 days, about 10 days, about 14 days, or about 21 days after viral exposure.

[0149] Non-human primate studies further confirmed that the compositions disclosed herein are protective. Ebola GP nanoparticles were evaluated without adjuvant and with either alum or Matrix M adjuvants. 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. Results in the macaque model were particularly unexpected. See Examples 24 and 24. Notably, the composition was not only protective against challenge with live Ebola vaccine, but the amount of Ebola RNA was undetectable 10 days after challenge with live virus. See Figure 48. Notably, in one macaque subject, there was a small signal at approximately day 7; however, by day 10, levels had returned to below the limit of detection. In contrast, exposure of untreated animals to live Ebola virus resulted in infection and disease, with the subject being euthanized on day 9.

[0150] Modified antigens The antigens disclosed herein encompass variants and mutants of these antigens. In certain embodiments, the antigen may share identity with the disclosed antigen. Generally, unless specifically defined in the context of a 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 alignment program ClustalW2, available at www.ebi.ac.uk / Tools / msa / clustalw2 / . The following default parameters 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 the protein. In other embodiments, the protein contained in the nanoparticle comprises the protein. Addition to the protein itself can be for various purposes. In some embodiments, the antigen can 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 can be a polyglutamic acid tag, a FLAG tag, an HA tag, a polyHis tag (having approximately 5-10 histidines), a Myc tag, a glutathione-S-transferase tag, a green fluorescent protein tag, a maltose-binding protein tag, a thioredoxin tag, or an Fc tag. In other embodiments, the extension can be an N-terminal signal peptide fused to the protein to enhance expression. While such signal peptides are often cleaved upon expression in cells, some nanoparticles may contain antigens with an intact signal peptide. Thus, if the nanoparticle contains an antigen, the antigen may contain an extension and thereby be a fusion protein when incorporated into the nanoparticle. For purposes of calculating sequence identity, the extension The 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 identity to a protein having a truncation, identity is measured over the remainder of the protein.

[0153] Combination Nanoparticles As used herein, a combination nanoparticle refers to a nanoparticle that induces an immune response against two or more different pathogens. Depending on the specific combination, the pathogens may be different strains or subtypes of the same species, or the pathogens may be different species. To prepare a combination nanoparticle, glycoproteins from multiple pathogens may be combined into a single nanoparticle by binding them in a detergent exchange step. The detergent exchange following glycoprotein binding to the column allows multiple glycoprotein types to form around the detergent core, providing a combination nanoparticle.

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

[0155] Such compositions are particularly desirable when the pathogens are related in some way. In one example, the composition may contain nanoparticles against strains identified by authorities each year as forming the seasonal influenza of a particular year. Typically, for seasonal influenza vaccines, vaccine compositions contain HA and / or NA nanoparticles that induce an immune response against strains of three, four, or five influenza subtypes. Thus, different strains of influenza may be combined in a vaccine composition. In some embodiments, the combination 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 HA 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, 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. 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 a native influenza HA protein and / or to an NA protein.

[0156] In another example, both influenza and RSV cause respiratory disease, and HA, NA, and / or RSV F may therefore be combined into a combination nanoparticle, or multiple nanoparticles may induce a response against RSV and one or more influenza strains. They may be combined in a vaccine composition to achieve the same.

[0157] Vaccine Composition The compositions disclosed herein may be used prophylactically or therapeutically, but are typically prophylactic. Accordingly, the present disclosure includes methods for treating or preventing infection. The method includes administering a therapeutic or prophylactic amount of the immunogenic composition of the present disclosure to a subject. Preferably, the pharmaceutical composition is a vaccine composition that provides a protective effect. In other embodiments, the protective effect may include an improvement in symptoms associated with infection in a percentage of an exposed population. For example, depending on the pathogen, the composition may prevent or reduce one or more viral disease symptoms selected from: fever, fatigue, muscle pain, headache, sore throat, vomiting, diarrhea, skin 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, particularly RSV F nanoparticles, have improved stability at slightly acidic pH levels. For example, the pH range for compositions containing nanoparticles may be about pH 5.8 to about pH 7.0, about pH 5.9 to about pH 6.8, about pH 6.0 to about pH 6.5, about pH 6.1 to about pH 6.4, about pH 6.1 to about pH 6.3, or about pH 6.2. Typically, compositions containing RSV F protein nanoparticles have a pH of about 6.2. For other nanoparticles, the composition may tend to be neutral; for example, influenza nanoparticles may have a pH of about 7.0 to 7.4, often about pH 7.2.

[0160] Adjuvants 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 adjuvants and are therefore available for administration as adjuvant-free compositions. Advantageously, the adjuvant-free compositions disclosed herein can provide a protective immune response when administered as a single dose. Alum-free compositions that induce a strong immune response are particularly useful in adults aged about 60 years and older.

[0161] Aluminum-based adjuvants In some embodiments, the adjuvant can be alum (e.g., AlPO4 or Al(OH)3). Typically, the nanoparticles are substantially bound to the alum. For example, the nanoparticles can be at least 80% bound, at least 85% bound, at least 90% bound, or at least 95% bound to the alum. Often, the nanoparticles are 92% to 97% bound to the alum in the composition. The amount of alum present per dose typically ranges between about 400 μg and about 1250 μg. For example, alum can 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 adjuvants Saponin-containing adjuvants may be combined with the immunogens disclosed herein. Saponins are glycosides derived from the bark of the tree Quillaja saponaria Molina. Typically, saponins are prepared using a multi-step purification process that results in multiple fractions. As used herein, the term "Quillaja saponaria Molina-derived saponin fraction" is used collectively to describe semi-purified or defined saponin fractions of Quillaja saponaria or substantially pure fractions 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 from Quil A, a 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 semi-preparative HPLC, eluting with a gradient of 25% to 60% acetonitrile in acidic water. The fraction referred to herein as "Fraction A" or "QH-A" is or corresponds to the fraction eluted at approximately 39% acetonitrile. The fraction referred to herein as "Fraction B" or "QH-B" is or corresponds to the fraction eluted at approximately 47% acetonitrile. The fraction referred to herein as "Fraction C" or "QH-C" is or corresponds to the fraction eluted at approximately 49% acetonitrile. Additional information regarding fraction purification can be found in U.S. Patent No. 5,057,540. When prepared as described herein, Quillaja saponaria Molina fractions A, B, and C each represent a group or family of closely chemically related molecules with definable properties. The chromatographic conditions under which they were obtained result in a high degree of batch-to-batch reproducibility with respect to elution profile and biological activity.

[0164] Other saponin fractions have been described. Fractions B3, B4 and B4b are described in EP 0 436 620. Fractions QA1 to QA22 are described in EP 0 3632 279 B2, Q-VAC (Nor-Feed, AS Denmark), Quillaja saponaria The compounds are described in Molina Spikoside (Isconova 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 EP 03632279B2 can be used, especially QA-7, QA-17, QA-18 and QA-21. They can be obtained as described in EP 03632279B2, especially in Example 1 on pages 6 and 8 and 9.

[0165] The saponin fractions described herein and used to form adjuvants are often substantially pure fractions; i.e., the fractions are substantially free of contaminants from other materials. In certain embodiments, a 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 is ISCOM (Immune Stimulating Complex ISCOMs may be administered in the form of cage-like particles called ISCOMs (ISCOMs). ISCOMs can be prepared as described in EP0109942B1, EP0242380B1, and EP0180546B1. In certain embodiments, transport and / or passenger antigens may be used as described in EP9600647-3 (PCT / SE97 / 00289).

[0167] Matrix adjuvants In some embodiments, 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 embodiments, the ISCOM matrix complex also contains a phospholipid. 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 EP 0 436 620 B1.

[0168] In other embodiments, the ISCOM is an ISCOM complex. An 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 detergent treatment, where a portion of the antigen is incorporated into the particle. In contrast, ISCOM matrices are formulated as a mixture with 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 ISCOM matrix complex or the saponin fraction incorporated into the ISCOM complex, or at least one additional adjuvant similarly 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 subfraction, such as QA1-21.

[0170] In certain embodiments, each ISCOM particle may contain at least two saponin fractions. Any combination of weight percents of different saponin fractions may be used. Any combination of weight percents of any two fractions may be used. For example, the particles may each contain any weight percent of Fraction A and any weight percent of another saponin fraction, such as a crude saponin fraction or Fraction C. Thus, in certain embodiments, each ISCOM matrix particle or each ISCOM complex particle may contain 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 of one saponin fraction, e.g., Fraction A, and the remaining up to 100% by weight of another saponin in each case, e.g., any coarse fraction or any other fraction, e.g., Fraction C. Weights are calculated as the total weight of the saponin fractions. Examples of ISCOM matrix complex and ISCOM complex adjuvants are disclosed in U.S. Patent Application Publication No. 2013 / 0129770.

[0171] In certain embodiments, the ISCOM matrix or ISCOM complex comprises 5-99% by weight of one fraction, e.g., Fraction A, and the remaining up to 100% by weight of another fraction, e.g., crude saponin fraction or Fraction C. Weights are calculated as the total weight of the saponin fractions.

[0172] In another embodiment, the ISCOM matrix or ISCOM complex comprises 40% to 99% by weight of one fraction, for example, Fraction A, and 1% to 60% by weight of another fraction, for example, the crude saponin fraction or Fraction C. Weights are calculated as the total weight of the saponin fractions.

[0173] In yet another embodiment, the ISCOM matrix or ISCOM complex comprises 70% to 95% by weight of one fraction, e.g., Fraction A, and 30% to 5% by weight of another fraction, e.g., crude saponin fraction or Fraction C. Weights are calculated as the total weight of the saponin fractions. In another embodiment, 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 multiple 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 the 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. A composition or vaccine may comprise at least two types of complexes or particles, each type having one type of saponin incorporated into a physically distinct particle.

[0176] In the composition, a mixture of ISCOM matrix complex particles and / or ISCOM complex particles may be used, where one saponin fraction Quillaja saponaria Molina and another saponin fraction 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 containing one saponin fraction may be present in the composition in any combination of weight percents. In certain embodiments, the composition may contain 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 of ISCOM matrices or complexes containing a first saponin fraction, with the remainder consisting of ISCOM matrices or complexes containing a different saponin fraction. In some embodiments, the remainder is one or more ISCOM matrices or complexes, each matrix or complex particle containing only one saponin fraction. In other embodiments, the ISCOM matrix or complex particle may contain more than one saponin fraction.

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

[0179] A preferred composition comprises a first ISCOM matrix containing Fraction A and a second ISCOM matrix containing Fraction C, where the Fraction A ISCOM matrix comprises about 70% by weight of the total saponin adjuvant and the Fraction C ISCOM matrix comprises about 30% by weight of the total saponin adjuvant. In another preferred composition, the Fraction A ISCOM matrix comprises about 85% by weight of the total saponin adjuvant and the Fraction C ISCOM matrix comprises about 15% by weight of the total saponin adjuvant, thereby providing certain particular In certain compositions, the Fraction A ISCOM matrix is ​​present in an amount ranging from about 70% to about 85% by weight of the total saponin adjuvant in the composition, and the Fraction C ISCOM matrix is ​​present in an amount ranging from about 15% to about 30%. Exemplary QS-7 and QS-21 fractions, their production, and their uses 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, the disclosures of which are incorporated by reference.

[0180] Other adjuvants Other adjuvants may be used in addition or as an alternative in some compositions. The inclusion of any adjuvant described in Vogel et al., "A Compendium of Vaccine Adjuvants and Excipients (2nd Edition)," incorporated herein by reference in its entirety for all purposes, is contemplated within the scope of this disclosure. Other adjuvants include complete Freund's adjuvant (a nonspecific stimulator of the immune response containing killed Mycobacterium tuberculosis bacteria), incomplete Freund's adjuvant, and aluminum hydroxide adjuvant. Other adjuvants include GMCSP, BCG, MDP compounds such as 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 paucilamellar lipid vesicles; for example, Novasome®. Novasome® are paucilamellar non-phospholipid vesicles ranging from about 100 nm to about 500 nm. They contain Brij 72, cholesterol, oleic acid, and squalene. Novasomes have been shown to be effective adjuvants (see U.S. Pat. Nos. 5,629,021, 6,387,373, and 4,911,928).

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

[0182] The compositions may be administered in a single-dose schedule or a multiple-dose schedule. Multiple doses may be used in a primary immunization schedule or a booster immunization schedule. In a multiple-dose schedule, various doses may be given by the same or different routes, such as a parenteral prime and mucosal boost, a mucosal prime and parenteral boost, etc. In some embodiments, subsequent boost 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 still provide a protective immune response.

[0183] In some embodiments, the dose measured in μg may be the total weight of the dose including solute, or the weight of RSV F nanoparticles, or the weight of 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 children ranges from about 30 μg to about 300 μg, about 90 μg The dose may range from about 100 μg to about 270 μg, 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 dose is about 120 μg and is administered with alum. In some aspects, pediatric doses may range from about 30 μg to about 90 μg. Certain populations may be administered with or without an adjuvant. For example, when administered to older adults, alum is preferably not included. In certain aspects, the composition may not include an additional adjuvant. In such circumstances, the dose may be increased by about 10%.

[0185] In some embodiments, the dose 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 volume of about 0.5 mL.

[0186] In certain embodiments, for a RSV vaccine, the dose may contain a concentration of RSV F protein 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 journal articles cited in this disclosure are hereby expressly incorporated by reference in their entirety for all purposes. [Example]

[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. The recombinant virus was then amplified by infection of Sf9 insect cells. Insect cell cultures were infected with baculovirus at approximately 3 MOI (multiplicity of infection = virus ffu or pfu / cell). Cultures and supernatants were harvested 48-72 hours post-infection. Crude cell harvests, approximately 30 mL, were 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 anion exchange chromatography, lentil lectin affinity / HIC, and cation exchange chromatography. Washed cells were lysed by detergent treatment and then subjected to a low pH treatment to precipitate BV and Sf9 host cell DNA and proteins. The neutralized low pH lysate was clarified and further purified by anion exchange and affinity chromatography before undergoing a second low pH treatment.

[0190] Affinity chromatography was used to remove Sf9 / BV proteins, DNA, and NP-9, and concentrate RSV F protein. Briefly, lentil lectin is a calcium- and manganese-containing metalloprotein that reversibly binds polysaccharides and glycosylated proteins containing 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). Non-glycosylated proteins and DNA were removed in the column flow-through, while glycosylated RSV F protein selectively bound to the resin. Weakly bound glycoproteins were removed using a buffer containing high salt and low molar concentration of methyl alpha-D-mannopyranoside (MMP).

[0191] Additionally, column washing was also used to exchange the NP-9 surfactant with 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 a high concentration of MMP. After elution, the RSV F protein trimer was assembled into micellar nanoparticles composed of the RSV F protein trimer and PS80 contained in the surfactant core. After the surfactant exchange, a low pH inactivation step was performed, followed by incubation on a sulfuric acid column in the presence of a buffer containing 0.1% PS80.

[0192] The eluted material was diluted with a solution containing PS80 appropriate for obtaining a bulk drug substance (DS) with a PS80:RSV F protein molar ratio of approximately 50. The appropriate composition of DS was obtained by combining RSV F nanoparticles with a solution containing 22 mM sodium phosphate, 0.03% PS80 in phosphate buffer pH 6.2. At each step during and after surfactant exchange, the antigen to PS80 ratio in the composition was maintained at a molar ratio of 35 to 60. The molar ratio was calculated using the PS80 and RSV F concentrations 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 vaccine compositions To obtain nanoparticles for the vaccine product to be administered, the drug substance was diluted to a drug product with a PS80:RSV protein molar ratio of approximately 50. The drug substance was thawed, diluted, and filled into glass vials or prefilled syringes for storage at 2-8°C prior to administration. The nanoparticles were bound to alum adjuvant. The alum adjuvant was added and mixed to ensure that approximately 95% of the nanoparticles were bound to alum, which represents approximately 0.4 mg of nanoparticles per 120 μg dose of RSV F nanoparticles in a 0.5 mL volume.

[0194] Example 3 Characterization of RSV F glycoprotein in nanoparticles The protein structure in the nanoparticles was analyzed using 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 lack any fatty acids (peak 5) and are soluble (peak 1). Analysis on SDS-PAGE gels separated additional variants, including the F1+2 protein, F1, F1A, F1B, and F1C moieties, and F2. See Figure 4. Analysis of the peptide structure was performed using peptide mapping. See Figure 5. RSV To evaluate the glycan structures on the F glycoprotein, HPLC-FLD was performed. The results demonstrated that the major glycan structures were fucosylated.

[0195] Example 4 Examination of RSV F nanoparticles by electron microscopy The nanoparticles prepared in Example 1 were visualized by electron microscopy. The results confirmed the formation of nanoparticles containing RSV F glycoprotein surrounding a surfactant core. The exact composition of the surfactant core remains unknown. Figure 6 illustrates the types of nanoparticles obtained. The RSV F protein maintained a trimeric structure even after surfactant exchange. Several types of nanoparticles were obtained, with varying numbers and morphologies of trimers / nanoparticles. Figure 6 shows that multiple trimers can be seen around the surfactant core. In the highlighted area, seven trimers are shown surrounding the surfactant. The main panel of Figure 6 illustrates the extent of the trimers around the surfactant core produced. The schematic structure of the RSV F protein trimer in the bottom left panel illustrates the orientation of the trimer with the base associated with the surfactant core. This 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 used to determine the size distribution profile of nanoparticles by measuring the change in the light scattering pattern of particles during Brownian motion. Nanoparticle size was determined as a linear function of the concentration of ionic surfactant relative to the concentration of RSV F nanoparticles (see Figures 7 and 8).

[0197] Analytical ultracentrifugation (AUC) was used to measure the course of sample concentration relative to the axis of the spin profile as a result of the applied centrifugal field. Figure 8 reveals that two main nanoparticle shapes emerge based on the concentration of nanoparticles present. The resulting nanoparticle types include monomeric and dimeric anisotropic rods and spherical oligomers. Structural intermediates between these two nanoparticle types are formed between concentrations that result in anisotropic rods and spherical oligomers. Figure 9 shows that the nanoparticle type can be controlled by modulating the concentration of RSV F protein; higher concentrations (1 mg / mL) result in a predominantly spherical oligomer population, while lower concentrations (0.22 mg / mL) result in a predominantly monomeric / dimeric anisotropic rod population. These data illustrate that controlling the amount of surfactant and RSV F concentration can result in nanoparticles with a specific diameter (z-average) of 20 nm to 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] Differences in primary structure were assessed after various stress treatments. Figure 10 shows a comparison of how specific regions withstood stress compared to the control. The data indicate that the nanoparticles had excellent stability throughout the protein, and only a particularly severe oxidation test using hydrogen peroxide was able to degrade the protein to any particular extent. However, even this treatment did not reduce the structural integrity of antigenic site II, the target of palivizumab. In fact, even with severe oxidation, the RSV-F protein only deteriorated structurally at positions 63-82, 237-258, and 360-364. Thus, even after severe stress, the nanoparticles remained substantially intact.

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

[0201] Example 7 Enhanced nanoparticle stability: maintained immunogenic properties The stressed vaccine compositions described in Example 6 were evaluated for immunogenicity in a mouse model. Mice were administered two intramuscular injections of vaccines 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 were administered along with a control (thawed from storage at -70°C): 50°C for 2 weeks, pH 10 at 25°C for 1 week, and 0.5% hydrogen peroxide at 25°C for 1 week.

[0202] The immune response was evaluated for the presence of anti-RSV F IgG, PCA titer, and RSV A neutralizing antibody. Physical and chemical stressors did not significantly affect RSV F protein immunogenicity in vivo. The stressed samples induced similar anti-RSV F IgG antibody titers and comparable functional PCA and RSV A neutralizing titers to those of the unstressed RSV F nanoparticle vaccine composition control (see Figures 12A-12D). Together, these forced degradation studies demonstrate that nanoparticles induce a strong immune response even when exposed to severe 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. Figure 13 illustrates the dramatic improvement in stability up to 18 months when nanoparticles were formed with 0.03% PS80 (i.e., a molar ratio of 55) compared to 0.015% (i.e., a molar ratio of 27). The left panel shows SDS-PAGE of nanoparticles produced at two concentrations at time 0. The data show that similar results to the reference preparation were obtained with both nanoparticle preparations. In particular, both show strong signals for F1 and F1+2, illustrating essentially no degradation. Aliquots of each preparation were then incubated at 4°C for 18 months, and then SDS-PAGE was performed again. The right panel and table data illustrate that nanoparticles containing only 0.015% PS80 in the particles yielded truncated F1. In contrast, nanoparticles prepared with 0.03% PS80 in the particles demonstrated excellent resistance to proteases. We believe that by maintaining the correct ratio of surfactant and protein, nanoparticles can be obtained with orientation of glycoproteins with protease-sensitive moieties likely protected by some steric hindrance mechanism. We further observed that concentrations of PS80 at or above 0.06% increased aggregate formation. Taken together, the data indicate that the optimal PS80 level for nanoparticle stability is about 0.03% to about 0.05%.

[0204] Example 9 Purification of HA nanoparticles The TMAE column was pre-equilibrated with 0.5 CV of Buffer A1 (25 mM Tris pH 7.5, 70 mM NaCl, 0.02% NP-9) at a flow rate of 91.7 cm 30 mL / min. The sample was loaded at 20 mL / min (25 min residence time) 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% NP-9) followed by 1.1 CV of 100% B. A representative chromatogram is shown in Figure 18B. The product from the TMAE column was applied to a lentil lectin affinity chromatography column pre-equilibrated with 3 CV of Buffer A11: 25 mM sodium phosphate pH 6.0, 10 mM NaCl, 0.05% PS80 (flow rate: 147 cm / hr, 13 mL / min). The sample was loaded at 6.5 mL / min, 73.5 cm / hr, for a residence time of 9.4 min. 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, detergent 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 eluted with Buffer A1 (25 mM sodium phosphate pH 6.0, 10 mM Add 3 CV of NaCl, 0.05% PS80 to the sulfuric acid column and add 2 CV of buffer A1. The column was washed and then eluted with 100% Buffer B1 (25 mM sodium phosphate pH 7.5, 500 mM NaCl, 0.05% PS80). The eluted product was then combined 1:1 with 50 mM sodium phosphate pH 9 and filter sterilized. The final product was pH 7.2. The chromatogram is shown in Figure 18D. Figure 18E provides a gel and Western blot of the various products obtained during the purification process 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 from various strains (A / New Hampshire / 1 / 2015, A / Switzerland / 9715293 / 2013, A / Hong Kong / 4801 / 2014, B / Phuket / 3073 / 2013, and B / Brisbane 60 / 2008). Data indicated that highly pure preparations were obtained in all cases. Analysis by gel densitometry showed purities greater than 93%, ranging from 93% to 97%. See Figures 19A-19H. The purity of the three A subtype strains was also analyzed by RP-HPLC, and was found to be 83% to 85% pure. See Figure 19I. Additionally, nanoparticle size was measured. The nanoparticles exhibited diameters ranging from 22.0 nm to 29.9 nm. See Figure 19.

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

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

[0208] Example 12 Immunogenicity analysis of HA nanoparticles co-administered with RSV F nanoparticles The immunogenicity of nanoparticles in vaccines was evaluated in a mouse model. A combination of nanoparticles containing two antigens (influenza HA protein from the A / Swiss H3 subtype and RSV F protein) was administered. Each nanoparticle was also administered separately. The vaccine was administered alone or with the adjuvant AlPO4 or matrix M saponin adjuvant. Figure 22 shows the treatments administered to groups 1 to 10. Group 10, the control, received no treatment. Treatments were administered on days 0 and 21. HAI responses to heterologous and homologous challenge were measured. See Figures 23A and 23B. Figure 23A shows that matrix-adjuvanted HA nanoparticles stimulated a particularly strong response to homologous challenge. Figure 23B shows that a strong HAI response was also obtained against the heterologous influenza strain A / Texas / 50 / 2012 when administered with matrix M, and this response was not affected by coadministration with RSV F nanoparticles.

[0209] The ability of RSV F nanoparticle components 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 of 80 μg / mL to approximately 700 μg / mL. When induced by both RSV F and influenza nanoparticles, a low response of approximately 20 μg / mL to 40 μg / mL was obtained in the absence of adjuvant or in the presence of AlPO4. However, when RSV F was 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, vaccine-induced T cell responses against RSV and against influenza A / Swiss / 9715293 / 2013 were measured (Figures 23E and 23F). The data show strong induction of IFNγ against both targets when Matrix M was used as an adjuvant.

[0211] Example 13 Trypsin-resistant nanoparticle production A specific approach to producing influenza A HA nanoparticles allows for the trypsin sensitivity of the HA protein, which alters its folding, resulting in reduced immunogenicity and stability of the vaccine formulation. To produce trypsin-resistant HA nanoparticles, a detergent exchange approach utilizing 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, 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 contained HA. After the anion exchange column, detergent exchange was performed using a Capto lentil lectin column (GE) with a high salt / detergent wash containing 0.01% PS80 (pH 7.2) and eluted in 0.01% PS80. Figure 24C shows an exemplary chromatogram of the detergent exchange step. Finally, tangential flow filtration (TFF): 50 kD MWCO filter (pH 7.2) was used to produce the bulk drug substance (BDS) used to store the product. PS80 was maintained at 0.05% in the pH 7.2 buffer during and after the TFF step.

[0213] Example 14 Trypsin-resistant nanoparticle analysis HA nanoparticles from various strains were evaluated using the process and production described in Example 13. 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 strain, B / Brisbane / 60 / 08 HA, evaluated as bulk drug substance. The yield was approximately 30 mg / L. Figure 25C shows the yield of the H3N2 strain, indicating that the process provided a purity of approximately 96% and a yield of approximately 20 mg / L.

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

[0215] Additionally, DSC provides information about protein conformation, allowing estimated stability profiles to be roughly extrapolated for each HA strain subjected to different process conditions. Differences between HA purified via a process step that exposes HA to pH 6.0 during purification and HA purified via a separate step (e.g., MMC resin or TFF membrane) are shown using B / Brisbane HA as an example. The data show higher Tm values ​​in intensity, a shift toward the appearance of a higher Tm for the main peak and sharper peaks, suggesting proper folding of HA when purified via a separate step. However, the data for HA exposed to pH 6.0 also show a significant drop in intensity, an earlier appearance of the Tm for the main peak, significantly broadened peaks indicative of slow asymmetric unfolding, and / or Tm values ​​containing misfolded protein and aggregation peaks at higher temperatures. Similar profiles were observed for other strains (A / Cal, A / Hong Kong, A / New Hampshire). Based on these three observations in the DSC data, we conclude that while low pH yields nanoparticles as described above, which may have certain applications, alternative process steps / conditions using neutral pH will yield HA protein with significantly better thermodynamic and potentially improved stability profiles.

[0216] Trypsin resistance Figure 26 illustrates the improved trypsin resistance obtained when nanoparticles are produced as described in Example 13. To test trypsin sensitivity, HA samples were diluted to 0.24 mg / mL and incubated in reduced trypsin at 37°C for 60 minutes. Digestion was stopped by adding trypsin inhibitor, followed by SDS-PAGE analysis. Comparison of the left and right panels of Figure 26 illustrates the enhanced trypsin resistance. Purified HA nanoparticles fabricated in Sf9 insect cells are HA0. When exposed to trypsin, HA0 is cleaved at Arg AA344 in H1 into HA1 and HA2. Correctly folded HA trimers will resist further cleavage when incubated with increasing concentrations of trypsin. B / Brisbane / 60 / 08 purified at neutral pH is resistant to trypsin and 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 can be achieved for various strains. Figure 26B shows strain A / Hong Kong / 4801 / 2014. A / Hong Kong / 4801 / 2014(H3N2) purified at neutral pH is trypsin resistant and therefore 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 for A / New Hampshire / 1 / 2015 and H1N1 subtypes: like other strains, acid-purified H1N1 was misfolded (data not shown), whereas neutral pH-purified protein was trypsin-resistant and correctly folded.

[0218] Comparison with commercially available influenza vaccines Previous approaches to producing recombinant influenza vaccines have not met with widespread success. To investigate whether egg-produced or recombinantly produced influenza vaccines exhibited trypsin resistance, egg-produced and recombinant influenza vaccines were tested using the same protocol described above. Trypsin sensitivity was compared in two vaccines (Fluzone® and Flublok®, respectively). Specifically, undiluted vaccine was incubated with various amounts of trypsin at 37°C for 60 minutes, followed by addition of trypsin inhibitor, 2x sample buffer, and heating at 70°C for 10 minutes before 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, which is cleaved into HA1, HA2, and HA1, is resistant to trypsin digestion. In contrast, the commercially available trivalent recombinant HA Flublok vaccine, when exposed to trypsin, is converted into HA1, HA2, and HA1 polypeptides and is trypsin-sensitive (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 poor immunogenicity and lack of widespread adoption to date.

[0221] In contrast, the methods disclosed herein confirm that purifying HA nanoparticles using a buffer of at least pH 7.0 reduces or eliminates the misfolding of HA protein that occurs when HA protein is exposed to acidic conditions during purification.

[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. Following expression, the N-terminal signal peptide was cleaved, and the mature protein was purified and formed into nanoparticles. Purified Ebola virus GP (EBOV / Mak GP) nanoparticles consist of multiple GP trimers assembled into spherical particles measuring 36 ± 4 nm (as measured by dynamic light scattering). Recombinant GP nanoparticles have a core region containing a glycoprotein 2 (GP2) "fusion subunit" along with two to nine or up to 15 outwardly extending "grail-like" glycoprotein 1 (GP1) trimer "attachment subunits."

[0223] For co-administration of Matrix M, a saponin-based adjuvant consisting of two populations of separately formulated 40 nm sized matrix particles was used: 85% Matrix A and 15% Matrix C. The matrix particles were formed by formulating purified saponin from Quillaja saponaria Molina with cholesterol and phospholipids.

[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 subcutaneously (SC) or intramuscularly (IM). Phosphate-buffered saline (PBS) was used as a placebo. Blood samples for serum were collected via the retroorbital route. Animals were anesthetized with isoflurane prior to blood collection.

[0225] On days 0 and 21, mice (n=10 per group) were treated with EBOV / Mak GP alone or with AlPO4 (50 μg) or Matrix M adjuvant (2.5 μg or 5 μg). g) and immunized via 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] Day 28 serum samples were evaluated for anti-EBOV / Mak neutralizing antibody responses using a pseudovirion neutralization reporter assay at the US Army Medical Research Institute of Infectious Diseases, Fredrick, MD. A hantavirus pulmonary syndrome (HPS) DNA vaccine delivered using a spring-powered jet injector elicits potent neutralizing antibody responses in rabbits and nonhuman 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-expressing core was pseudotyped using the plasmid pWRG / EBOV-Z76(opt) expressing Zaire Ebola virus 1976 (Mayinga) GP. The plasmid used to obtain pseudotyped Ebola GP was pWRG / EBOV / Mak-Z76(opt) expressing Zaire Ebola virus 1976 (Mayinga) GP. PsVs were prepared in 293T cells. Mouse serum was heat-inactivated at 56°C for 30 minutes, and then an initial 1:20 dilution was prepared, followed by five-fold serial dilutions in Eagle's minimum essential medium (EMEM) (Life Technologies) supplemented with 10% (vol / vol) heat-inactivated FBS, 100 IU / mL penicillin, and 100 μg / mL streptomycin (cEMEM). Ebola GP PsVs were diluted in cEMEM. An equal volume of 4 × 10 3PsVs solution containing focus-forming units and 10% 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 black, clear-bottom 96-well plates (Corning) were infected with 50 μl of each PsVs-serum mixture and then incubated for an additional 18–24 h at 37°C. The medium was discarded, the cells were lysed, and luciferase substrate was added according to the Renilla Luciferase Assay System (Promega #E2820) protocol. Flash luciferase signals were measured using a Tecan M200 microplate reader. Raw values ​​were transferred to GraphPad Prism version 6.04, and the data were baseline-corrected for the untreated PsVs signal. Data were fitted to a four-parameter logistic nonlinear regression model using GraphPad Prism, and PsVNA 50% (PsVNA50) neutralization titers were then 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), Zaire Ebola virus 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 EBOV / Mak GP (Novavax). Unreacted surfaces were blocked with StartingBlock Blocking Buffer (Pierce) for 1 h at room temperature (RT). Plates were sequentially reacted with 5-fold serial dilutions of serum samples starting at 1:100 (2 h), goat anti-mouse IgG (or IgG1 and IgG2a) conjugated to horseradish peroxidase (HRP) (Southern Biotech) (1 h), peroxidase substrate 3,3,5,5-tetramethylbenzidine (TMB) (Sigma) (10 min), and TMB Stop Buffer (ScyTek Laboratories) at RT. Plates were washed three times with PBS / Tween (Quality Biologicals) before the addition of the HRP conjugate and TMB reagent.

[0228] Plates were read at 450 nm on a SpectraMax plus plate reader (Molecular Devices). Concentration responses were fitted to a four-parameter curve fit using SoftMax Pro software (Molecular Devices). Antibody titers were calculated as 50% of maximal antibody binding (EC 50 ) was defined as the reciprocal of the highest dilution at which there was a response. If serum IgG titers were outside the low detection range, a titer (starting dilution) <100 was recorded, a value of 50 was assigned to the sample, and the group geometric mean titer (GMT) 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 assess IFNγ- and EBOV / Mak GP-specific IgG-secreting cells Single cell suspensions were prepared from individual spleens by gently triturating the tissue with the plunger of a syringe. Single bone marrow cell suspensions were prepared by triturating the tissue with a 21-gauge needle syringe at 2% The bone was prepared by flushing with PBS containing FBS. The cells were washed twice with PBS containing 2% FBS and counted. IFNγ ELISPOT assay was performed using a mouse IFNγ ELISPOT kit (eBioscience, San Diego, CA) according to the manufacturer's protocol. Anti-IFNγ antibody (15 μg / ml in PBS) was used to coat ELISPOT plates (Millipore, Darmstadt, Germany) at 100 μl / well overnight at 4°C. Plates were washed four times with PBS and blocked with RPMI 1640 medium + 5% FBS for 1-2 hours at room temperature. A total of 3 × 10 cells in a volume of 200 μl were used. 5 Splenocytes were stimulated with a 15-mer EBOV GP peptide pool (2.5 μg / ml) containing 11 overlapping amino acids spanning the entire EBOV GP sequence. Phorbol myristate acetate (PMA) (50 ng / ml) plus ionomycin (200 ng / ml) was used as a positive control, and medium was used as a negative control. Each stimulation condition was performed in triplicate. Assay plates were incubated overnight at 37°C in a 5% CO2 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). Ebola GP-specific spot counts were obtained by subtracting the background counts of the medium control from the GP peptide-stimulated wells. Data shown on the graph are the average of triplicate wells. To measure GP-specific IgG-secreting cells, ELISPOT plates were incubated with EBOV / Mak Plates were coated with GP (2.5 μg / ml in PBS) and incubated overnight at 4°C. Plates were washed and blocked as above. 3-5 × 10 cells per well were added. 5Triplicate splenocytes or bone marrow cells were plated and the plates were incubated overnight at 37°C. On day 2, the plates were washed, and goat anti-mouse IgG-HRP was added and incubated for 1.5 hours. Spots were developed and counted as above. The average number of spots from triplicate wells was calculated 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 fresh splenocytes were used. 6 The cells 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) at 4°C for 30 minutes. The cells were washed twice and suspended in PBS containing 2% FBS for analysis. 1 × 10 cells were used to stain follicular helper T cells. 6 Fresh splenocytes were incubated with CXCR5-biotin, washed twice, and then Cells were 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). Cells were washed twice and suspended in PBS containing 2% FBS for analysis.

[0231] For intracellular staining for cytokines, splenocytes were cultured at 1 x 10 per well. 6Cells were cultured in 96-well U-bottom plates at single cells. Peptide stimulation was performed as described for ELISPOT culture. Plates were incubated for 6 hours at 37°C in the presence of BD GolgiPlug™ and BD GolgiStop™ (BD Biosciences). Cells were washed twice and incubated with CD3-BV 650, CD4-PerCP, CD8-FITC, CD44-APC-Cy7, and CD62L-PE-Cy7 (BD Biosciences). The cells were incubated with a mixture of antibodies against cell surface markers, including antibodies against chromatin-specific markers (Pharmingen, CA) and yellow LIVE / DEAD® dye (Life Technologies, NY) for 20 min at 4°C. After two washes, the cells were fixed with Cytofix / Cytoperm (BD Biosciences) for 30 min at 4°C, followed by BD Biosciences fixation. The cells were washed twice with 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 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's adjustment from ANOVA determined significance between groups using group as the independent variable and 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 adjuvant. Mice were vaccinated by SC injection with 5 μg of EBOV / Mak GP alone or EBOV / Mak GP formulated in Matrix M or AlPO4 adjuvants on days 0, 14, and 28. 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 AlPO4 (GMT = 436, p = 0.0001) (Figure 28A). The AlPO4 adjuvant produced 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 adjuvant, the neutralizing GMT titer in sera from mice immunized with EBOV / Mak GP alone was 197, and a lower titer was observed when EBOV / Mak GP was adjuvanted with AlPO4 (GMT = 49, p = 0.1). The neutralizing titer observed in sera from mice immunized with EBOV / Mak GP containing matrix M had a GMT of 6,463, 32-fold higher than that obtained with EBOV / Mak GP alone. In this assay, EBOV / Mak 2014 strain GP expressing EBOV / Mak GP was used. Because PsVs expressing EBOV 1976 Mayinga strain GP were not available, PsVs expressing 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, two weeks after the third vaccination administered on day 28, mice were challenged intraperitoneally with 1,000 pfu of mouse-adapted Zaire Ebola virus strain 1976 Mayinga. Control mice began dying of infection after 3 days, while mice vaccinated with EBOV / Mak GP alone or EBOV / Mak GP adjuvanted with AlPO4 died on days 5 or 6, respectively. Twenty-one days after challenge, all mice vaccinated with Matrix M-adjuvanted EBOV / Mak GP and one mouse vaccinated with EBOV / Mak GP alone were alive and healthy. In contrast, all other mice died of Ebola virus infection or were euthanized by day 8 (Figure 28C).

[0236] Example 18 Kinetics of Ebola GP IgG, IgG1, and IgG2a responses To further characterize the immune response to Matrix M-adjuvanted EBOV / Mak GP in more detail, two groups of Balb / c mice (10 mice / group) were treated with Matrix M. Mice were injected with 5 μg of EBOV / Mak GP adjuvanted with either 2.5 or 5 μg. Groups of mice injected with PBS, EBOV / Mak GP alone, or EBOV / Mak GP containing AlPO4 served as controls. EBOV / Mak GP-specific IgG and IgG subclasses (IgG1 and IgG2a) were measured by ELISA on days 14, 21, 28, and 60.

[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). None of the 10 mice in the EBOV / Mak GP group or the EBOV / Mak GP with AlPO4 group 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). There was still no response in the groups given EBOV / Mak GP alone or EBOV / Mak GP with AlPO4. On day 21, all mice received a second injection. On day 28, the IgG response increased to 3.0 × 10 in mice receiving Matrix M (2.5 or 5 μg), respectively. 5 and 4.9 × 10 5 There was a strong increase in the IgG response in the ELISA GMT titers (Fig. 29A). On days 28 and 60, specific IgG responses were detected in several mice in the EBOV / Mak GP alone and AlPO4-containing groups, but were significantly lower than those in mice immunized with EBOV / Mak GP containing matrix M (Fig. 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- and 139-fold higher, respectively, than those in the EBOV / Mak GP with AlPO4 group (Fig. 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 of 10 mice given EBOV / Mak GP alone and 4 of 10 mice given EBOV / Mak GP with AlPO4 produced low levels of IgG1 on day 28 (Figure 29B). On day 60, antigen-specific IgG1 was detected in the serum from all 5 remaining mice in the group given EBOV / Mak GP with AlPO4, although the mean titer was 2.5 or 5. The EBOV / Mak GP group was 51-fold lower and 41-fold lower, respectively, than the group receiving 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 polyfunctional T cell responses T cell responses to the different EBOV / Mak GP formulations were then assessed by measuring the number of IFNγ-secreting T cells after ex vivo stimulation of splenocytes with EBOV / Mak GP peptides in an ELISPOT assay. On day 28, IFNγ-secreting cells increased in spleens from mice immunized with EBOV / Mak GP containing Matrix M in a Matrix M dose-dependent manner (Figs. 30A and 30B). The mean numbers of IFNγ-secreting cells in the groups receiving EBOV / Mak GP containing 5.0 and 2.5 μg of Matrix M were 17- and 10-fold higher, respectively, than the group receiving EBOV / Mak GP alone, and 8- and 5-fold higher, respectively, than the group receiving EBOV / Mak GP containing AlPO4 (Fig. 30A).

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

[0241] Matrix M-induced CD4+ and CD8+ T cell responses were further evaluated by intracellular staining for cytokines in combination with cell surface markers. Analysis of splenocytes by flow cytometric staining at day 28 showed that both CD4+ and CD8+ T cells from the EBOV / Mak GP group with Matrix M secreted IFNγ, TNFα, and IL-2 (Figures 30C and 30D). The frequency of cytokine-secreting CD4+ and CD8+ T cells was significantly higher in spleens from the EBOV / Mak GP group with Matrix M than the baseline or minimal responses observed in control mice, mice receiving EBOV / Mak GP alone, or EBOV / Mak GP with AlPO4 (Figures 30C and 30D). The frequency of T cells simultaneously producing two or more cytokines (IFNγ, TNFα, and IL-2) was also evaluated at day 28. Significant levels of both CD4+ and CD8+ T cells producing either two or three cytokines were detected only in spleens from mice immunized with EBOV / Mak GP containing matrix M.

[0242] Example 20 Germinal centers and follicular helper T cell responses The frequency and absolute number of GC B cells in the spleen were analyzed by flow cytometry staining (Figure 31A). 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 a response with a GC frequency of 1.22 and 2.12%, respectively, compared with placebo, EBOV / Mak GP alone, or EBOV / Mak GP with AlPO (0.38, 0.41, and 0.44%, respectively) (Figure 31B). Accordingly, the absolute number of GC cells in the spleen also increased in the group receiving Matrix M (Figure 31C). By day 60, the frequency and absolute number returned to background levels (Figures 31D and 31E).

[0243] T on day 28 FH Cell frequency analysis included 2.5 or 5 μg of matrix M EBOV / Mak GP has a higher T than EBOV / Mak GP alone or with AlPO4. FH The results showed that T FH The absolute number of cells was also enhanced by EBOV / Mak GP with matrix M compared to EBOV / Mak GP alone or with AlPO4 (Fig. 32C). FH The frequency and absolute number of cells regressed to near background levels (FIGS. 32D and 32E).

[0244] Example 21 EBOV / Mak GP-specific plasma cells To assess 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 60 days after immunization. Analysis at day 60 revealed very few EBOV / Mak GP-specific IgG-secreting cells (<6 / 10) in the spleens from mice immunized with Matrix M-adjuvanted EBOV / Mak GP vaccine. 6We demonstrated the formation of long-lived plasma B cells (spleen cells) in the spleens from mice immunized with EBOV / Mak GP alone or with EBOV / Mak GP containing AlPO4 (Fig. 33A). IgG-secreting cells were not detected in the spleens from mice immunized with EBOV / Mak GP alone or with EBOV / Mak GP containing AlPO4 (Fig. 33A). In contrast, numerous EBOV / Mak GP-specific IgG-secreting cells were found in the bone marrow from mice that received matrix M-adjuvanted EBOV / Mak GP (Fig. 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 to bind to nanoparticles was tested. The antibodies were 13C6, 13F6, 6D8, and KZ52. EC50 curves and values ​​are shown in Figure 36, and additional binding kinetics data is shown in Figure 37. Figure 38 shows efficacy data using 13C6 as a reference. Three of the four antibodies exhibited excellent binding to GP.

[0246] Example 23 Non-human primate research: baboons 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 antigen with AlPO4. Groups 2 and 3 received 60 μg and 5 μg of antigen, respectively, along with 50 μg of Matrix M. Baboons were immunized on days 0 and 21. Strong responses were obtained against both Makona GP and Mayinga GP. See Figure 40. Additional analysis confirmed that the responses were long-lasting. Figure 41 shows the EC50 values ​​of IgG against Makona at later time points. The data establish that the responses were durable.

[0247] Additional studies confirm that IFNγ levels are substantially increased after immunization. Figure 42 shows that matrix M combined with GP increased IFNγ levels even more than combined with alum adjuvant. Interestingly, a lower dose of 5 μg of GP resulted in a more pronounced increase in IFNγ levels. TNFα and IFNγ responses in T cells are shown in Figure 43, and cytokine responses are shown in Figure 44. The responses are also more pronounced with GP and matrix M than with alum in each case. These data highlight the robust immune response of the disclosed formulations in the baboon model.

[0248] Example 24 Non-human primate research: Macaque research 1 To further confirm the protective effect of the nanoparticles, a macaque study was conducted as shown in Figure 45. Macaques were immunized intramuscularly with the vaccine on days 0 and 21 as indicated and challenged on day 42. Anti-GP responses were 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 in response to various peptide pools were measured at weeks 0, 3, and 5. The results demonstrate that immunization induced IFNγ-secreting cells in the immunized macaques.

[0249] Animal survival is remarkable. Figure 48. By day 7, the Ebola viral load in placebo-treated macaques was 10 7 By day 9, the placebo animals were euthanized. In contrast, 100% of the treated animals survived. Remarkably, the immune response was able to render viral loads undetectable by RT-PCR in almost all animals at almost all time points. Animal 33362 exhibited a viral load approximately 10% above the limit of detection on day 7. However, by day 10, the levels had fallen to levels beyond the ability of the assay to detect it.

[0250] Example 25 Non-human primate research: Macaque research 2 A second study was conducted in macaques. At week 0, animals were dosed with 5 μg of GP plus 50 μg of Matrix M, with additional boosts at either week 3 or week 6. 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 sustained 3 weeks after the first injection. The right panel illustrates results in animals with a 6-week gap between doses. These animals showed a substantial increase 2 weeks after the second booster dose, illustrating the beneficial effect of the prime-boost approach.

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

Claims

1. (i) nanoparticles comprising a non-ionic surfactant core and a trypsin-resistant influenza glycoprotein, wherein the influenza glycoprotein is associated with the core, and the non-ionic surfactant is PS80 and is present at about 0.03% to about 0.05%; and (ii) a pharmaceutically acceptable buffer solution A composition comprising:

2. 2. The composition of claim 1, wherein the influenza glycoprotein is selected from the group consisting of an influenza HA protein and an influenza NA protein.

3. 3. The composition of claim 2, wherein the influenza glycoprotein is an influenza HA protein.

4. 4. The composition of claim 3, wherein the influenza HA protein subtype is selected from the group consisting of H1, H3, H4, H5, and H7.

5. 3. The composition of claim 2, wherein the influenza glycoprotein is an influenza NA protein.

6. 4. The composition of claim 3, wherein the influenza NA protein subtype is selected from the group consisting of N1, N2, N3 and N5.

7. The pharmaceutically acceptable buffer solution is (i) 15 mM to 50 mM sodium phosphate; (ii) 150 mM to about 250 mM NaCl; 10. The composition of claim 1, wherein the pH of the composition is between 7.0 and 7.

5.

8. 8. The composition of claim 7, wherein the pharmaceutically acceptable buffer comprises: (i) 25 mM sodium phosphate; (ii) about 150 mM NaCl, and the pH of the composition is about 7.

5.

9. (i) nanoparticles comprising a PS80 surfactant core and trypsin-resistant influenza HA protein; (ii) a buffer containing 25 mM sodium phosphate pH 7.5, and 150 mM sodium chloride; and (iii) adjuvant 10. A vaccine composition comprising:

10. 10. The vaccine composition of claim 9, wherein the adjuvant is alum.

11. 10. The vaccine composition of claim 9, wherein the adjuvant is an ISCOM matrix adjuvant comprising a first ISCOM matrix containing fraction A and a second ISCOM matrix containing fraction C.

12. 12. The vaccine composition of claim 11, wherein the first ISCOM matrix comprises 70% to 95% by weight of the total ISCOM matrix, and the second ISCOM matrix comprises the remainder.

13. 13. The vaccine composition of claim 12, wherein the first ISCOM matrix comprises 85% by weight of the total ISCOM matrix, and the second ISCOM matrix comprises the remainder.

14. 10. The vaccine composition of claim 9, wherein the influenza HA protein subtype is selected from the group consisting of H1, H3, H4, H5 and H7.

15. 1. A method for preparing recombinant trypsin-resistant influenza nanoparticles, comprising: (i) binding a protein extract comprising a first detergent and an influenza glycoprotein to a protein purification column, wherein the column binds the influenza glycoprotein; (ii) performing a surfactant exchange by substantially replacing the first surfactant with a second surfactant; and (iii) eluting the bound influenza glycoprotein from the column in the presence of the second detergent to provide the nanoparticles. Including, the nanoparticles have a transition midpoint (Tm) of at least about 60 as measured by differential scanning calorimetry, and no buffers having a pH of less than 7.0 are used during preparation; method.

16. 16. The method of claim 15, wherein the influenza glycoprotein is an HA glycoprotein.

17. 16. The method of claim 15, wherein the influenza glycoprotein is an NA glycoprotein.

18. 16. The method of claim 15, wherein the first surfactant is NP-9 and the second surfactant is PS-80.

19. 20. The method of claim 18, wherein the PS80 in step (iii) is from about 0.03% to about 0.05%.

Images