Methods and compositions for treating respiratory diseases

Abstract

Disclosed herein are methods and nanoparticles suitable for use in reducing exacerbations in COPD patients.SOLUTION: The methods and compositions advantageously reduce the incidence of hospitalization of COPD patients that occurs in response to an environmental challenge, such as exposure to or infection by RSV. Also disclosed are dosages, formulations, and methods for preparing the vaccines and nanoparticles.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 536,235, filed July 24, 2017, the disclosure of which is incorporated herein for all purposes.

[0002] This application incorporates in its entirety for all purposes the disclosures of U.S. Patent Application No. 15 / 257,436, filed September 6, 2016, and U.S. Provisional Patent 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.

[0003] Description of electronically submitted text files The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety. A computer-readable copy of the Sequence Listing (Filename: NOVV_075_01WO_SeqList_ST25.txt, Date of Record: July 19, 2018, File Size: Approximately 67 KB).

[0004] [Technical field] The present disclosure generally relates to nanoparticles and immunogenic compositions containing them that are useful for treating respiratory diseases and preventing exacerbations associated with respiratory diseases. The nanoparticles provide antigens, such as glycoprotein antigens, bound to a surfactant core and are typically produced using recombinant approaches. The nanoparticles have reduced chronic obstructive pulmonary disease (COPD) exacerbations and associated hospitalizations, particularly in elderly populations. The present disclosure also provides compositions containing the nanoparticles, methods for producing them, and methods for treating respiratory diseases (e.g., COPD) and preventing further exacerbations. [Background technology]

[0005] Respiratory and infectious diseases remain a problem worldwide. While progress has been made in developing vaccines against some pathogens, many remain a threat to human health. The most notable is HIV, for which a vaccine remains elusive. Attempts to generate vaccines against specific pathogens have been unsuccessful, resulting in further pathology.

[0006] The World Health Organization estimates that 6% of all deaths worldwide, or more than 3 million deaths annually, are attributable to chronic obstructive pulmonary disease (COPD), one of the world's leading respiratory diseases. COPD exacerbations have a significant negative impact on patients and place a significant strain on healthcare resources. There is clear evidence of an irreversible decline in lung function after each exacerbation. Prevention and treatment of exacerbations are the primary goals of clinical management of COPD (see Decramer M, et al., (2008) "Targeting the COPD Exacerbation," J Respir Med 102:S3-S15, and Rabe KF, et al., (2007) "Global strategy for diagnosis, management, and prevention of chronic obstructive pulmonary disease: GOLD Executive Summary," Am J Respir Crit Care Med 176:532-555).

[0007] Among other treatment options, the American Lung Association recommends vaccinations for COPD patients, particularly annual influenza and pneumonia vaccinations. Thus, there is ongoing interest in producing therapeutic and preventative medications to reduce or eliminate exacerbations in the COPD population, thereby reducing the disease burden and associated costs. Summary of the Invention

[0008] The present disclosure provides nanoparticles suitable for treating respiratory diseases and disorders. In certain embodiments, the nanoparticles treat chronic obstructive pulmonary disorder (COPD). In other embodiments, the nanoparticles prevent COPD exacerbations in response to respiratory irritation, such as exposure to pathogens.

[0009] In some embodiments, the nanoparticles induce an immune response against a pathogen, hi some embodiments, the pathogen is a virus, and typically the antigen used to generate the viral nanoparticles is a viral glycoprotein.

[0010] In one aspect, the present disclosure provides nanoparticles comprising viral proteins with improved 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 exemplary 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 comprising a non-ionic surfactant, providing a nanoparticle structure that promotes immunogenicity and inhibits antigen degradation.

[0011] In some embodiments, the antigen is selected from the group consisting of a RSV F protein, an influenza HA protein, an influenza NA protein, and combinations thereof. Typically, the antigen is a glycoprotein.

[0012] 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 and / or pre-fusion states. In a further aspect, each PS80 particle may contain 4 to 7 RSV F proteins.

[0013] In some embodiments, the RSV F composition may contain sodium phosphate at a concentration of 15 mM to 25 mM, NaCl at a concentration of 125 mM to 175 mM, and histidine at a concentration of 0.25% to 2% w / v, and the pH of the composition is 5.8 to 7.2.

[0014] In some embodiments, an HA or NA influenza composition may comprise sodium phosphate at a concentration of 15 mM to 25 mM, NaCl at a concentration of 125 mM to 300 mM, and histidine at 0.25% to 2% w / v, and the pH of the composition is greater than about pH 6.8, and typically less than about pH 8.0.

[0015] In some embodiments, the composition comprises an adjuvant. In further embodiments, the adjuvant is an alum or saponin-based matrix adjuvant. In some embodiments, the composition does not comprise an adjuvant.

[0016] In some embodiments, the method for preventing infection comprises administering one or more doses of a vaccine composition. In some embodiments of the method, a single dose of the composition is administered to induce a protective immune response. In some embodiments of the method, each dose comprises about 100 μg to about 150 μg of protein antigen. In further embodiments of the method, the one or more doses are administered subcutaneously. In some embodiments of the method, the composition comprises an adjuvant. In further embodiments of the method, the adjuvant is alum. In some embodiments of the method, the composition does not comprise an adjuvant.

[0017] In further 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 over 75 years old, over 70 years old, over 65 years old, over 60 years old, over 55 years old, over 50 years old, or over 45 years old. Thus, in certain aspects, the adult can be about 45 to about 75 years old.

[0018] In some embodiments of the RSV vaccine, 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, wherein the first RSV F nanoparticle type comprises anisotropic rods, the second RSV F nanoparticle type comprises spherical oligomers, and the third RSV F nanoparticle type comprises an intermediate between the anisotropic rods and the spherical oligomers.

[0019] In some embodiments, the 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, where the second surfactant is PS80, thereby improving the stability of the nanoparticles. In a further embodiment of the method, the first surfactant is NP-9. In some embodiments of the method, the improved 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.

[0020] In some embodiments, the RSV F nanoparticles comprise one or more RSV F protein trimers bound to 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 RSV F nanoparticles, each RSV F protein trimer comprises 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 comprises 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 primary fusion cleavage site.

[0021] In some embodiments of the RSV F nanoparticles, the RSV F protein comprises a deletion of 10 amino acids corresponding to residues 137-166 of SEQ ID NO: 2, and inactivation of the primary furin cleavage site by mutation of arginine residues at positions 133, 135, and 136 to glutamine. In further embodiments of the RSV F nanoparticles, the RSV F protein comprises or consists of 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. Vaccine formulations comprising RSV F nanoparticles consist essentially of mature peptides, including several full-length peptides (SEQ ID NO: 8). Over time, proteolysis may result in small amounts of cleaved RSV F peptides. However, advantageously, the RSV F nanoparticles disclosed herein minimize such degradation and provide long-term stability.

[0022] Also provided are nanoparticles comprising the RSV F protein in combination with influenza proteins HA, NA, or both. [Brief explanation of the drawings]

[0023] [Figure 1] The amino acid sequence of the modified RSV F protein (SEQ ID NO: 19) is shown, including the F1 domain in light text (residues 1-84), the F2 domain in dark text (residues 85-539), black lines connecting cysteines that form disulfide bonds, underlined asparagines indicating N-linked glycosylation sites, light vertical dotted lines indicating furin cleavage sites, and black vertical dotted lines indicating the major cleavage site. [Figure 2] Electron micrographs of RSV F nanoparticles with RSV F protein trimers bound to the core of PS80 show multiple RSV F trimers bound to each particle (40-50 nm in diameter). [Figure 3] 1 shows an analysis of hospitalizations for all-cause (not related to RSV detection) COPD exacerbations in the E-301 and E201 studies. [Figure 4]1 is a graph detailing the decline in the COPD exacerbation-free hospitalization population over time in the E-301 study. DETAILED DESCRIPTION OF THE INVENTION

[0024] Disclosed herein are nanoparticles for treating respiratory diseases and disorders, methods for producing and administering them, and vaccine compositions containing them. The nanoparticles surround a surfactant core and provide bound antigens, resulting in a structure that provides enhanced stability by multiple means. Without being bound by theory, the immune response and associated protection induced by the disclosed nanoparticles results in reduced exacerbations of respiratory diseases or disorders (e.g., COPD). The surfactant core and antigen bind through physicochemical interactions mediated by the properties of the antigen and surfactant. Furthermore, the nanoparticles provide particularly excellent antigen presentation to the immune system, which is believed, without being bound by theory, to be due to the orientation of the antigen around the surfactant core.

[0025] In one aspect, the present disclosure provides a composition comprising a recombinant viral glycoprotein nanoparticle. In a particular 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.

[0026] In certain embodiments, the present disclosure provides immunogenic compositions comprising one or more viral glycoprotein species in a nanoparticle structure, wherein the glycoproteins are in the form of trimers and each nanoparticle comprises at least one trimer bound to a non-ionic surfactant. In certain embodiments, the nanoparticles consist of antigens, such as viral glycoproteins, from only one pathogen.

[0027] The nanoparticles can be used to treat respiratory diseases or disorders. In some embodiments, the nanoparticles are used to treat COPD. In some embodiments, the nanoparticles reduce the incidence of COPD exacerbations in response to respiratory stimuli, such as pathogens or other environmental COPD triggers.

[0028] The nanoparticles can be used to prevent and / or treat 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 comprising nanoparticles.

[0029] The present disclosure provides vaccine compositions comprising nanoparticles. The compositions may include nanoparticles having antigens from multiple pathogens. In some aspects, the vaccine compositions may include nanoparticles having antigens from more than one viral strain from the same species of virus. In some aspects, the vaccine compositions may include nanoparticles containing antigens from different viral species. In another embodiment, the present disclosure provides pharmaceutical packs or kits including one or more containers filled with one or more of the components of the vaccine composition.

[0030] In another embodiment, the present disclosure provides a method for formulating a vaccine composition for inducing immunity to an infection or at least one disease symptom thereof in a mammal, comprising adding an effective amount of nanoparticles to the composition. The disclosed nanoparticles are useful in preparing compositions that stimulate an immune response that confers immunity or substantial immunity to an infectious agent. Thus, in one embodiment, the present disclosure provides a method for inducing immunity to an infection or at least one disease symptom thereof in a subject, comprising administering at least one effective dose of nanoparticles.

[0031] 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 the adjuvant and the nanoparticles do not substantially interact.

[0032] Also provided herein are methods for producing nanoparticles and vaccine compositions, which advantageously provide nanoparticles that are substantially free of contamination with other proteins, such as proteins associated with recombinant expression of proteins in a baculovirus / Sf9 system.

[0033] definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a protein" may refer to one protein or a mixture of such proteins.

[0034] As used herein, the term "adjuvant" refers to an agent that, when used in combination with an immunogen, enhances 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. Vaccine compositions may include an effective amount of one or more adjuvants.

[0035] As used herein, the term "about" or "approximately," when preceding a numerical value, indicates a range of plus or minus 10% of the value. For example, "about 100" includes 90 and 110.

[0036] As used herein, the terms "immunogen," "antigen," and "epitope" refer to substances such as proteins, including glycoproteins, and peptides that are capable of eliciting an immune response.

[0037] As used herein, an "immunogenic composition" is a composition comprising an antigen such that 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.

[0038] As used herein, a "subunit" composition, e.g., a vaccine, contains one or more selected antigens from a pathogen, rather than all antigens. Such compositions are substantially free of intact virus or lysates of such cells or particles, and are typically prepared from at least partially purified, and often substantially purified, immunogenic polypeptides from the pathogen. The antigens in the subunit compositions disclosed herein are typically prepared recombinantly, often using a baculovirus system.

[0039] As used herein, "substantially" refers to a substance (e.g., a compound, polynucleotide, or polypeptide) that forms the majority of the sample in which it is contained, or a process step that is nearly complete. For example, in a sample, a substantially purified component constitutes 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 is substantially exchanged, the amount remaining in the sample is about 0.5% to about 10% or less, preferably about 0.5% to about 1.0%.

[0040] As used herein, the terms "treat," "treatment," and "treating" refer to an approach for obtaining beneficial or desired results, e.g., clinical results. For purposes of this disclosure, a beneficial or desired result may include inhibiting or suppressing the onset or progression of an infection or disease, ameliorating symptoms of an infection or disease, or reducing its occurrence, or a combination thereof.

[0041] 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 an infection or disease or its symptoms that subsequently develops.

[0042] As used herein, "effective dose" or "effective amount" refers to an amount of an immunogen sufficient to induce an immune response that alleviates at least one symptom of a pathogen infection. An effective dose or amount can be determined, for example, by measuring the amount of neutralizing secretory and / or serum antibodies, for example, by plaque neutralization, complement fixation, enzyme-linked immunosorbent (ELISA), or microneutralization assays.

[0043] As used herein, the term "vaccine" refers to an immunogenic composition, such as an immunogen derived from a pathogen, used to induce an immune response against a pathogen that provides protective immunity (e.g., immunity that protects a subject from infection of the subject by the pathogen and / or reduces the severity of a disease or condition caused by infection with the pathogen). A protective immune response may include the formation of antibodies and / or a cell-mediated response. Depending on the context, the term "vaccine" may refer to a suspension or solution of immunogens administered to a subject to generate protective immunity.

[0044] As used herein, the term "subject" includes humans and other animals. Typically, the subject is a human. For example, the subject can be an adult, teenager, child (2-14 years old), infant (1-24 months old), or newborn (up to 1 month old). In some embodiments, an adult is about 65 years old or older, or an elderly person about 60 years old or older. In some embodiments, the subject is a pregnant woman or a woman who is about to become pregnant. In other embodiments, the subject is not a human, for example, a non-human primate, such as a baboon, chimpanzee, gorilla, or macaque. In certain embodiments, the subject can be a pet, such as a dog or cat.

[0045] 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 United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias for use in mammals, especially humans. Pharmaceutically acceptable carriers or excipients may be included in the compositions disclosed herein.

[0046] As used herein, "respiratory disease" or "respiratory disorder" refers to a disease or disorder of the airways and / or other structures of the lungs, including, but not limited to, chronic obstructive pulmonary disease (COPD), asthma, allergic rhinitis and sinusitis, bronchiectasis, and pulmonary hypertension. COPD refers to a group of diseases that share symptoms including airway obstruction and one or more of the breathing-related problems, including emphysema, chronic bronchitis, and in some cases asthma (see www.cdc.gov / tobacco / campaign / tips / diseases / copd.html).

[0047] As used herein, the term "about" means plus or minus 10% of the indicated numerical value.

[0048] overview COPD symptoms typically appear when a subject already has significant lung damage, and symptoms often worsen over time. COPD is a common consequence of subjects suffering from emphysema or chronic bronchitis. In chronic bronchitis, the primary symptoms are daily coughing and mucus (phlegm) production for at least three months per year for two consecutive years. Emphysema is a condition in which the alveoli at the end of the smallest airways (bronchioles) in the lungs are destroyed, typically by smoking. Other symptoms of COPD may include shortness of breath, especially during physical activity, wheezing, chest tightness, a chronic cough that often produces mucus, and blue lips or nail beds (cyanosis).

[0049] Subjects with COPD are also likely to experience episodes called exacerbations, during which these symptoms worsen beyond normal daily fluctuations and persist for at least several days. Exacerbations typically occur in response to environmental stimuli encountered by the subject. A variety of biological and non-biological environmental stimuli, including passive smoking, gasoline fumes, bacterial, and viral infections, can trigger exacerbations in COPD patients.

[0050] The methods and compositions used herein can be used to reduce or eliminate the severity of an exacerbation or one or more symptoms associated with an exacerbation. The immunogenic compositions of the present disclosure comprise one or more antigens derived from a pathogen in nanoparticle form.

[0051] Antigens derived from pathogens are combined with non-ionic surfactants to provide nanoparticles with improved stability, excellent immunogenicity, and surrounding surfactant cores that treat respiratory diseases and disorders. The present disclosure also provides methods and compositions for vaccinating subjects against pathogens to treat respiratory diseases and disorders. The antigens are typically viral proteins, often glycoproteins. Compositions containing nanoparticles that find use as vaccine compositions for treating respiratory diseases and disorders are also disclosed.

[0052] In certain embodiments, the composition comprises nanoparticles that induce an immune response against RSV alone. In other embodiments, the composition comprises nanoparticles against RSV that comprise glycoproteins from RSV, such as an RSV-F protein and one or more influenza HA glycoprotein strains (e.g., nanoparticles comprising HA glycoproteins from one, two, three, or four different influenza strains).

[0053] In addition to the nanoparticles, the subject may be administered an additional immunogenic composition, for example, a composition that induces an immune response against one or more of Streptococcus pneumoniae, Bordetella pertussis, Haemophilus influenzae type b (Hib), and measles.

[0054] A notable result of the immunogenic compositions disclosed herein is that administration of nanoparticle compositions comprising the RSV F glycoprotein reduces hospitalizations for "all-cause" COPD exacerbations, meaning that the response to the composition not only reduces RSV-stimulated exacerbations but also has a more general benefit, reducing all-cause exacerbations. See, e.g., Figures 4 and 5. Thus, compositions comprising nanoparticles are useful for administering to subjects with COPD to prevent exacerbations in response to environmental stimuli.

[0055] Nanoparticle structure and morphology The nanoparticles of the present disclosure comprise glycoprotein antigens bound to a non-ionic surfactant core. Figure 2 illustrates an example of multiple RSV F antigens bound to a surfactant core.

[0056] 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 2-9 trimers. In certain embodiments, each nanoparticle contains 2-6 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 2-9, and in other embodiments, the nanoparticles in the composition may contain 2-6 trimers. In certain embodiments, the composition comprises a heterogeneous population of nanoparticles with 2-6 trimers per nanoparticle or 2-9 trimers per nanoparticle. In other embodiments, the composition may comprise a substantially homogeneous population of nanoparticles. For example, the population may comprise approximately 95% of nanoparticles with 5 trimers.

[0057] The antigen is bound to the nonionic 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 comprise an antigen assembled into a multi-oligomeric glycoprotein-PS80 protein-surfactant nanoparticle, with a head region protruding outward and a hydrophobic region and PS80 surfactant forming a central core surrounded by the antigen.

[0058] The nanoparticles disclosed herein have a Z-ave size range of about 20 nm to about 60 nm, about 20 nm to about 50 nm, about 20 nm to about 45 nm, or about 25 nm to about 45 nm. Particle size (Z-ave) is measured by dynamic light scattering (DLS) using a Malvern Zetasizer unless otherwise specified.

[0059] The vaccine compositions disclosed herein may contain several nanoparticle types. In some embodiments, the nanoparticle types are in the form of anisotropic rods and can be dimeric or monomeric. In other embodiments, the nanoparticle types are spherical oligomers. In still other embodiments, the nanoparticles can be described as intermediate nanoparticles with sedimentation properties intermediate between the first two types. The formation of nanoparticle types can be tuned by controlling the surfactant and protein concentrations during the production process. The nanoparticle type can be determined by measuring the sedimentation coefficient.

[0060] Nanoparticle generation 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 surfactant exchange approach, in which a first surfactant is used to isolate proteins, and then the first surfactant is exchanged with a second surfactant to form nanoparticles.

[0061] 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 a 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 of both cathepsin-L and chitinase. High levels of expression may be obtained in insect cell expression systems. Non-limiting examples of insect cells include Spodoptera frugiperda (Sf) cells, such as Sf9 and Sf21, Trichoplusia ni cells, such as High Five cells (also known as BTI-TN-5B1-4), and Drosophila S2 cells.

[0062] For cell culture, general transfection and cell growth methods can be used.Vector, for example, vector containing polynucleotide encoding fusion protein, can be transfected into host cell according to the method well known in the art.For example, introduction of nucleic acid into eukaryotic cell can be achieved by calcium phosphate coprecipitation, electroporation, microinjection, lipofection and transfection using polyamine transfection reagent.In one embodiment, the vector is recombinant baculovirus.

[0063] 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 are grown and express proteins (e.g., recombinant proteins) for purification and isolation. Typically, cell culture is carried out in a bioreactor under sterile, controlled temperature and atmospheric conditions. A bioreactor is a chamber used for culturing cells 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 sterilized plastic bag (e.g., Cellbag®, Wave Biotech, Bridgewater, NJ). In another embodiment, the sterilized plastic bag is a bag of approximately 50 L to 3500 L.

[0064] Surfactant extraction and purification of nanoparticles After host cell growth, proteins can be recovered from the host cells using detergents and purification protocols. After the host cells have grown for 48 to 96 hours, the cells are isolated from the medium and a detergent-containing solution is added to solubilize the cell membrane and release the protein in a detergent extract. Triton X-100 and tergitol, also known as NP-9, are suitable detergents for extraction. The detergent may be added to a final concentration of about 0.1% to about 1.0%. For example, concentrations 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%.

[0065] 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, Brij® 72, Brij® 76, Brij® 92V, Brij® 97, Brij® 58P, Cremophor® EL, decaethylene glycol monododecyl ether, N-decanoyl-N-methylglucan, or the like. Mine, 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, n-dodecyl beta-D-maltoside, heptaethylene glycol monodecyl ether, heptaethylene glycol monododecyl ether, heptaethylene glycol monotetradecyl ether, n-hexadecyl beta-D-maltoside, hexaethylene glycol monododecyl ether, hexaethylene Glycol monohexadecyl ether, hexaethylene glycol monooctadecyl ether, hexaethylene glycol monotetradecyl ether, Igepal CA-630, Igepal CA-630, methyl-6-0-(N-heptylcarbamoyl)-alpha-D-glucopyranoside, nonaethylene glycol monododecyl ether, N-nonanoyl-N-methylglucamine, N-nonanoyl N-methylglucamine, octaethylene glycol monodecyl ether, octaethylene glycol monododecyl ether, octaethylene glycol ethylene 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 monohexadecyl ether, pentaethylene glycol monohexadecyl ether, pentaethylene glycol monooctadecyl ether,Pentaethylene glycol monooctyl ether, polyethylene glycol diglycidyl ether, polyethylene glycol ether W-1, polyoxyethylene 10 tridecyl ether, polyoxyethylene 100 stearate, polyoxyethylene 20 isohexadecyl ether, polyoxyethylene 20 oleyl ether, polyoxyethylene 40 stearate, polyoxyethylene 50 stearate, polyoxyethylene 8 stearate, polyoxyethylene bis(imidazolylcarbonyl), polyoxyethylene 25 propylene glycol stearate, Quillaja bark saponin, Span® 20, Span® 40, Span® 60, Span® 65, Span® 80, Span® 85, Tergitol Type 15-S-12, Tergitol Type 15-S-30, Tergitol Type 15-S-5, Tergitol Type 15-S-7, Tergitol Type 15-S-9, Tergitol Type NP-10, Tergitol Type NP-4, Tergitol Type NP-40, Tergitol Type NP-7, Tergitol Type NP-9, Tergitol Type TMN-10, Tergitol Type TMN-6, Triton X-100, or a combination thereof.

[0066] The nanoparticles can then be isolated from the cell debris using centrifugation. In some embodiments, gradient centrifugation, such as using cesium chloride, sucrose, and iodixanol, can be used. Other techniques, such as standard purification techniques including ion exchange, affinity, and gel filtration chromatography, can alternatively or additionally be used.

[0067] 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 are often used to remove detergents during purification, but can adversely affect the methods disclosed herein.

[0068] Surfactant Exchange To form nanoparticles, the first surfactant used to extract proteins from host cells is essentially replaced with a second surfactant to achieve the nanoparticle structure. NP-9 is the 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 specific range.

[0069] In certain embodiments, detergent exchange is performed using affinity chromatography, which binds glycoproteins via sugar moieties. For example, affinity chromatography may use a legume lectin column. Legume lectins were the first proteins identified in plants and have been shown to interact specifically and reversibly with sugar residues. See, for example, Sharon and Lis, "Legume lectins—a large family of homologous proteins," FASEBJ. 1990 November;4(14):3198-208; Liener, "The Lectins: Properties, Functions, and Applications in Biology and Medicine," Elsevier, 2012. Suitable lectins include concanavalin A (con A), pea lectin, sainfoin lectin, and lentil lectin. Lentil lectin is a suitable column for detergent exchange due to its binding properties. See, for example, Example 10. Lectin columns are commercially available, e.g., Capto Lentil Lectin is available from GE Healthcare. In certain embodiments, lentil lectin columns may use recombinant lectins. At the molecular level, it is believed that sugar moieties bind to lentil lectins, liberating amino acids of the protein to coalesce around the surfactant, resulting in the formation of a surfactant core, providing nanoparticles bearing multiple copies of the antigen, e.g., glycoprotein oligomers that may be dimers, trimers, or tetramers immobilized on the surfactant.

[0070] Detergents may be present at up to about 0.1% (w / v) during the initial purification step when incubated with proteins to form nanoparticles during detergent exchange; this amount can be lowered to achieve final nanoparticles with optimal stability. For example, nonionic surfactants (e.g., PS80) may be present at about 0.03% to about 0.1%. Preferably, to improve stability, nanoparticles contain about 0.03% to about 0.05% PS80. Amounts of PS80 less than about 0.03% in the formulation do not exhibit good stability. Furthermore, when PS80 is present at more than about 0.05%, aggregates form. Therefore, about 0.03% to about 0.05% PS80 provides structural and stability advantages that allow for long-term stability of nanoparticles with reduced degradation.

[0071] Detergent exchange can be performed using proteins purified as described above, purified, frozen for storage, and then thawed for detergent exchange.

[0072] Enhanced stability and enhanced immunogenicity of nanoparticles Without being bound by theory, it is believed that conjugating an antigen to a non-ionic surfactant core provides superior stability and antigen presentation. The nanoparticles disclosed herein provide surprisingly good stability and immunogenicity.

[0073] The location of the glycoprotein anchored in the detergent core is believed to enhance stability by reducing undesired interactions. For example, improved protection against protease-based degradation can be achieved by anchoring the glycoprotein in the core at the molar ratios disclosed herein through a shielding effect whereby steric hindrance blocks protease access.

[0074] Nanoparticle antigens In typical embodiments, the antigen used to generate 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 to facilitate binding of the protein to the surfactant core. In many cases, the protein is necessarily a glycoprotein.

[0075] 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. Suitable RSV F proteins for use in the compositions described herein can 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 properties, such as improved protein expression and enhanced immunogenicity. Additional information describing the structure of the RSV-F protein can be found in Swanson et al. A Monomeric Uncleaved Respiratory Syncytial Virus F Antigen Retains Prefusion-Specific Neutralizing Epitopes Journal of Virology, 2014, 88, 11802-11810. Jason S. McLellan et al. Structure of RSV Fusion Glycoprotein Trimer Bound to a Prefusion-Specific Neutralizing Antibody. Science, 2013, 340, 1113-1117.

[0076] The primary fusion cleavage site is located at residues 131-136, corresponding to SEQ ID NO: 2. Inactivation of the primary fusion cleavage site is achieved by mutating residues within the site, resulting in furin no longer recognizing the consensus site. For example, inactivation of the primary 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).

[0077] In certain embodiments, 1 to 10 amino acids corresponding to amino acids 137-145 of SEQ ID NO:2 can be deleted, including specific examples of suitable RSV F proteins shown below. Each of SEQ ID NOs:3-13 can optionally be prepared with an active primary fusion cleavage site KKRKRR (SEQ ID NO:18). The wild-type strain of SEQ ID NO:2 has sequence 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 can be isolated from the host cell and then the signal peptide cleaved. The N-terminal RSV F signal peptide consists of the amino acids of SEQ ID NO:20 (MELLILKANAITTILTAVTFCFASG). 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 a RSV F nanoparticle vaccine. See FIG. 1. Optionally, one or more to all 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]

[0078] In some embodiments, the RSV F protein disclosed herein is altered from the wild-type strain only by deletion of the fusion domain, optionally with inactivation of the primary cleavage site. In other embodiments, additional alterations can be made to the RSV F protein. Typically, cysteine ​​residues are mutated. Typically, the N-linked glycosylation site is not mutated. See Figure 1. Additionally, antigen site II, also referred to herein as the palivizumab site, is conserved due to the ability of the palivizumab antibody to bind to that site. The motavizumab antibody also binds to site II. Additional suitable RSV-F proteins can be found in U.S. Patent Publication No. 2011 / 0305727, which is incorporated by reference, including RSV-F proteins comprising sequences spanning residues 100-150 as disclosed in Figure 1C.

[0079] In certain other embodiments, the RSV F1 or F2 domain may be modified compared to the wild-type strain, as 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 changes, which may be mutations or deletions. Similarly, the F2 domain may have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 changes, 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.

[0080] In certain examples, the RSV nanoparticle formulation may comprise about 0.025% to about 0.03% PS80 with RSV F ranging from about 270 μg / mL to about 300 μg / mL, or from about 60 μg / mL to about 300 μg / mL. In other embodiments, the nanoparticle formulation may comprise about 0.035% to about 0.04% PS80 in a composition comprising 300 μg / mL to about 500 μg / mL of RSV F. In yet other embodiments, the nanoparticle formulation may comprise about 0.035% to about 0.04% PS80 in a composition comprising 350-500 μg / mL of RSV F.

[0081] Because the concentrations of antigen and surfactant may vary, the respective amounts are sometimes referred to as the molar ratio of nonionic surfactant to protein. For example, the molar ratio of PS80 to protein is calculated using the PS80 concentration, the protein concentration of the antigen measured by ELISA / A280, and their respective molecular weights. The molecular weight of PS80 used in the calculation is 1310, and for RSV F, the molecular weight of RSV F is 65 kD. The molar ratio is calculated as follows: (PS80 concentration × 10 × 65,000) ÷ (1310 × RSV F concentration in mg / mL). Therefore, for example, if the nanoparticle concentration measured by protein is 270 μg / mL and the PS80 concentrations are 0.015% and 0.03%, respectively, the PS80:RSV F protein molar ratios are 27:1 (i.e., 0.015 × 10 × 65,000 / (1310 × 0.27)) and 55:1, respectively.

[0082] In certain embodiments, the molar ratio is within the range of 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, an alternative nonionic surfactant is PS80, with a molar ratio of PS80:protein of about 30:1 to about 50:1. In the case of RSV-F glycoprotein, nanoparticles having a molar ratio ranging from 35:1 to about 65:1, particularly a ratio of about 45:1, are particularly stable.

[0083] Modified antigen The antigens disclosed herein encompass variations and variants of these antigens. In certain embodiments, an antigen may share identity with a disclosed antigen. Generally, and unless otherwise specified for a particular antigen, the percentage of identity may be at least 80%, at least 90%, at least 95%, at least 97%, or at least 98%. The percentage of identity can be calculated using the alignment program ClustalW2, available at www.ebi.ac.uk / Tools / msa / clustalw2 / . The following default parameters can be used for pairwise alignment: Protein Weight Matrix=Gonnet; Gap Open=10; Gap Extension=0.1.

[0084] 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. Additions to the protein itself can be made 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 comprises an epitope. For example, the tag can be a polyglutamate 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 during expression in cells, some nanoparticles may contain an antigen with an intact signal peptide. Thus, when a nanoparticle contains an antigen, the antigen may include an extension and, therefore, when incorporated into the nanoparticle, may form a fusion protein. For the purpose of calculating sequence identity, the extensions are not included.

[0085] 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. Instead of or in addition to the N-terminus, the C-terminus may be truncated. 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. To calculate the identity of the truncated protein, the identity is measured relative to the remainder of the protein.

[0086] 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 different pathogen species. To prepare a combination nanoparticle, glycoproteins from multiple pathogens can be combined into a single nanoparticle by conjugating them in a detergent exchange step. Binding of the glycoproteins to a column followed by detergent exchange results in the formation of multiple glycoprotein types around a surfactant core, providing a combination nanoparticle.

[0087] 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 include 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.

[0088] Such compositions are particularly desirable when the pathogens are related in some way. In one example, the composition may contain nanoparticles directed against the strains identified by authorities each year as forming the seasonal influenza of a particular year. Typically, in the case of seasonal influenza vaccines, the vaccine composition includes RSV antigens combined with HA and / or NA nanoparticles that induce an immune response against three, four, or five influenza subtype strains. Thus, different influenza strains can be combined in a vaccine composition. In some embodiments, the combined nanoparticles may contain an HA protein from a first strain and an NA protein from a second strain. In other embodiments, the nanoparticles may contain one or more HA and one or more NA proteins from the same or different subtypes. For example, the nanoparticles may comprise 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 includes H1, H2, H5, H6, H8, H9, H11, H12, H13, and H16, and group 2 includes H3, H4, H7, H10, H14, and H15. NA proteins also form two groups: group 1 includes N1, N4, N5, and N8, and group 2 includes 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 NA protein.

[0089] In another example, as described above, because both influenza and RSV cause respiratory disease exacerbations, HA, NA, and / or RSV F may be combined into a nanoparticle or multiple nanoparticles may be combined into an immunogenic composition to induce an immune response against RSV and one or more influenza strains.

[0090] Vaccine Composition The compositions disclosed herein can be used prophylactically or therapeutically, but are typically prophylactic. Accordingly, the present disclosure includes a method for treating or preventing infection. The method comprises 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 aspects, 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 alleviate one or more viral disease symptoms selected from heat exhaustion, muscle pain, headache, sore throat, vomiting, diarrhea, rash, symptoms of kidney and liver dysfunction, internal bleeding, and external bleeding, compared to untreated subjects.

[0091] The nanoparticles may be formulated for administration as a vaccine in the presence of various excipients, buffers, etc. For example, a 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, with about 22 mM sodium phosphate being present in certain cases. 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). Sodium chloride, when present, may be about 150 mM. In certain compositions, such as influenza vaccines, sodium chloride may be present in higher amounts, including about 200 mM, about 300 mM, or about 350 mM.

[0092] Certain nanoparticles, particularly RSV F nanoparticles, have improved stability at slightly acidic pH levels. For example, the pH range of a composition containing nanoparticles can 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, RSV F protein nanoparticle compositions have a pH of about 6.2. Other nanoparticles tend to be more neutral; for example, influenza nanoparticles can have a pH of about 7.0 to about pH 7.4, often about pH 7.2.

[0093] 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 over the age of about 60.

[0094] Aluminum-based adjuvants In some embodiments, the adjuvant may be alum (e.g., AlPO4 or Al(OH)3). Typically, the nanoparticles are substantially bound to the alum. For example, the nanoparticles may 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 per dose typically ranges from about 400 μg to about 1250 μg. For example, alum may be present in an amount of about 300 μg to about 900 μg, about 400 μg to about 800 μg, about 500 μg to about 700 μg, about 400 μg to about 600 μg, or about 400 μg to about 500 μg per dose. Typically, alum is present at about 400 μg for a 120 μg dose of protein nanoparticles.

[0095] Matrix adjuvants The matrix particles of the present disclosure are formed using only one saponin fraction. Several approaches for generating saponin fractions and forming matrix particles 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 of Quil A, a crude aqueous extract of Quilja saponaria Molina, is separated by chromatography and eluted with 70% aqueous acetonitrile to recover the lipophilic fraction. This lipophilic fraction is then separated by semi-preparative HPLC using a gradient of 25% to 60% acetonitrile in acidic water. The fraction referred to herein as "Fraction A" or "QH-A" is the fraction eluted at approximately 39% acetonitrile. 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. Further information regarding fraction purification can be found in U.S. Pat. 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 are obtained are such that batch-to-batch reproducibility is highly consistent with respect to elution profile and biological activity.

[0096] Other usable saponin fractions have been described. Fractions B3, B4, and B4b are described in EP 0436620. Fractions QA1 to QA22 are described in EP 03632279B2. Q-VAC (Nor-Feed, AS Denmark), Quillaja saponaria Molina Spikoside (Isconova AB, Ultunaallen 2B, 756 51 Uppsala, Sweden) are also described. 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 0 3632 279 B2 may be used, particularly QA-7, QA-17, QA-18, and QA-21, which are obtained as described in Example 1 of EP 0 3632 279 B2, particularly pages 6, 8, and 9. In an embodiment, the saponin fraction from Quillaja saponaria Molina is selected from any one of QA1 to 21.

[0097] Other saponin fractions, such as 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. These fractions can be used to produce matrices for use in the methods and compositions disclosed herein.

[0098] In some embodiments, the composition comprises matrix particles having only one saponin fraction, while in other embodiments, the composition may comprise multiple types of matrix particles, each having one saponin fraction for each particle type, but with different fractions.

[0099] Matrix particles having one saponin fraction each may be present in the composition in any combination of weight percents. In certain embodiments, the composition may contain 0.1% to 99.9%, 5% to 95%, 10% to 90%, 15% to 85%, 20% to 80%, 25% to 75%, 30% to 70%, 35% to 65%, 40% to 60%, 45% to 55%, 40% to 60%, or 50% by weight of matrix particles containing a first saponin fraction, with the remainder made up of matrix particles containing a different saponin fraction.

[0100] The amount of each matrix in the composition can vary as a percentage of the total composition. For example, the amount of Fraction A matrix can be about 80% (w / w), about 85% (w / w), about 90% (w / w), about 92% (w / w), or about 95% (w / w), with the remainder being Fraction C matrix. Typically, Fraction A matrix ranges from about 80% (w / w) to about 95% (w / w), with the remainder of the composition being Fraction C matrix. Usually, about 85% (w / w) of Fraction A matrix and the remainder Fraction C matrix (i.e., 85:15) are used. A specific example of an 85:15 combination of Fraction A matrix and Fraction C matrix is ​​Matrix-M™ (Novavax AB, Uppsala, Sweden), which is a mixture of Fraction A matrix and Fraction C matrix in a ratio of about 85:15.

[0101] Other adjuvants In some compositions, other adjuvants may be used in addition or instead. 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 Freund's complete adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), Freund's incomplete 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, a three-component compound 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, such as Novasomes®. Novasomes® are paucilamellar non-phospholipid vesicles ranging from about 100 nm to about 500 nm in size. They contain Brij 72, cholesterol, oleic acid, and squalene. Novasomes have been shown to be effective adjuvants (see US Pat. Nos. 5,629,021, 6,387,373, and 4,911,928).

[0102] Administration and Dosage The compositions disclosed herein can be administered via systemic, mucosal, or transdermal routes, or directly to specific tissues.As used herein, the term "systemic administration" includes parenteral administration.In particular, parenteral administration includes subcutaneous, intraperitoneal, intravenous, intraarterial, intramuscular, or intrasternal injection, intravenous, or kidney dialysis infusion techniques.Typically, systemic parenteral administration is intramuscular injection.As used herein, the term "mucosal administration" includes oral, intranasal, intravaginal, intrarectal, intratracheal, intestinal, and intraocular administration.Preferably, administration is intramuscular.

[0103] The compositions can be administered in a single-dose or multiple-dose schedule. Multiple doses can be used in a primary immunization schedule or a booster schedule. In a multiple-dose schedule, various doses can be administered by the same or different routes, such as parenteral prime and mucosal boost, mucosal prime and parenteral boost, etc. In some embodiments, subsequent booster doses are administered about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or about 6 weeks after the previous administration. However, typically, the compositions disclosed herein are administered only once and still provide a protective immune response.

[0104] In some embodiments, the dose measured in μg can 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.

[0105] Antigen doses, including those for pediatric administration, can range from about 30 μg to about 300 μg, about 90 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 administered with alum. Certain populations may be administered with or without an adjuvant. For example, when administered to elderly individuals, the absence of alum is preferred. In certain aspects, the composition may not include an added adjuvant. In such situations, the dose may be increased by about 10% or about 20%.

[0106] 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.

[0107] In certain embodiments of the RSV vaccine, the dose may contain a RSV F protein concentration of about 175 μg / mL to about 325 μg / mL, about 200 μg / mL to about 300 μg / mL, about 220 μg / mL to about 280 μg / mL, or about 240 μg / mL to about 260 μg / mL.

[0108] Incorporation by Reference All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entirety for all purposes. [Example]

[0109] 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 picked 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. Approximately 30 mL of crude cell harvest was clarified by centrifugation at approximately 800 x g for 15 minutes. The resulting crude cell harvest containing the RSV F protein was purified as described below.

[0110] The nonionic detergent Tergitol® NP-9 (nonylphenol ethoxylate) was used in the membrane protein extraction protocol. The NP-9 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, resulting in precipitation of BV and Sf9 host cell DNA and proteins. The neutralized low pH-treated lysate was clarified and further purified by anion exchange and affinity chromatography before a second low pH treatment was performed.

[0111] Affinity chromatography was used to remove Sf9 / BV proteins, DNA, and NP-9 and enrich for 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). Glycosylated RSV F protein selectively binds to the resin, while nonglycosylated proteins and DNA are removed in the column flow-through. Weakly bound glycoproteins were removed with a buffer containing high salt and low molar concentrations of methyl α-D-mannopyranoside (MMP).

[0112] Additionally, column washes were used to exchange the NP-9 detergent with the detergent polysorbate 80 (PS80). To perform the detergent exchange, the RSV F glycoprotein was bound to a lentil lectin column, followed by incubation 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 trimers assembled into micellar nanoparticles composed of PS80 and RSV F protein trimers contained in the detergent core. Following detergent exchange, a low-pH inactivation step was performed, followed by incubation on a sulfate column in the presence of a buffer containing 0.1% PS80.

[0113] The eluted material was diluted with a solution containing sufficient PS80 to provide a bulk storage drug substance (DS) with a PS80:RSV F protein molar ratio of approximately 50. The appropriate composition of the DS was achieved by combining RSV F nanoparticles with a solution containing 22 mM sodium phosphate, 0.03% PS80, and phosphate buffer at pH 6.2. During the surfactant exchange and at each subsequent step, the PS80-to-antigen ratio in the composition was maintained at a molar ratio of 35-60. 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 kDa.

[0114] Example 2 Preparation of vaccine compositions To provide nanoparticles for the administered vaccine formulation, the drug substance was diluted into the formulation at a PS80:RSV protein molar ratio of approximately 50. The drug substance was thawed, diluted, and filled into glass vials or prefilled syringes, then stored at 2-8°C prior to administration. The nanoparticles were bound to alum adjuvant. Alum adjuvant was added and mixed so that approximately 95% of the nanoparticles were bound to the alum, representing approximately 0.4 mg of RSV F nanoparticles per 120 μg dose in a 0.5 mL volume.

[0115] Example 3 RSV F in nanoparticles reduces COPD exacerbation hospitalizations In a randomized, placebo-controlled study of 11,856 subjects aged 60 years and older, a vaccine containing 135 μg RSV F nanoparticles without alum reduced all-cause COPD exacerbation hospitalizations (referred to as "E-301" in Figures 3 and 4). Subjects were followed for one year to confirm vaccine safety, immunogenicity, and efficacy. Surprisingly, administration of the RSV F nanoparticle vaccine reduced COPD exacerbation hospitalizations in both the general study population (p=0.017) and the subpopulation of subjects previously identified as having baseline COPD (p=0.14).

[0116] In a smaller, comparable study of 1,600 subjects aged 60 years and older, the group receiving the RSV F nanoparticle vaccine experienced no COPD exacerbation hospitalizations (referred to as "E-201" in Figure 3). The data showed that in the E-201 study, 0 of 798 subjects receiving the vaccine experienced a hospitalization event compared to 4 of 801 subjects receiving a placebo, resulting in a 100% vaccine effectiveness rate (VE%).

Claims

1. 1. A method of reducing an exacerbation of chronic obstructive pulmonary disease (COPD) in a human subject, comprising:

1. A method comprising administering to the subject a nanoparticle vaccine, wherein the nanoparticles comprise a non-ionic surfactant core and a viral glycoprotein, wherein the viral glycoprotein is associated with the core, and wherein the surfactant is present at about 0.03% to about 0.05%.

2. 10. The method of claim 1, wherein the exacerbation is caused by an environmental stimulus.

3. The method of claim 2 , wherein the environmental stimulus is a pathogen.

4. The method of claim 3 , wherein the pathogen is a viral pathogen.

5. 5. The method of claim 4, wherein the viral pathogen is selected from the group consisting of RSV virus and influenza virus.

6. 10. The method of claim 1, wherein the incidence of exacerbations as determined by hospitalization rate is reduced by about 50%.

7. 7. The method of claim 6, wherein the human is at least 75 years old, at least 65 years old, preferably at least 60 years old.

8. 2. The method of claim 1, wherein the nonionic surfactant is selected from the group consisting of PS20, PS40, PS60, PS65, and PS80.

9. 9. The method of claim 8, wherein the viral glycoprotein is selected from the group consisting of an RSV F protein, an influenza HA protein, an influenza NA protein, and combinations thereof.

10. 10. The method of claim 9, wherein the viral glycoprotein is an RSV F protein and the molar ratio of non-ionic detergent to viral glycoprotein is from about 30:1 to about 60:

1.

11. 11. The method of claim 10, wherein the RSV F protein comprises a deletion of 1 to 10 amino acids corresponding to amino acids 137-146 of SEQ ID NO:2 and an inactivated primary furin cleavage site corresponding to amino acids 131-136 of SEQ ID NO:2, wherein the primary furin cleavage site is inactivated by mutation.

12. The method of claim 11 , wherein the composition is substantially free of any other surfactants.

13. 12. The method of claim 11, 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.

14. 14. The method of claim 13, wherein the RSV F protein comprises SEQ ID NO:

19.

15. 14. The method of claim 13, wherein the RSV F protein consists of SEQ ID NO:

19.

16. 16. The method of claim 15, wherein the nonionic surfactant is PS80.

17. 1. A method of reducing chronic obstructive pulmonary disease (COPD) exacerbations in response to an environmental stimulus in a human having COPD, comprising administering to the human an immunogenic composition, the immunogenic composition comprising first nanoparticles, the first nanoparticles comprising a PS80 non-ionic surfactant core and a first viral glycoprotein, the viral glycoprotein being bound to the core, and the surfactant being present at about 0.03% to about 0.05%.

18. 18. The method of claim 17, wherein the first viral glycoprotein is an RSV F glycoprotein.

19. 19. The method of claim 18, wherein the immunogenic composition comprises second nanoparticles, the second nanoparticles comprising a PS80 non-ionic surfactant core and a second viral glycoprotein, and the second viral protein is an influenza HA protein.

20. 1. An immunogenic composition for use in a method of treating an exacerbation of COPD in a human subject with COPD, wherein the vaccine composition comprises first nanoparticles, the first nanoparticles comprising a PS80 non-ionic surfactant core and a first viral glycoprotein, the viral glycoprotein being bound to the core, and the surfactant being present at about 0.03% to about 0.05%.

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