Methods of preventing diseases or disorders caused by RSV infection

Abstract

To provide a pharmaceutical composition for maternal immunization.SOLUTION: The invention provides a pharmaceutical composition for maternal immunization, wherein the pharmaceutical composition comprises an RSVF protein and an adjuvant, wherein the pharmaceutical composition is administered to a pregnant female carrying a gestational infant, wherein the maternal immunization induces an immune response against at least one symptom associated with RSV lower respiratory tract infection (LRTI) in the infant after birth, wherein the pregnant female is at about 28 weeks to about 33 weeks of gestation, and wherein the RSVF protein comprises SEQ ID NO:

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 811,945, filed February 28, 2019, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] Description of electronically submitted text files

[0002] The contents of the text file submitted electronically herewith are incorporated by reference in their entirety into this specification: Computer-readable format copy of sequence listing (file name: NOVV_084_01WO_SeqList_ST25.txt, date of recording: February 24, 2020; file size: 90 kilobytes).

[0003] Technical Field

[0003] The present invention relates generally to modified or mutated respiratory syncytial virus fusion (F) proteins, including immunogenic compositions, e.g., vaccines, for the treatment and / or prevention of RSV infection, as well as methods for making and using the same. [Background technology]

[0004] background

[0004] Respiratory syncytial virus (RSV) is a member of the Pneumovirus genus in the Paramyxoviridae family. Human RSV (HRSV) is a leading cause of severe lower respiratory tract disease in young children and is responsible for significant morbidity and mortality in humans. RSV is also recognized as an important cause of disease in immunocompromised adults and in the elderly. Due to incomplete resistance to RSV in infected hosts after natural infection, RSV can cause multiple infections during childhood and adulthood.

[0005]

[0005] Developing 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 to be used in difficult environments where refrigeration may not be available. Therefore, there is ongoing interest in generating a vaccine against the RSV virus. Summary of the Invention [Means for solving the problem]

[0006] overview

[0006] The present disclosure provides a method of maternal immunization comprising administering to a pregnant woman carrying a term infant a composition comprising an RSV F protein and an adjuvant, wherein the method induces an immune response in the postnatal infant against at least one symptom associated with RSV lower respiratory tract infection (LRTI), wherein the pregnant woman is between about 28 and about 33 weeks pregnant. DETAILED DESCRIPTION OF THE INVENTION

[0007] Detailed Description definition As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to a "protein" may refer to a single protein or a mixture of such proteins, a reference to a "process" is a reference to equivalent steps and / or methods known to those skilled in the art, and so forth.

[0008]

[0008] 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 increasing or broadening the specificity of either or both the antibody and cellular immune responses.

[0009]

[0009] As used herein, the terms "about" or "approximately," when preceding a numerical value, indicate a range of the value plus or minus 10%. For example, "about 100" includes 90 and 110.

[0010]

[0010] As used herein, the terms "immunogen," "antigen," and "epitope" refer to substances that are capable of eliciting an immune response, such as proteins, including glycoproteins, and peptides.

[0011]

[0011] As used herein, an "immunogenic composition" is a composition containing 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 against the antigen.

[0012] As used herein, a "subunit" composition, e.g., a vaccine, comprises one or more selected antigens, but not all antigens, from a pathogen. Such compositions are substantially free of intact virus or lysates of such cells or particles, and are typically prepared from immunogenic polypeptides 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.

[0013]

[0013] As used herein, "substantially" refers to the isolation of a substance (e.g., a compound, polynucleotide, or polypeptide) such that the substance forms the majority percent of the sample in which it is contained. For example, in a sample, the 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%. If the component is substantially replaced, the remaining amount in the sample is about 0.5% or less to about 1.0%, preferably less than about 0.5% to about 10%.

[0014]

[0014] As used herein, the terms "treat" and "treatment" refer to an approach to 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 onset or progression of an infection or disease; ameliorating or reducing the progression, symptoms of an infection or disease; or a combination thereof.

[0015]

[0015] As used herein, "prevention" is used interchangeably with "prophylaxis" and can mean the complete prevention of infection or disease, the prevention of the progression of symptoms of infection or disease; the delay in the onset of infection or disease or its symptoms; or the reduction in the severity of infection or disease or its symptoms that have subsequently progressed.

[0016]

[0016] 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 effective 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 assay.

[0017]

[0017] As used herein, the term "vaccine" refers to an immunogenic composition, e.g., 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 against infection by the pathogen and / or reduces the severity of disease or symptoms caused by infection by 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 also refer to a suspension or solution of immunogen administered to a vertebrate to provide protective immunity.

[0018] As used herein, the term "subject" includes humans and other animals. Typically, the subject is a human. For example, the subject may be an adult, a teenager, a child (2-14 years old), or an infant (0-2 years old). In some embodiments, an adult is an adult 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 plans 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.

[0019] In some embodiments, the subject is a female who is about 28 to about 33 weeks pregnant. In some embodiments, the subject is a female who is greater than 33 weeks pregnant. As used herein, the term "gestational infant" refers to a fetus or developing fetus of a pregnant female.

[0020] As used herein, the term "pharmacologically 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, and more particularly in humans. The composition may be useful as a vaccine and / or antigenic composition for inducing a protective immune response in a vertebrate.

[0021]

[0021] As used herein, the term "about" means plus or minus 10% of the indicated numerical value.

[0022] Overview

[0022] The RSV virus has a genome composed of single-stranded negative-sense RNA, which is tightly bound to viral proteins to form a nucleocapsid. The viral envelope is composed of a lipid bilayer derived from the plasma membrane that contains virally encoded structural proteins. Viral polymerase is packaged by the virion and transcribes the genomic RNA into mRNA. The RSV genome encodes three transmembrane structural proteins, F, G, and SH, two matrix proteins, M and M2, three nucleocapsid proteins, N, P, and L, and two nonstructural proteins, NS1 and NS2.

[0023] Fusion of HRSV and cell membranes is thought to occur at the cell surface and is an essential step for the import of viral ribonucleoproteins into the cytoplasm during the early pathological stages of infection. This process is mediated by the fusion (F) protein, which also promotes fusion of the membrane of infected cells with that of adjacent cells to form characteristic syncytia, a prominent cytopathological effect and additional mechanism of viral spread. Therefore, neutralization of fusion activity is important in host immunity. Indeed, monoclonal antibodies developed against the F protein have been shown to neutralize viral infectivity and inhibit membrane fusion (Calder et al., 2000, Virology 271:122-131).

[0024] The RSV F protein shares structural features and limited, but important, amino acid sequence identity with the F glycoproteins of other paramyxoviruses. The F protein is synthesized as a 574-amino acid inactive precursor (F0) that is cotranslationally glycosylated to asparagine in the endoplasmic reticulum, where it assembles into homo-oligomers. Before reaching the cell surface, the F0 precursor is cleaved by proteases from the N-terminus to F2 and from the C-terminus to F1. The F2 and F1 chains remain covalently linked by one or more disulfide bonds.

[0025] Immunoaffinity-purified full-length F protein was found to accumulate in the form of micelles (also characterized as rosettes), which is similar to that observed with other full-length viral membrane glycoproteins (Wrigley et al., 1986 (Electron Microscopy of Proteins, Vol. 5, pp. 103-163, Academic Press, London)). Under electron microscopy, molecules within the rosettes appear as either inverted conical rods (approximately 70%) or lollipop-shaped (approximately 30%) structures, the wider ends of which protrude away from the center of the rosette. The rod conformational state binds F glycoprotein in the pre-fusion inactive state, while the lollipop conformational state binds F glycoprotein in the post-fusion active state.

[0026] Electron microscopy can be used to distinguish pre-fusion and post-fusion (also referred to as pre-fusion and fusogenic) conformations, as demonstrated by Calder et al., 2000, Virology 271:122-131. Pre-fusion conformations can also be distinguished from fusogenic (post-fusion) conformations by liposome binding assays. Additionally, pre-fusion and fusogenic conformations can be distinguished using antibodies (e.g., monoclonal antibodies) that specifically recognize conformational epitopes present on one or the other of the pre-fusion or fusogenic forms of the RSV F protein (but not on the other form). Such conformational epitopes may result from preferential exposure of antigenic determinants on the surface of the molecule. Alternatively, conformational epitopes may arise from the juxtaposition of non-contiguous amino acids in a linear polypeptide.

[0027] Previously, it was shown that the F precursor is cleaved at two sites (site I after residue 109 and site II after residue 136), both preceded by motifs recognized by furin-like proteases. Site II is adjacent to the fusion peptide, and cleavage of the F protein at both sites is required for membrane fusion (Gonzalez-Reyes et al., 2001, PNAS 98(17):9859-9864). If cleavage is completed at both sites, there is thought to be a transition from a cone-shaped to a lollipop-shaped rod.

[0028] Nanoparticle Structure and Morphology

[0028] The nanoparticles of the present disclosure comprise an antigen bound to a non-ionic surfactant core. Advantageously, the nanoparticles have improved resistance to environmental stresses so as to achieve enhanced stability.

[0029] 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 varying numbers of trimers. For example, the composition may contain nanoparticles with a number of trimers 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% of the nanoparticles with 5 trimers.

[0030]

[0030] The antigen is associated with 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 assembling the nanoparticles to form a core that presents the antigen. Thus, in certain embodiments, the nanoparticles may contain an antigen that assembles into a multi-oligomeric glycoprotein-PS80 protein-surfactant nanoparticle in which the head region protrudes outward and the hydrophobic region and the PS80 surfactant form a central core surrounded by the antigen.

[0031] The nanoparticles disclosed herein have a Z-ave size ranging 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-ave) is measured by dynamic light scattering (DLS) using a Malvern Zetasizer unless otherwise specified.

[0032]

[0032] Several nanoparticle types may be included in the vaccine compositions disclosed herein. In some embodiments, the nanoparticle type is in the form of anisotropic rods, which may be dimeric or monomeric. In other embodiments, the nanoparticle type is a spherical oligomer. 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 may be regulated by controlling the concentrations of surfactants and proteins during the manufacturing process. The nanoparticle type may be determined by measuring the sedimentation coefficient.

[0033] Nanoparticle production The nanoparticles of the present disclosure are non-naturally occurring products, and their components do not occur together in nature. Typically, 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.

[0034] Antigens contained within the nanoparticles are typically produced by recombinant expression in host cells. Standard recombinant techniques can 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 double knocked out 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, e.g., Sf9, Sf21, Trichoplusia ni cells, e.g., High Five cells, and Drosophila S2 cells.

[0035]

[0035] Typical transfection and cell growth methods can be used to culture cells. A vector, for example, a vector containing a polynucleotide encoding a fusion protein, can be transfected into host cells according to methods well known in the art. For example, introducing nucleic acid into eukaryotic cells can be achieved by calcium phosphate co-precipitation, electroporation, microinjection, lipofection, and transfection using polyamine transfection reagent. In one embodiment, the vector is a recombinant baculovirus.

[0036] Methods for growing host cells include, but are not limited to, batch cell culture techniques, batch-fed cell culture techniques, continuous cell culture techniques, and perfusion cell culture techniques. Cell culture refers to the growth and propagation of cells in a bioreactor (fermentation chamber) where the cells grow 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 culture cells, and 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 another embodiment, the pre-sterilized plastic bag is an approximately 50 L to 3500 L bag.

[0037] Surfactant extraction and nanoparticle purification Following host cell growth, proteins can be harvested 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 into a detergent extract. Triton X-100 and tergitol (also known as NP-9) are preferred detergents for extraction. The detergent may be added to 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%.

[0038] In other embodiments, various first detergents can be used to isolate proteins from host cells. For example, the first detergent can 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-methylglucamine, n-decyl alpha-D-glucopyranoside, decyl beta-D-maltopyranoside, or the like. Do, n-Dodecanoyl-N-methylglucamide, n-Dodecyl alpha-D-maltoside, n-Dodecyl beta-D-maltoside, n-Dodecyl beta-D-maltoside, Heptaethylene glycol monodecyl ether, Heptaethylene glycol monododecyl ether, Heptaethylene glycol monotetradecyl ether, n-Hexadecyl beta-D-maltoside, Hexaethylene glycol monododecyl ether, Hexaethylene glycol monohexadecyl ether, Hexaethylene glycol monooctadecyl ether, Hexaethylene glycol monotetradecyl ether, Igepal CA-630, IgepalCA-630, Methyl-6-O-(N-heptylcarbamoyl)-alpha-D-glucopyranoside, Nonaethylene Glycol Monododecyl Ether, N-Nonanyl-N-methylglucamine, N-Nonanyl-N-methylglucamine, Octaethylene Glycol Monodecyl Ether, Octaethylene Glycol Monododecyl Ether, Octaethylene Glycol Monohexadecyl Ether, Octaethylene Glycol Monooctadecyl Ether, Octaethylene Glycol Monotetradecyl Ether, Octyl-beta-D-glucopyranoside, Pentaethylene Glycol Monodecyl Ether, Pentaethylene Glycol Monododecyl Ether, Pentaethylene Glycol Monohexadecyl Ether, Pentaethylene Glycol Monohexyl Ether, Pentaethylene Glycol Monooctadecyl Ether, Pentaethylene Glycol Monooctyl Ether, Polyethylene Glycol Diglycidyl Ether, Polyethylene Glycol Ether W-1, Polyoxyethylene 10 Tridecyl Ether, Polyoxyethylene 100 Stearate, Polyoxyethylene 20 Isohexadecyl Ether, Polyoxyethylene 20 Oleyl Ether, Polyoxyethylene 40 Stearate, Polyoxyethylene 50 Stearate, Polyoxyethylene 8 Stearate, Polyoxyethylene Bis(imidazolylcarbonyl), Polyoxyethylene 25 Propylene Glycol Stearate, Saponin from Quillaja Bark, Span® 20, Span® 40, Span® 60, Span® 65, Span® 80, Span® 85, Tergitol The soluble ...

[0039] The nanoparticles may then be isolated from the cellular debris using centrifugation. In some embodiments, gradient centrifugation (e.g., using cesium chloride, sucrose, and iodixanol) may be used. Other techniques may be used alternatively or in addition, including standard purification techniques such as ion exchange chromatography, affinity chromatography, and gel filtration chromatography.

[0040] 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.

[0041] Surfactant Exchange To form nanoparticles, the first surfactant used to extract proteins from host cells is substantially replaced with a second surfactant to achieve a nanoparticle structure. NP-9 is a preferred extraction surfactant. Typically, the nanoparticles do not contain detectable NP-9 as measured by HPLC. The second surfactant is typically selected from the group consisting of PS20, PS40, PS60, PS65, and PS80. Preferably, the second surfactant is PS80. To maintain the stability of the nanoparticle formulation, the ratio of the second surfactant to the protein is maintained within a specific range.

[0042] In certain embodiments, detergent exchange is performed to bind glycoproteins via their carbohydrate moieties using affinity chromatography. For example, affinity chromatography may use a legume lectin column. Legume lectins are proteins originally identified in plants and found to interact specifically and reversibly with carbohydrate residues. See, for example, Sharon and Lis, "Legume lectins—a large family of homologous proteins," FASEB J. 1990 Nov; 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 preferred column for detergent exchange due to its binding properties. See, for example, Example 10. Lectin columns are commercially available; for example, 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 carbohydrate moieties bind to lentil lectins, liberating amino acids of the protein that coalesce around the surfactant, resulting in the formation of a surfactant core, to provide nanoparticles bearing multiple copies of the antigen, e.g., glycoprotein oligomers that may be dimers, trimers, or tetramers immobilized within the surfactant.

[0043]

[0043] When surfactants are incubated with proteins to form nanoparticles during detergent exchange, they may be present at up to about 0.1% (w / v) during the initial purification steps, and this amount can be reduced to achieve final nanoparticles with optimal stability. For example, a nonionic surfactant (e.g., PS80) may 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. Amounts of PS80 less than about 0.03% in the formulation do 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 enable long-term stability of the nanoparticles with reduced degradation.

[0044]

[0044] Detergent exchange may be performed on purified proteins as discussed above, purified, frozen for storage, and then thawed for detergent exchange.

[0045] Enhanced stability and immunogenicity of nanoparticles

[0045] Without wishing to be bound by theory, it is believed that combining antigens with a nonionic surfactant core results in superior stability and antigen presentation. The nanoparticles disclosed herein surprisingly achieve good stability and immunogenicity. Advantageous stability is particularly useful for vaccines used in countries lacking adequate storage; for example, some locations in Africa may lack refrigeration, so vaccines for diseases endemic in areas facing difficult storage conditions, such as Ebola virus and RSV, would particularly benefit from improved stability. Furthermore, HA influenza nanoparticles produced using a neutral pH approach exhibit superior folding compared to known recombinant influenza vaccines.

[0046]

[0046] In particular, previous approaches using surfactants to generate RSV vaccines, including the split vaccine described in U.S. Patent Application Publication No. 2004 / 0028698 to Colau et al., have not resulted in effective structures. Unlike the nanoparticles with proteins surrounding a surfactant core disclosed herein, the composition of Colau et al. contained amorphous material lacking discernible viral structure, likely preventing effective presentation of epitopes to the immune system. Furthermore, the disclosed nanoparticles have particularly enhanced stability because the orientation of antigens (often glycoproteins) around the surfactant core sterically hinders access for enzymes and other chemicals that cause proteolysis.

[0047] 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 prepared to determine the integrity of antigenic proteins after various treatments designed to stress the nanoparticles by mimicking harsh storage conditions. Thus, a measure of stability is the relative abundance of antigenic peptides in stressed samples compared to control samples. A robust immune response is achieved even after various different stresses against the RSV F nanoparticle composition. The nanoparticles improved protease resistance using PS80 levels greater than 0.015%. Notably, at 18 months, 0.03% PS80 showed a 50% reduction in the formation of truncated species compared to 0.015% PS80. The nanoparticles disclosed herein are stable at 2-8°C. However, advantageously, they are also stable at 25°C for at least two 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. In the case of RSV-F nanoparticles, stability may be determined by measuring the formation of truncated F1 protein. Advantageously, the RSV-F nanoparticles disclosed herein retain intact antigenic site II at 90-100% abundance, as measured by peptide mapping, compared to a control RSV-F protein in response to various stresses, including pH (pH 3.7), high pH (pH 10), high temperature (50°C for 2 weeks), and even peroxide oxidation.

[0048]

[0048] The location of the glycoprotein anchored in the surfactant core is believed to provide enhanced stability by reducing undesired interactions. For example, improved protection from protease-based degradation can be achieved through a shielding effect, whereby anchoring the glycoprotein in the core at the molar ratios disclosed herein provides steric hindrance that blocks protease access.

[0049] Thus, in certain embodiments, disclosed herein are RSV-F nanoparticles and compositions containing the same, which retain 90% to 100% of the intact Site II peptide 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 at pH 3.7 for 1 week, incubation at 25°C at pH 10 for 1 week, stirring at 25°C for 1 week, and incubation with an oxidizing agent, such as hydrogen peroxide, for 1 week at 25°C. In addition, composition functionality is maintained after such treatment. For example, neutralizing antibody, anti-RSV IgG, and PCA titers are preserved compared to controls.

[0050]

[0050] Enhanced immunogenicity is exemplified by the cross-neutralization achieved by influenza nanoparticles: the orientation of influenza antigens protruding from the core is thought to provide more effective presentation of epitopes to the immune system.

[0051] Nanoparticle RSV antigen

[0051] In a typical embodiment, the antigen used to generate nanoparticles is a viral protein. In some aspects, the protein may be modified, but retains the ability to stimulate an immune response to the native peptide. In some aspects, the protein essentially contains or is suitable for containing a transmembrane domain to facilitate the binding of the protein into the surfactant core. In many cases, the protein is a glycoprotein in nature.

[0052] 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 a natural variant. The mutations confer desirable characteristics, such as improved protein expression and enhanced immunogenicity. Additional information describing RSV-F protein structure can be found in Swanson et al. A Monomeric Uncleaved Respiratory Syncytial Virus F Antigen Retains Prefusion-Specific Neutralizing Epitopes. Journal of Virology, 2014, 88, 11802-11810. Jason S. McLellan et al. Structure of RSV Fusion Glycoprotein Trimer Bound to a Prefusion-Specific Neutralizing Antibody. Science, 2013, 340, 1113-1117.

[0053] The primary fusion cleavage site is located at residues 131-136, which correspond to SEQ ID NO: 2. Inactivation of the primary fusion cleavage site can be achieved by mutating residues within the site, such that furin can no longer recognize 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 of the 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).

[0054] In certain embodiments, 1 to 10 amino acids corresponding to amino acids 137-146 of SEQ ID NO: 2 may be deleted, with specific examples of suitable RSV F proteins shown below. Each of SEQ ID NOs: 3-13 may 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 sequencing errors (A to P, V to I, and V to M), which are corrected in SEQ ID NOs: 3-13. After 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 and then the signal peptide is cleaved. The N-terminal RSV F signal peptide consists of the amino acids of SEQ ID NO: 26 (MELLILKANAITTILTAVTFCFASG). Thus, for example, after 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. In some cases, to enhance expression, one or up to all of the RSV F signal peptide amino acids can be deleted or mutated, or the entire signal peptide can be deleted and replaced with a different signal peptide. The initial methionine residue is maintained to initiate expression.

[0055] [Table 1]

[0056] In some embodiments, the RSV F proteins disclosed herein are merely altered from wild-type strains by deletions in the fusion domain, possibly resulting in inactivation of the primary 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. 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) are found in U.S. Patent Application Publication No. 2011 / 0305727, particularly the RSV-F protein containing the sequence spanning residues 100-150 disclosed in FIG. 1C therein.

[0057] In certain other embodiments, the RSV F1 or F2 domain may have a modification relative 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 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.

[0058] In certain examples, the RSV nanoparticle formulation may contain about 0.025% to about 0.03% PS80, and the RSV F ranges 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 contain about 0.035% to about 0.04% PS80 in the composition, and the RSV F ranges from 300 μg / mL to about 500 μg / mL. In yet other embodiments, the nanoparticle formulation may contain about 0.035% to about 0.04% PS80 in the composition, and the RSV F ranges from 350 to 500 μg / mL.

[0059]

[0058] Because the concentrations of antigen and surfactant can vary, the amount of each may be referred to as the molar ratio of nonionic surfactant to protein. For example, the molar ratio of PS80 to protein is calculated by using the PS80 concentration and 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 using RSV F as an example, the molecular weight of RSV F is 65 kD. The molar ratio is calculated as follows: (PS80 concentration x 10 x 65000) ÷ (1310 x RSV F concentration (mg / mL)). Thus, for example, the nanoparticle concentration measured by protein is 270 μg / mL and the PS80 concentration is 0.015% and 0.03%, which corresponds to a molar ratio of PS80 to RSV F protein of 27:1 (i.e., 0.015 × 10 × 65000 / (1310 × 0.27)) and 55:1, respectively.

[0060] 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, the substituted nonionic surfactant is PS80, and the molar ratio is about 30:1 to about 50:1 PS80:protein. In the case of RSV-F glycoprotein, nanoparticles with a molar ratio within the range of 35:1 to about 65:1, particularly a ratio of about 45:1, are particularly stable.

[0061] modified antigen

[0060] The antigens disclosed herein encompass variations and mutants of the antigens. In certain embodiments, an antigen may share identity with a disclosed antigen. Typically, and unless specifically defined in the context of a specifically identified antigen, the percentage identity may be at least 80%, at least 90%, at least 95%, at least 97%, or at least 98%. 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.

[0062] 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 may be made for various purposes. In some embodiments, the antigen may be extended at the N-terminus, C-terminus, or both. In some embodiments, the extension is a tag useful for functions such as purification or detection. In some embodiments, the tag contains an epitope. For example, the tag may be a 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 may 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 antigens with an intact signal peptide. Thus, if the nanoparticle comprises an antigen, the antigen may contain an extension and therefore may be a fusion protein when incorporated into the nanoparticle, and the extension is not included in calculating identity for the sequence.

[0063] 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. To calculate identity to a protein having a truncation, identity is measured over the remainder of the protein.

[0064] 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 particular 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 by binding to a single nanoparticle in a detergent exchange step. Binding of the glycoproteins to a column followed by detergent exchange allows multiple glycoprotein types to form around a surfactant core, providing a combination nanoparticle.

[0065]

[0064] The present disclosure also provides vaccine compositions that induce immune responses against two or more different pathogens by combining two or more nanoparticles, each of which induces a response against a different pathogen. In some cases, the vaccine composition may contain one or more combined 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.

[0066] In another example, influenza and RSV both cause respiratory disease. Thus, to induce a response against RSV and one or more influenza strains, HA, NA, and / or RSV F may be mixed into a combination nanoparticle, or multiple nanoparticles may be combined in a vaccine composition.

[0067] Vaccine Composition The compositions disclosed herein may be used prophylactically or therapeutically, but will typically be prophylactic. Accordingly, the present disclosure includes methods of treating or preventing an infection. In some embodiments, the infection is caused by RSV. In some embodiments, the infection is a lower respiratory tract infection (LRTI). The method includes administering to a subject a therapeutic or prophylactic amount of an immunogenic composition of the present disclosure. Preferably, the pharmaceutical composition is a vaccine composition that provides protection. In other embodiments, the protection may include amelioration of symptoms associated with the infection in a certain percentage of an exposed population. For example, depending on the pathogen, the composition may prevent or alleviate one or more symptoms of viral disease selected from: fatigue, fever, muscle aches, headache, sore throat, vomiting, diarrhea, rash, symptoms of impaired kidney and liver function, internal bleeding, and external bleeding, compared to an untreated subject.

[0068]

[0067] 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). Sodium chloride, if 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.

[0069] Certain nanoparticles, particularly RSV F nanoparticles, have improved stability at slightly acidic pH levels. For example, the pH range for a composition 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, RSV F protein nanoparticle compositions have a pH of about 6.2. For other nanoparticles, the composition may be more neutral; for example, influenza nanoparticles may have a pH of about 7.0 to about pH 7.4, often about pH 7.2.

[0070] 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 an adjuvant and are thereby available for administration as adjuvant-free compositions. Advantageously, the adjuvant-free compositions disclosed herein can achieve a protective immune response when administered as a single dose. Alum-free compositions that induce a robust immune response are particularly useful in adults aged about 60 years or older.

[0071] 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%, at least 85%, at least 90%, 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 is typically in the range of about 400 μg to about 1250 μg. For example, the 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 protein nanoparticle dose.

[0072] Saponin adjuvants

[0071] Adjuvants containing saponins may also be combined with the immunogens disclosed herein. Saponins are glycosides derived from the bark of the Quillaja saponaria Molina tree. Typically, saponins are prepared using a multi-step purification process that yields multiple fractions. As used herein, the term "saponin fraction from Quillaja saponaria Molina" is used generally to describe a semi-purified or defined saponin fraction of Quillaja saponaria, or a substantially pure fraction thereof.

[0073] Saponin fraction Several approaches to producing saponin fractions are suitable. Fractions A, B, and C, as described in U.S. Patent No. 6,352,697, may be prepared as follows: A lipophilic fraction from Quil 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 from 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. 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 with respect to elution profile and biological activity is highly consistent.

[0074] 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 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 0 3632 279 B2 may also be used, especially QA-7, QA-17, QA-18, and QA-21, which are obtained as described in EP 0 3632 279 B2, especially on page 6, and in Example 1 on pages 8 and 9.

[0075] The saponin fractions described herein and used to form adjuvants are often substantially pure fractions; that is, the fractions are substantially free of contaminants from other materials. In certain embodiments, a substantially pure saponin fraction may contain up to 40%, up to 30%, up to 25%, up to 20%, up to 15%, up to 10%, up to 7%, up to 5%, up to 2%, up to 1%, up to 0.5%, or up to 0.1% by weight of other compounds, such as other saponins or other adjuvant materials.

[0076] ISCOM Structure The saponin fraction may be administered in the form of cage-like particles called ISCOMs (immunostimulating complexes). ISCOMs may be prepared as described in EP 0109942 B1, EP 0242380 B1, and EP 0180546 B1. In certain embodiments, carrier and / or passenger antigens may be used as described in EP 9600647-3 (PCT / SE97 / 00289).

[0077] 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 a 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 (not necessarily glycosides) and may be produced as described in EP 0 436 620 B1.

[0078] In another embodiment, 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 an antigen bound by detergent treatment such that a portion of the antigen is integrated into the particle. In contrast, an ISCOM matrix is ​​formulated as a mixture with the antigen, and the binding between the ISCOM matrix particle and the antigen is mediated by electrostatic and / or hydrophobic interactions.

[0079]

[0078] According to one embodiment, the saponin fraction integrated into the ISCOM matrix complex or ISCOM complex, or at least one additional adjuvant (which is also integrated 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 (e.g., QA 1-21).

[0080] 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, a particle may contain any weight percent of Fraction A and any weight percent of another saponin fraction, such as a crude saponin fraction or Fraction C, respectively. Thus, in certain embodiments, each ISCOM matrix particle or each ISCOM complex particle may contain 0.1-99.9%, 5-95%, 10-90%, 15-85%, 20-80%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 40-60%, or 50% by weight of one saponin fraction, e.g., fraction A, and in each case the remainder, up to 100%, of another saponin, 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.

[0081] In certain embodiments, the ISCOM matrix or ISCOM complex comprises 5 to 99% by weight of one fraction, such as fraction A, and the remainder up to 100% by weight of another fraction, such as the crude saponin fraction or fraction C. Weights are calculated as the total weight of the saponin fractions.

[0082] In another embodiment, the ISCOM matrix or ISCOM complex comprises 40% to 99% by weight of one fraction, such as fraction A, and 1% to 60% by weight of another fraction, such as the crude saponin fraction or fraction C. Weights are calculated as the total weight of the saponin fractions.

[0083] In yet another embodiment, the ISCOM matrix or ISCOM complex comprises 70% to 95% by weight of one fraction, such as fraction A, and 30% to 5% by weight of another fraction, such as the 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 QA 1 to 21.

[0084] 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, each containing 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, each containing one saponin fraction from Quillaja saponaria Molina, and the saponin fraction within one complex particle being different from the saponin fraction within another complex particle.

[0085] 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.

[0086]

[0085] In the composition, a mixture of ISCOM matrix complex particles and / or ISCOM complex particles may be used in which one saponin fraction Quillaja (Quillaja saponaria Molina) and another saponin fraction Quillaja (Quillaja saponaria Molina) are separately incorporated into different ISCOM matrix complex particles and / or ISCOM complex particles.

[0087] ISCOM matrix or ISCOM complex particles, each having 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%, 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% ISCOM matrix or complex particles containing a first saponin fraction (the remainder, comprised of ISCOM matrix or complex particles, contains a different saponin fraction). In some embodiments, the remainder is one or more ISCOM matrix or complex particles, and each matrix or complex particle contains only one saponin fraction. In other embodiments, the ISCOM matrix or complex particles may contain multiple saponin fractions.

[0088]

[0087] 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.

[0089] A preferred composition comprises a first ISCOM matrix containing Fraction A and a second ISCOM matrix containing Fraction C, with the Fraction A ISCOM matrix comprising about 70% by weight of the total saponin adjuvant and the Fraction C ISCOM matrix comprising 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. Thus, in certain compositions, the Fraction A ISCOM matrix is ​​present in a range of about 70% to about 85% and the Fraction C ISCOM matrix is ​​present in a range of about 15% to about 30% by weight of the total saponin adjuvant in the composition. Exemplary QS-7 and QS-21 fractions, their production, and their uses are described in U.S. Pat. Nos. 5,057,540; 6,231,859; 6,352,697; 6,524,584; 6,846,489; 7,776,343; and 8,173,141, which are incorporated by reference for their disclosures.

[0090] Other adjuvants In some embodiments, other adjuvants may be used in addition to or as an alternative to the composition. 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), 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 a paucilamellar lipid vesicle; e.g., Novasomes®. Novasomes® are paucilamellar nonphospholipid 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).

[0091] Administration and Dosage

[0090] The compositions disclosed herein may be administered via systemic, mucosal, or transdermal routes, or directly into specific tissues. As used herein, the term "systemic administration" includes parenteral routes of administration. In particular, parenteral administration includes subcutaneous, intraperitoneal, intravenous, intraarterial, intramuscular, or intrasternal injection, intravenous infusion, 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 ocular administration. Preferably, administration is intramuscular.

[0092] 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, the various doses may be given by the same route or by different routes (e.g., parenteral prime and mucosal boost, mucosal prime and parenteral boost, etc.). In some embodiments, a subsequent boost dose is administered about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or about 6 weeks after the previous dose. Typically, however, the compositions disclosed herein are administered only once and still provide a protective immune response.

[0093] In some embodiments, the dose measured in μg may be the total weight of the dose including solutes, the weight of RSV F nanoparticles, or the weight of RSV F protein. The dose is measured using protein concentration assay A280 or ELISA.

[0094]

[0093] The dose of antigen, including those for pediatric administration, may be within the range of 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 and is administered with alum. In some aspects, pediatric doses may be within the range of about 30 μg to about 90 μg. Certain populations may be administered with or without an adjuvant. For example, when administered to the elderly, alum is preferably absent. In certain aspects, the composition may lack an added adjuvant. In such circumstances, the dose may be increased by about 10%.

[0095]

[0094] 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 about 0.5 mL, which are typical amounts.

[0096]

[0095] In specific embodiments for RSV vaccines, the dose may include an 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.

[0097] RSV F protein-containing compositions, e.g., vaccine compositions, and nanoparticles, are further described in U.S. Patent Application No. 16 / 009,257 and U.S. Patent Application No. 15 / 819,962, both of which are incorporated herein by reference in their entirety for all purposes.

[0098]

[0097] All patents, patent applications, references, and journal articles cited in this disclosure are expressly incorporated herein by reference in their entirety for all purposes. [Example]

[0099] Example Example 1 - Maternal vaccination during pregnancy protects infants from RSV lower respiratory tract infection (LRTI) A vaccine composition containing aluminum-adjuvanted RSV fusion (F) protein recombinant nanoparticles was administered to women between approximately 28 and 33 weeks of pregnancy. Results showed that the vaccine protected infants from severe consequences of RSV infection, including severe hypoxemia. The protective effect was associated with a shorter hospital stay.

[0100] Through the first 90 days of an infant's life, vaccine efficacy against RSV LRTI hospitalization was 53 percent and vaccine efficacy against severe RSV hypoxemia was 70 percent. In sharp contrast, administration of the vaccine to women greater than 33 weeks of gestation demonstrated a substantial reduction in vaccine efficacy. Administration beyond 33 weeks resulted in efficacy rates of only 26 percent for LRTI hospitalization and 44% for severe RSV hypoxemia, measured through the infant's first 90 days of life.

[0101] This study highlights the surprising finding that administering a vaccine to women during a narrow window of pregnancy can result in significantly beneficial outcomes for the infant after birth. This result marks the first time that a vaccine composition against RSV has demonstrated a high efficacy rate against severe hypoxemia caused by RSV infection in a Phase III clinical trial.

Claims

1. 1. A method of maternal immunization comprising administering to a pregnant woman carrying a term infant a composition comprising an RSV F protein and an adjuvant, wherein the method induces an immune response in the infant after birth against at least one symptom associated with RSV lower respiratory tract infection (LRTI), wherein the pregnant woman is between about 28 and about 33 weeks pregnant.

2. 10. The method of claim 1, wherein the at least one symptom is hypoxemia.

3. 3. The method of claim 1 or 2, wherein the adjuvant is an aluminum-based adjuvant.

4. 4. The method of any one of claims 1 to 3, wherein the composition comprises nanoparticles comprising a non-ionic surfactant core and an RSV F protein, wherein the RSV F protein is associated with the core, and wherein the surfactant is present at about 0.03% to about 0.05%.

5. 5. The method of claim 4, wherein the surfactant is selected from the group consisting of PS-20, PS-40, PS-60, PS-65, and PS-80.

6. 6. The method of any one of claims 1-5, wherein the RSV F protein comprises a deletion of 1-10 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.

7. The method of any one of claims 1 to 5, wherein the RSV-F protein is selected from the group consisting of SEQ ID NOs: 3 to 12.

8. The method of claim 7, wherein the RSV-F protein is encoded by SEQ ID NO:

8.

9. The method of any one of claims 1 to 5, wherein the RSV-F protein comprises SEQ ID NO: 19.

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