Use of a composition to prevent disease or disorder caused by rsv infection

Description

1 / 35 USE OF A COMPOSITION TO PREVENT DISEASE OR DISORDER CAUSED BY RSV INFECTION (Split Application BR 11 2021 017074 0) CROSS-REFERENCED WITH RELATED APPLICATIONS

[001] This application claims the benefit of priority under U.S. Provisional Application No. 62 / 811,945, filed February 28, 2019, the contents of which are incorporated by reference herein in their entirety for all purposes. DESCRIPTION OF THE TEXT FILE SENT ELECTRONICALLY

[002] The contents of the text file sent electronically with this document are incorporated herein by reference in their entirety: A machine-readable copy of the Sequence Listing (filename: NOVV_084_01WO_SeqList_ST25.txt, registration date: February 24, 2020; file size: 90 kilobytes). TECHNICAL FIELD

[003] The present invention is generally related to modified or mutated respiratory syncytial virus (F) fusion proteins and methods for preparing and using them, including immunogenic compositions, such as vaccines for the treatment and / or prevention of RSV infection. BACKGROUND

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

[005] The deployment of an effective vaccine depends on a combination of achievements. The vaccine must stimulate a response. Petition 870260074126, dated 07 / 24 / 2026, page 11 / 51 2 / 35 effective immunological response that reduces infection or disease by a sufficient amount to be beneficial. A vaccine must also be stable enough to be used in challenging environments where refrigeration may not be available. Therefore, there is ongoing interest in producing vaccines against RSV viruses. SUMMARY

[006] This disclosure provides methods of maternal immunization comprising administering a composition comprising an RSV F protein and an adjuvant to a pregnant woman carrying a gestational baby, wherein the method induces an immune response against at least one symptom associated with RSV lower respiratory tract infection (LRTI) in the baby after birth and wherein the pregnant woman is about 28 weeks to about 33 weeks pregnant. DETAILED DESCRIPTION Definitions

[007] As used in this document and the accompanying claims, the singular forms a, an and the include plural referents, unless the context clearly dictates otherwise. Thus, for example, reference to a protein may refer to a protein or to mixtures of such protein, and reference to the method includes reference to equivalent steps and / or methods known to those skilled in the art, and so forth.

[008] As used in this document, the term adjuvant refers to a compound that, when used in combination with an immunogen, enhances or otherwise alters or modifies the induced immune response against the immunogen. Modification of the immune response may include enhancing or broadening the specificity of one or both of the antibodies and cellular immune responses.

[009] As used in this document, the term about or approximately when preceding a numerical value indicates the Petition 870260074126, dated 07 / 24 / 2026, page 12 / 51 3 / 35 is a value that is more or less a range of 10%. For example, "about 100" covers 90 and 110.

[0010] As used in this document, the terms “immunogen”, “antigen” and “epitope” refer to substances such as proteins, including glycoproteins and peptides, that are capable of inducing an immune response.

[0011] As used in this document, an “immunogenic composition” is a composition comprising an antigen where administration of the composition to an individual results in the development in the individual of a humoral and / or cellular immune response to the antigen.

[0012] As used in this document, a “subunit” composition, for example, a vaccine, which includes one or more of the selected antigens, but not all of the antigens of a pathogen. Such a composition is substantially free of intact virus or lysate of such cells or particles and is typically prepared from immunogenic polypeptides, at least partially purified, often substantially purified of the pathogen. The antigens in the subunit composition disclosed in this document are typically recombinantly prepared, often using a baculovirus system.

[0013] As used in this document, “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, a substantially purified component comprises 85%, preferably 85%–90%, more preferably at least 95%–99.5%, and most preferably at least 99% of the sample. If a component is substantially replaced, the amount remaining in a sample is less than or equal to about 0.5% to about 10%, preferably less than about 0.5% to about 1.0%. Petition 870260074126, dated 07 / 24 / 2026, p. 13 / 51 4 / 35

[0014] The terms treat, treatment and “treating”, as used in this document, refer to an approach to obtain beneficial or desired results, for example, clinical results. For the purposes of this disclosure, beneficial or desired results may include inhibiting or suppressing the initiation or progression of an infection or disease; improving or reducing the development of symptoms of an infection or disease; or a combination thereof.

[0015] Prevention, as used in this document, is used interchangeably with prophylaxis and may mean the complete prevention of an infection or disease, or the prevention of the development of symptoms of that infection or disease; delay in the onset of an infection or disease or of its symptoms; or a decrease in the severity of a subsequently developed infection or disease or of its symptoms.

[0016] As used in this document, an effective dose or effective amount refers to an amount of an immunogen sufficient to induce an immune response that reduces at least one symptom of pathogen infection. An effective dose or effective amount can be determined, for example, by measuring amounts of secretory and / or serum neutralizing antibodies, for example, by plaque neutralization, complement fixation, enzyme-linked immunosorbent assay (ELISA) or microneutralization.

[0017] As used in this document, the term vaccine refers to an immunogenic composition, such as an immunogen derived from a pathogen, that is used to induce an immune response against the pathogen that provides protective immunity (e.g., immunity that protects an individual against infection with the pathogen and / or reduces the severity of the disease or condition caused by infection with the pathogen). The protective immune response may include antibody formation and / or a cell-mediated response. Depending on the context, the term vaccine may also Petition 870260074126, dated 07 / 24 / 2026, p. 14 / 51 5 / 35 may refer to a suspension or solution of an immunogen that is administered to a vertebrate to produce protective immunity.

[0018] As used in this document, the term individual includes humans and other animals. Typically, the individual is a human. For example, the individual may be an adult, an adolescent, a child (2 to 14 years of age), or an infant (0 to 2 years). In some respects, adults are elderly, around 65 years of age or older, or around 60 years of age or older. In some respects, the individual is a pregnant woman or a woman intending to become pregnant. In other respects, the individual is not a human; for example, a non-human primate; for example, a baboon, a chimpanzee, a gorilla, or a monkey. In certain respects, the individual may be a pet, such as a dog or cat.

[0019] In some respects, the individual is a woman who is about 28 to about 33 weeks pregnant. In some respects, the individual is a woman who is more than 33 weeks pregnant. As used in this document, the term gestational infant means the fetus or developing fetus of a pregnant woman.

[0020] As used in this document, the term pharmaceutically acceptable means approved by a U.S. federal or state government regulatory agency or listed in the U.S. Pharmacopoeia, European Pharmacopoeia, or other generally recognized pharmacopoeia for use in mammals, and more particularly in humans. These compositions may be useful as vaccines and / or antigenic compositions to induce a protective immune response in a vertebrate.

[0021] As used in this document, the term about means approximately 10% of the indicated numerical value. Outline

[0022] The RSV virus has a genome comprised of a single-stranded negative-sense RNA, which is strongly associated with Petition 870260074126, dated 07 / 24 / 2026, page 15 / 51 6 / 35 viral protein to form the nucleocapsid. The viral envelope is composed of a lipid bilayer derived from the plasma membrane that contains virus-encoded structural proteins. A viral polymerase is packaged with 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 non-structural proteins, NS1 and NS2.

[0023] It is thought that the fusion of HRSV and cell membranes occurs at the cell surface and is a necessary step for the transfer of viral ribonucleoprotein into the cell cytoplasm during the early stages of infection. This process is mediated by the fusion protein (F), which also promotes the fusion of the membrane of infected cells with that of adjacent cells to form a characteristic syncytium, which is both a prominent cytopathic effect and an additional mechanism of viral dissemination. Consequently, neutralizing fusion activity is important in host immunity. In fact, 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 limited but significant structural features and amino acid sequence identity with F glycoproteins of other paramyxoviruses. It is synthesized as an inactive 574-amino acid precursor (F0) that is cotranslationally glycosylated into asparagines in the endoplasmic reticulum, where it assembles into homo-oligomers. Before reaching the cell surface, the F0 precursor is cleaved by a protease into N-terminus F2 and C-terminus F1. The F2 and F1 chains remain covalently linked by one or more disulfide bonds.

[0025] Full-length F proteins purified by Petition 870260074126, dated 07 / 24 / 2026, page 16 / 51 7 / 35 immunoaffinity molecules were found to accumulate in the form of micelles (also characterized as rosettes), similar to those observed with other full-length viral membrane glycoproteins (Wrigley et al., 1986, in Electron Microscopy of Proteins, Vol. 5, pp. 103-163, Academic Press, London). Under electron microscopy, the molecules in the rosettes appear as inverted cone-shaped rods (~70%) or lollipop-shaped structures (~30%) with their wider ends projecting away from the rosette centers. The rod conformation is associated with an F glycoprotein in the inactivated pre-fusion state, while the lollipop conformation is associated with an F glycoprotein in the active post-fusion state.

[0026] Electron micrographs can be used to distinguish between pre-fusion and post-fusion conformations (alternatively designated prefusogenic and fusogenic), as demonstrated by Calder et al., 2000, Virology 271:122-131. The pre-fusion conformation can also be distinguished from the fusogenic (post-fusion) conformation by liposome association assays. Additionally, pre-fusion and fusogenic conformations can be distinguished using antibodies (e.g., monoclonal antibodies) that specifically recognize conformational epitopes present in either the pre-fusion or fusogenic form of the RSV F protein, but not in the other form. These conformational epitopes may be due to the preferential exposure of an antigenic determinant on the surface of the molecule. Alternatively, the conformational epitopes may arise from the juxtaposition of amino acids that are not contiguous in the linear polypeptide.

[0027] It has been previously demonstrated 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 a Petition 870260074126, dated 07 / 24 / 2026, p. 17 / 51 8 / 35 Fusion peptide and F protein cleavage at both sites is required for membrane fusion (Gonzalez-Reyes et al., 2001, PNAS 98(17): 9859-9864). When cleavage is completed at both sites, a transition from cone-shaped rods to lollipop-shaped rods is believed to occur. Structure and morphology of nanoparticles

[0028] The nanoparticles of the present disclosure comprise antigens associated with a nonionic detergent core. Advantageously, the nanoparticles have improved resistance to environmental stresses, thus providing enhanced stability.

[0029] In particular embodiments, nanoparticles are composed of multiple protein trimers surrounding a nonionic detergent 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 particular embodiments, each nanoparticle contains 2 to 6 trimers. The compositions disclosed in this document may contain nanoparticles having different numbers of trimers. For example, a composition may contain nanoparticles in which the number of trimers ranges from 2-9; in other embodiments, the nanoparticles in a composition may contain from 2-6 trimers. In particular embodiments, the compositions contain a heterogeneous population of nanoparticles having 2 to 6 trimers per nanoparticle or 2 to 9 trimers per nanoparticle. In other embodiments, the compositions may contain a substantially homogeneous population of nanoparticles.For example, the population may contain approximately 95% nanoparticles having 5 trimers.

[0030] The antigens are associated with the non-ionic detergent-containing core of the nanoparticle. Typically, the detergent is selected from polysorbate-20 (PS20), polysorbate-40 (PS40), polysorbate-60 (PS60), polysorbate-65 Petition 870260074126, dated 07 / 24 / 2026, page 18 / 51 9 / 35 (PS65) and polysorbate-80 (PS80). The presence of the detergent facilitates the formation of nanoparticles, forming a core that organizes and presents the antigens. Thus, in certain embodiments, the nanoparticles may contain antigens mounted on multioligomeric PS80 glycoprotein detergent nanoparticles with head regions projecting outwards and hydrophobic regions, and the PS80 detergent forming a central core surrounded by the antigens.

[0031] The nanoparticles disclosed in that document range in Z-bird size from 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. The particle size (Z-bird) is measured by dynamic light scattering (DLS) using a Malvern Zetasizer, unless otherwise specified.

[0032] Several types of nanoparticles can be included in the vaccine compositions disclosed in this document. In some aspects, the nanoparticle type is in the form of an anisotropic rod, which can be a dimer or monomer. In other aspects, the nanoparticle type is a spherical oligomer. In still other aspects, the nanoparticle can be described as an intermediate nanoparticle, having sedimentation properties intermediate between the first two types. The formation of nanoparticle types can be regulated by controlling the concentration of detergent and protein during the production process. The nanoparticle type can be determined by measuring the sedimentation coefficient. Nanoparticle production

[0033] The nanoparticles in this disclosure are products of non-natural occurrence, whose components do not occur together in nature. Generally, the methods disclosed in this document use a detergent exchange approach in which a first detergent is used to isolate a protein and then this first detergent is exchanged for a second detergent to Petition 870260074126, dated 07 / 24 / 2026, page 19 / 51 10 / 35 form nanoparticles.

[0034] The antigens contained in the nanoparticles are typically produced by recombinant expression in host cells. Conventional recombinant techniques can be used. Typically, the proteins are expressed in insect host cells using a baculovirus system. In preferred embodiments, the baculovirus is a cathepsin-L inactivated baculovirus. In other preferred embodiments, the baculovirus is a chitinase inactivated baculovirus. In still other preferred embodiments, the baculovirus has dual cathepsin-L and chitinase inactivation. High-level expression can be achieved in insect cell expression systems. Non-limiting examples of insect cells are Spodoptera frugiperda (Sf) cells, e.g., Sf9, Sf21, Trichoplusia ni cells, e.g., High Five cells, and Drosophila S2 cells.

[0035] Typical methods of transfection and cell growth can be used to culture the cells. Vectors, for example, vectors comprising polynucleotides encoding fusion proteins, can be transfected into host cells according to methods well known in the art. For example, the introduction of nucleic acids into eukaryotic cells can be achieved by calcium phosphate coprecipitation, electroporation, microinjection, lipofection, and transfection using polyamine transfection reagents. In one embodiment, the vector is a recombinant baculovirus.

[0036] Methods for culturing host cells include, but are not limited to, batch, fed-batch, continuous, and perfusion cell culture techniques. Cell culture means the growth and propagation of cells in a bioreactor (a fermentation chamber), where the cells propagate and express proteins (e.g., recombinant proteins) for purification and isolation. Petition 870260074126, dated 07 / 24 / 2026, page 20 / 51 11 / 35 Typically, cell culture is performed under sterile and controlled temperature and atmospheric conditions in a bioreactor. A bioreactor is a chamber used to grow cells in which environmental conditions, such as temperature, atmosphere, agitation, and / or pH, can be monitored. In one embodiment, the bioreactor is a stainless steel chamber. In another embodiment, the bioreactor is a pre-sterilized plastic bag (e.g., Cellbag®, Wave Biotech, Bridgewater, NJ). In yet another embodiment, the pre-sterilized plastic bags are bags ranging in size from approximately 50 L to 3500 L. Detergent extraction and nanoparticle purification

[0037] After the host cells have grown, the protein can be harvested from the host cells using detergents and purification protocols. Once 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, releasing the protein into a detergent extract. Each of Triton X-100 and tergitol, also known as NP-9, is a preferred detergent for extraction. The detergent can be added at a final concentration of about 0.1% to about 1.0%. For example, the concentration can 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 can be from about 0.1% to about 0.3%. Ideally, the concentration should be around 0.5%.

[0038] In other respects, different first detergents can be used to isolate the protein from the host cell. 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, Petition 870260074126, dated 07 / 24 / 2026, page 21 / 51 12 / 35 Decyl beta-D-maltopyranoside, n-Dodecanoyl-N-methylglucamide, n-Dodecyl alpha-D-maltoside, n-Dodecyl beta-D-maltoside, nDodecyl 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-O-(N-heptylcarbamoyl)alpha-D-glucopyranoside, nonaethylene glycol monododecyl ether, N-Nonanoyl-N-methylglucamine, N-Nonanoyl-Nmethylglucamine, octaethyleneglycol monodecyl ether, octaethyleneglycol monododecyl ether, octaethyleneglycol monododecyl ether, octaethyleneglycol monohexadecyl ether, octaethyleneglycol monooctadecyl ether, octaethyleneglycol monotetradecyl ether, octyl-beta-D glucopyranoside, pentaethyleneglycol monodecyl ether,pentaethylene glycol monododecyl ether, pentaethylene glycol monohexadecyl ether, pentaethylene glycol monohexyl ether, pentaethylene glycol monooctadecyl ether, pentaethylene glycol mono-octyl ether, polyethylene glycol diglycidyl ether, polyethylene glycol monohexyl ether, pentaethylene glycol mono-octadecyl ether, pentaethylene glycol mono-octyl ether, polyethylene glycol diglycidyl ether, polyethylene glycol W-1 ether, polyoxyethylene tridecyl ether 10, polyoxyethylene stearate 100, polyoxyethylene isohexadecyl ether 20, polyoxyethylene oleyl ether 20, polyoxyethylene stearate 40, polyoxyethylene stearate 50, polyoxyethylene stearate 8, polyoxyethylene bis(imidazolyl carbonyl) stearate polyoxyethylene 25 propylene barcolinate 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, Petition 870260074126, dated 07 / 24 / 2026, page 22 / 51 13 / 35 Type NP-4, Tergitol Type NP-40, Tergitol, Type NP-7, Tergitol Type NP-9, Tergitol Type TMN-10, Tergitol Type TMN-6, Triton X100 or combinations thereof.

[0039] The nanoparticles can then be isolated from cellular debris using centrifugation. In some embodiments, gradient centrifugation, such as using cesium chloride, sucrose, and iodixanol, can be used. Other techniques can be used as alternatives or in addition, such as standard purification techniques including, for example, ion-exchange chromatography, affinity chromatography, and gel filtration.

[0040] For example, the first column could be an ion-exchange chromatography resin, such as Fractogel® EMD TMAE (EMD Millipore), the second column could be a lentil (Lens culinaris) lectin affinity resin, and the third column could be a cation-exchange column, such as a Fractogel® EMD SO3 resin (EMD Millipore). In other respects, the cation-exchange column could be an MMC column or a Nuvia C Prime column (Bio-Rad Laboratories, Inc.). Preferably, the methods disclosed in this document do not use a detergent extraction column; for example, a hydrophobic interaction column. This column is frequently used to remove detergents during purification, but it can negatively impact the methods disclosed in this document. Detergent change

[0041] To form nanoparticles, the first detergent, used to extract the protein from the host cell, is substantially replaced by a second detergent to achieve the nanoparticle structure. NP-9 is a preferred extraction detergent. Typically, the nanoparticles do not contain detectable NP-9 when measured by HPLC. The second detergent is typically selected from the group consisting of PS20, PS40, PS60, PS65, and PS80. Preferably, the second detergent is PS80. To maintain the stability of the nanoparticle formulations, the Petition 870260074126, dated 07 / 24 / 2026, page 23 / 51 The ratio of detergent to protein (14 / 35) is maintained within a certain range.

[0042] In particular aspects, detergent exchange is performed using affinity chromatography to link glycoproteins through their carbohydrate moiety. For example, affinity chromatography can use a legume lectin column. Legume lectins are proteins originally identified in plants that 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, sanfena 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 aspects, the lentil lectin column may use a recombinant lectin. At the molecular level, it is believed that the carbohydrate moieties bind to the lentil lectin, releasing amino acids from the protein to coalesce around the detergent, resulting in the formation of a detergent core delivering nanoparticles having multiple copies of the antigen, for example, glycoprotein oligomers that may be dimers, trimers, or tetramers anchored in the detergent.

[0043] The detergent, when incubated with the protein to form nanoparticles during detergent exchange, can be present at up to about 0.1% (w / v) during the initial purification steps, and this amount is reduced to achieve the final nanoparticles with ideal stability. For example, the non-ionic detergent (e.g., PS80) can be about Petition 870260074126, dated 07 / 24 / 2026, page 24 / 51 15 / 35 0.03% to approximately 0.1%. Preferably, to improve stability, the nanoparticle contains approximately 0.03% to approximately 0.05% PS80. Amounts below approximately 0.03% PS80 in formulations do not show such good stability. Furthermore, if PS80 is present above approximately 0.05%, aggregates are formed. Consequently, approximately 0.03% to approximately 0.05% PS80 provides structural and stability benefits that allow for long-term stability of nanoparticles with reduced degradation.

[0044] Detergent exchange can be performed with purified proteins as discussed above and purified, frozen for storage and then thawed for detergent exchange. Enhanced Stability and Enhanced Immunogenicity of Nanoparticles

[0045] Without being limited by theory, it is thought that associating the antigen with a nonionic detergent core offers superior stability and antigen presentation. The nanoparticles disclosed in this paper provide surprisingly good stability and immunogenicity. The advantageous stability is especially useful for vaccines used in countries without adequate storage; for example, certain locations in Africa may lack refrigeration, and therefore vaccines for diseases prevalent in areas facing difficult storage conditions, such as Ebola virus and RSV, particularly benefit from improved stability. Furthermore, HA nanoparticles against influenza produced using the pH-neutral approach exhibit superiority to known recombinant influenza vaccines.

[0046] Notably, previous approaches to using detergents to produce RSV vaccines, including split vaccines, as described in US 2004 / 0028698 for Colau et al., failed to produce effective structures. Instead of Petition 870260074126, dated 07 / 24 / 2026, page 25 / 51 16 / 35 nanoparticles containing proteins around a detergent core, as disclosed in this document, the compositions of Colau et al. contained amorphous material without identifiable viral structures, presumably resulting in a failure to effectively present epitopes to the immune system. Furthermore, the disclosed nanoparticles have particularly enhanced stability because the orientation of the antigens, often glycoproteins, around the detergent core sterically prevents access by enzymes and other chemicals that cause protein degradation.

[0047] Nanoparticles have enhanced stability, as determined by their ability to maintain immunogenicity after exposure to varying stress. Stability can be measured in several ways. In one approach, a peptide map can be prepared to determine the integrity of the antigen protein after various treatments designed to stress the nanoparticles, simulating adverse storage conditions. Thus, a measure of stability is the relative abundance of antigen peptides in a stressed sample compared to a control sample. Even after several different stresses for an RSV F nanoparticle composition, robust immune responses are achieved. Nanoparticles improved protease resistance using PS80 levels above 0.015%. Notably, at 18 months, PS80 at 0.03% shows a 50% reduction in the formation of truncated species compared to PS80 at 0.015%.The nanoparticles disclosed in this document are stable at 2-8°C. Advantageously, however, they are also stable at 25°C for at least 2 months. In some embodiments, the compositions are stable at 25°C for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months. For RSV F nanoparticles, stability can be determined by measuring the formation of the truncated F1 protein. Advantageously, RSV F nanoparticles... Petition 870260074126, dated 07 / 24 / 2026, p. 26 / 51 The 17 / 35 proteins disclosed in this document advantageously retain an intact antigenic site II at an abundance of 90 to 100%, as measured by peptide mapping, compared to the 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] It is thought that the position of the glycoprotein anchored in the detergent core provides improved stability, reducing undesirable interactions. For example, improved protection against protease-based degradation can be achieved through a protective effect whereby the anchoring of the glycoproteins in the core at the molar ratios disclosed in this document results in steric hindrance blocking protease access.

[0049] Thus, in particular aspects, RSV F nanoparticles and compositions containing them are disclosed in this document, which retain 90% to 100% of the intact Site II peptide, compared to the untreated control, in response to one or more treatments selected from the group consisting of incubation at 50°C for 2 weeks, incubation at pH 3.7 for 1 week at 25°C, incubation at pH 10 for 1 week at 25°C, agitation for 1 week at 25°C and incubation with an oxidant, such as hydrogen peroxide, for 1 week at 25°C. Additionally, after such treatments, the functionality of the compositions is maintained. For example, neutralizing antibody titers, anti-RSV IgG and PCA are preserved compared to the control.

[0050] Enhanced immunogenicity is exemplified by the cross-neutralization achieved by influenza nanoparticles. It is thought that the orientation of influenza antigens projecting from the core provides a more effective presentation of epitopes to the immune system. RSV nanoparticle antigens

[0051] In typical modalities, the antigens used for Petition 870260074126, dated 07 / 24 / 2026, page 27 / 51 18 / 35 producing the nanoparticles are viral proteins. In some respects, the proteins may be modified, but retain the ability to stimulate immune responses against the natural peptide. In some respects, the protein inherently contains or is adapted to contain a transmembrane domain to promote the association of the protein into a detergent core. Frequently, the protein is naturally a glycoprotein.

[0052] In one aspect, the virus is respiratory syncytial virus (RSV) and the viral antigen is the fusion glycoprotein (F). The structure and function of RSV F proteins are well characterized. Suitable RSV F proteins for use in the compositions described in this document 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, RSV F proteins are mutated compared to their natural variants. These mutations confer desirable characteristics such as enhanced protein expression, potentiated immunogenicity, and the like. Additional information describing the structure of RSV F proteins 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 to 136 corresponding to SEQ ID NO: 2. Inactivation of the primary fusion cleavage site can be achieved by mutating residues at 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 the positions corresponding to arginine 133, Petition 870260074126, dated 07 / 24 / 2026, p. 28 / 51 19 / 35 arginine 135 and arginine 136 of the F protein of wild-type RSV (SEQ ID NO: 2). In particular aspects, one, two, or all three arginines are mutated to glutamine. In other aspects, inactivation is achieved by mutation of 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 particular aspects, 1 to 10 amino acids corresponding to acids 137 to 146 of SEQ ID NO: 2 can be eliminated, including the particular 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 in SEQ ID NO: 2 has sequencing errors (A to P, V to I, and V to M) that are corrected in SEQ ID NOS: 3-13. After expression of the RSV F protein in a host cell, the N-terminal signal peptide is cleaved to provide the final sequences. Typically, the signal peptide is cleaved by host cell proteases. In other aspects, however, the full-length protein can be isolated from the host cell and the signal peptide subsequently cleaved. The N-terminal F signal peptide of RSV consists of amino acids with SEQ ID NO: 26 (MELLILKANAITTILTAVTFCFASG).Thus, for example, after cleavage of the signal peptide with SEQ ID NO: 8 during expression and purification, a mature protein having the sequence SEQ ID NO: 19 is obtained and used to produce an RSV F nanoparticle vaccine. Optionally, one or more of all amino acids in the RSV F signal peptide may be eliminated, mutated, or the entire signal peptide may be eliminated and replaced with a different signal peptide to enhance expression. An initiation methionine residue is retained to initiate expression. SEQ ID NO: Protein Elimination of Fusion Domain Primary Fusion Cleavage Site Sequence Petition 870260074126, dated 07 / 24 / 2026, page 29 / 51 20 / 35 Expressed 1 Wild-type strain A2 (nucleic) KKRKRR (active) 2 Wild-type strain A2 (protein) KKRKRR (active) 3 137-138 deletion (Δ1) KKQKQQ (inactive) 4 137-138 deletion (Δ2) KKQKQQ (inactive) 5 137-139 deletion (Δ3) KKQKQQ (inactive) 6 137-140 deletion (Δ4) KKQKQQ (inactive) 7 137-141 deletion (Δ5) KKQKQQ (inactive) 8 137-146 deletion (Δ10) KKQKQQ (inactive) 9 137-142 deletion (Δ6) KKQKQQ (inactive) 10 137-143 (Δ7) KKQKQQ (inactive) 11 Elimination of 137-144 (Δ8) KKQKQQ (inactive) 12 Elimination of 137-145 (Δ9) KKQKQQ (inactive) 13 Elimination of 137-145 (Δ9) KKRKRR (active)

[0055] In some respects, the RSV F protein disclosed in this document is altered only from a wild-type strain by deletions in the fusion domain, optionally with inactivation of the primary cleavage site. In other respects, additional alterations to the RSV F protein may be made. Typically, cysteine ​​residues are mutated. Typically, N-linked glycosylation sites are not mutated. Additionally, antigenic site II, also referred to in this document as the Palivizumab site due to the ability of the palivizumab antibody to bind to this site, is preserved. The Motavizumab antibody also binds to site II. Additional suitable RSV F proteins, incorporated by reference, are found in US Publication 2011 / 0305727, including, in particular, RSV F proteins containing the sequences spanning residues 100 to 150, as disclosed in Figure 1C in that document.

[0056] In certain other respects, the F1 or F2 domains of RSV may have modifications relative to the wild-type strain as shown in SEQ ID NO: 2. For example, the F1 domain may have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 alteration(s), which may be mutations or deletions. Similarly, the F2 domain may have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 alteration(s), which may be mutations or deletions. The F1 and F2 domains may each independently retain at least 90%, at least 94%, by Petition 870260074126, dated 07 / 24 / 2026, page 30 / 51 21 / 35 minus 95%, at least 96%, at least 98%, at least 99%, or 100% identity with the wild-type sequence.

[0057] In a particular example, a pharmaceutical product of RSV nanoparticles may contain about 0.025% to about 0.03% of PS80 with F of RSV in a range of about 270 μg / mL to about 300 μg / mL, or about 60 μg / mL to about 300 μg / mL. In other respects, the drug in nanoparticles may contain about 0.035% to about 0.04% of PS80 in a composition with F of RSV at 300 μg / mL to about 500 μg / mL. In still other aspects, the nanoparticle pharmaceutical product may contain approximately 0.035% to approximately 0.04% PS80 in a composition with an F concentration of RSV at 350-500 μg / mL.

[0058] Since antigen and detergent concentrations can vary, the amounts of each can be referred to as a molar ratio of non-ionic detergent:protein. For example, the molar ratio of PS80 to protein is calculated using the PS80 concentration and the antigen protein concentration measured by ELISA / A280 and their respective molecular weights. The molecular weight of PS80 used for the calculation is 1310, and using F of RSV as an example, the molecular weight of F of RSV is 65 kD. The molar ratio is calculated as follows: (PS80 concentration x 10 x 65000) / (1310 x F of RSV concentration in mg / mL). Thus, for example, the concentration of nanoparticles, measured by the protein, is 270 μg / mL and the PS80 concentrations are 0.015% and 0.03%. These have a molar ratio of PS80 to RSV F protein of 27:1 (i.e., 0.015 x 10 x 65000 / (1310 x 0.27)) and 55:1, respectively.

[0059] In particular aspects, the molar ratio is in a 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. Frequently, the non-ionic replacement detergent is PS80 and the molar ratio is about 30:1 to about 50:1, PS80: protein. For the Petition 870260074126, dated 07 / 24 / 2026, p. 31 / 51 22 / 35 RSV F glycoprotein nanoparticles, having a molar ratio in the range of 35:1 to about 65:1 and, particularly, a ratio of about 45:1, are especially stable. Modified Antigens

[0060] The antigens disclosed in this document encompass variations and mutants of these antigens. In certain respects, the antigen may share identity with a disclosed antigen. Generally, and unless specifically defined in the context of specifically identified antigens, the percentage of identity may be at least 80%, at least 90%, at least 95%, at least 97%, or at least 98%. The percentage identity can be calculated using the ClustalW2 alignment program, available at www.ebi.ac.uk / Tools / msa / clustalw2 / . The following default parameters can be used for pairwise alignment: Protein Weight Matrix = Gonnet; Penalty Opening = 10; Penalty Extension = 0.1.

[0061] In particular aspects, the protein contained in the nanoparticles consists of this protein. In other aspects, the protein contained in the nanoparticles comprises this protein. Additions to the protein itself may be for various purposes. In some aspects, the antigen may be extended at the N-terminus, the C-terminus, or both. In some aspects, the extension is a useful tag for a function, such as purification or detection. In some aspects, the tag contains an epitope. For example, the tag may be a polyglutamate tag, a FLAG tag, an HA tag, a polyHis tag (having about 510 histidines), a Myc tag, a Glutathione-Stransferase tag, a green fluorescent protein tag, a maltose-binding protein tag, a thioredoxin tag, or an Fc tag. In other aspects, the extension may be an N-terminal signal peptide fused to the protein to enhance expression.Although these signal peptides are frequently cleaved during expression in the cell, some... Petition 870260074126, dated 07 / 24 / 2026, page 32 / 51 23 / 35 nanoparticles can contain the antigen with an intact signal peptide. Thus, when a nanoparticle comprises an antigen, the antigen may contain an extension and therefore may be a fusion protein when incorporated into nanoparticles. For identity calculation purposes for the sequence, extensions are not included.

[0062] In some respects, the antigen may be truncated. For example, the N-terminus may be truncated by about 10 amino acids, about 30 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids, or about 200 amino acids. The C-terminus may be truncated instead of or in addition to the N-terminus. For example, the C-terminus may be truncated by about 10 amino acids, about 30 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids, or about 200 amino acids. For purposes of calculating identity with the protein having truncations, identity is measured over the remaining portion of the protein. Combination Nanoparticles

[0063] A combination nanoparticle, as used in this document, refers to a nanoparticle that induces immune responses 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 several pathogens can be combined into a single nanoparticle by linking them at the detergent exchange stage. The linking of the glycoproteins to the column followed by detergent exchange allows various types of glycoproteins to form around a detergent core, to provide a combination nanoparticle.

[0064] The disclosure also provides vaccine compositions that induce immune responses against two or more different pathogens by combining two or more nanoparticles, each Petition 870260074126, dated 07 / 24 / 2026, page 33 / 51 24 / 35 inducing a response against a different pathogen. Optionally, vaccine compositions may contain one or more combination nanoparticles alone or in combination with additional nanoparticles in order to maximize the immune response against multiple pathogens while reducing the number of vaccine compositions administered to the individual.

[0065] In another example, influenza and RSV cause respiratory disease and HA, NA and / or F of RSV can therefore be mixed in a nanoparticle combination or multiple nanoparticles can be combined in a vaccine composition to induce responses against RSV and one or more influenza strains. Vaccine compositions

[0066] The compositions disclosed in this document may be used prophylactically or therapeutically, but will typically be prophylactic. Consequently, the disclosure includes methods for treating or preventing infection. In some respects, the infection is caused by RSV. In some respects, the infection is a lower respiratory tract infection (LRTI). The methods involve administering to the individual a therapeutic or prophylactic amount of the immunogenic compositions of the disclosure. Preferably, the pharmaceutical composition is a vaccine composition that provides a protective effect. In other respects, the protective effect may include the improvement of a symptom associated with the infection in a percentage of the exposed population.For example, depending on the pathogen, the composition may prevent or reduce one or more of the following selected viral disease symptoms: fever, fatigue, muscle pain, headache, sore throat, vomiting, diarrhea, rash, symptoms of kidney and liver failure, internal bleeding, and external bleeding, compared to an untreated individual.

[0067] Nanoparticles can be formulated for administration as vaccines in the presence of various excipients, Petition 870260074126, dated 07 / 24 / 2026, page 34 / 51 25 / 35 buffers and the like. For example, vaccine compositions 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 particular cases, about 22 mM of 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, when present, may be about 150 mM. In certain compositions, for example, influenza vaccines, sodium chloride may be present in higher quantities, including approximately 200 mM, approximately 300 mM, or approximately 350 mM.

[0068] Certain nanoparticles, particularly RSV F nanoparticles, have enhanced stability at slightly acidic pH levels. For example, the pH range for the composition containing the nanoparticles can be from 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, the composition for RSV F protein nanoparticles is around pH 6.2. In other nanoparticles, the composition may tend towards neutral; for example, influenza nanoparticles may have a pH of about 7.0 to pH 7.4; often around pH 7.2. Adjuvants

[0069] In certain embodiments, the compositions disclosed in this document may be combined with one or more adjuvants to enhance an 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 in this document may provide protective immune responses when administered as a single dose. The Petition 870260074126, dated 07 / 24 / 2026, p. 35 / 51 26 / 35 aluminum-free compositions that induce robust immune responses are especially useful in adults aged 60 and over. Aluminum-based adjuvants

[0070] In some embodiments, the adjuvant may be alum (e.g., AlPO4 or Al(OH)s). Typically, the nanoparticle is substantially bound to the alum. For example, the nanoparticle may be at least 80% bound, at least 85% bound, at least 90% bound, or at least 95% bound to the alum. Frequently, the nanoparticle is 92% to 97% bound to the alum in a composition. The amount of alum present per dose is typically in a range between about 400 μg to about 1250 μg. For example, alum may be present in a dose quantity of approximately 300 μg to approximately 900 μg, approximately 400 μg to approximately 800 μg, approximately 500 μg to approximately 700 μg, approximately 400 μg to approximately 600 μg, or approximately 400 μg to approximately 500 μg. Typically, alum is present at approximately 400 μg for a 120 μg dose of the protein nanoparticle. Saponin Adjuvants

[0071] Adjuvants containing saponin may also be combined with the immunogens disclosed in this document. Saponins are glycosides derived from the bark of the quillaja tree Quillaja saponaria. Typically, saponin is prepared using a multi-step purification process, resulting in various fractions. As used in this document, the term “a quillaja saponaria saponin fraction” is used generically to describe a semi-purified or defined saponin fraction of Quillaja saponaria or a substantially pure fraction thereof. Saponin fractions

[0072] Several approaches to the production of saponin fractions are suitable. Fractions A, B, and C are described in US Pat. No. 6,352,697 and can be prepared as follows. A lipophilic fraction of Quil A, a crude aqueous extract of quillaja Petition 870260074126, dated 07 / 24 / 2026, page 36 / 51 27 / 35 Quillaja saponaria 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 with elution using a gradient of 25% to 60% acetonitrile in acidic water. The fraction referred to in this document as “Fraction A” or “QH-A” is the fraction that is eluted in approximately 39% acetonitrile. The fraction referred to in this document as “Fraction B” or “QH-B” is the fraction that is eluted in approximately 47% acetonitrile. The fraction referred to in this document as “Fraction C” or “QH-C” is the fraction that is eluted in approximately 49% acetonitrile. Additional information regarding the purification of fractions can be found in U.S. Patent No. 5,057,540.When prepared as described in this document, fractions A, B, and C of quillaja saponaria each represent groups or families of chemically related molecules with definable properties. The chromatographic conditions under which they are obtained are such that batch-to-batch reproducibility in terms of elution profile and biological activity is highly consistent.

[0073] Other saponin fractions have been described. Fractions B3, B4 and B4b are described in EP 0436620. Fractions QA1-QA22 are described in EP03632279 B2, Q-VAC (Nor-Feed, AS Denmark), quillaja Quillaja saponaria Spikoside (Isconova AB, Ultunaallén 2B, 756 51 Uppsala, Sweden). Fractions QA-1, QA-2, QA-3, QA-4, QA5, 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, especially QA-7, QA-17, QA-18 and QA-21 can be used. They are obtained as described in EP 0 3632 279 B2, especially on page 6 and in Example 1 on pages 8 and 9.

[0074] The saponin fractions described in this document and used to form adjuvants are frequently substantially pure fractions; that is, the fractions are Petition 870260074126, dated 07 / 24 / 2026, page 37 / 51 28 / 35 substantially free from contamination by other materials. In particular aspects, a substantially pure saponin fraction may contain up to 40% by weight, up to 30% by weight, up to 25% by weight, up to 20% by weight, up to 15% by weight, up to 10% by weight, up to 7% by weight, up to 5% by weight, up to 2% by weight, up to 1% by weight, up to 0.5% by weight, or up to 0.1% by weight of other compounds, such as other saponins or other adjuvant materials. ISCOM structures

[0075] Saponin fractions can be administered in the form of a cage-like particle, called an ISCOM (Immunostimulating Complex). ISCOMs can be prepared as described in EP0109942B1, EP0242380B1 and EP0180546 B1. In particular embodiments, a carrier and / or passenger antigen may be used, as described in EP 9600647-3 (PCT / SE97 / 00289). Matrix adjuvants

[0076] In some respects, ISCOM is an ISCOM matrix complex. An ISCOM matrix complex comprises at least a saponin moiety and a lipid. The lipid is at least a sterol, such as cholesterol. In particular aspects, the ISCOM matrix complex also contains a phospholipid. ISCOM matrix complexes may also contain one or more other immunomodulatory (adjuvant-active) substances, not necessarily a glycoside, and may be produced as described in EP0436620B1.

[0077] In other respects, ISCOM is an ISCOM complex. An ISCOM complex contains at least one saponin, at least one lipid, and at least one type of antigen or epitope. The ISCOM complex contains antigen associated with detergent treatment in such a way that a portion of the antigen integrates into the particle. In contrast, the ISCOM matrix is ​​formulated as a mixture with the antigen, and the association between the ISCOM matrix particles and the antigen is mediated by electrostatic and / or electrostatic interactions. Petition 870260074126, dated 07 / 24 / 2026, page 38 / 51 29 / 35 hydrophobic.

[0078] According to one embodiment, the saponin fraction integrated into an ISCOM matrix complex or an ISCOM complex, or at least one additional adjuvant, which is also integrated into the ISCOM matrix complex or ISCOM or mixed with it, is selected from fraction A, fraction B or fraction C of Quillaja saponaria, a semi-purified preparation of Quillaja saponaria, a purified preparation of Quillaja saponaria or any purified sub-fraction, for example, QA 1-21.

[0079] In particular aspects, each ISCOM particle may contain at least two saponin fractions. Any combinations of % by weight of different saponin fractions may be used. Any combination of % by weight of any two fractions may be used. For example, the particle may contain any % by weight of fraction A and any % by weight of another saponin fraction, such as a crude saponin fraction or fraction C, respectively. Therefore, in particular aspects, each ISCOM matrix particle or each ISCOM complex particle may contain from 0.1 to 99.9 by weight, 5 to 95% by weight, 10 to 90% by weight, 15 to 85% by weight, 20 to 80% by weight, 25 to 75% by weight, 30 to 70% by weight, 35 to 65% by weight, 40 to 60% by weight, 45 to 55% by weight, 40 to 60% by weight or 50% by weight of a saponin fraction, for example fraction A, and the remainder up to 100% in each case of another saponin, for example, any crude fraction or any other fraction, for example, fraction C.The weight is calculated as the total weight of the saponin fractions. Examples of ISCOM matrix complex and ISCOM complex adjuvants are disclosed in US Public Application No. 2013 / 0129770.

[0080] In particular embodiments, the ISCOM matrix or ISCOM complex comprises 5-99% by weight of one fraction, for example, fraction A, and the remainder up to 100% by weight of another fraction, for example, a crude saponin fraction or fraction C. The weight is calculated as the total weight of the saponin fractions. Petition 870260074126, dated 07 / 24 / 2026, pp. 39 / 51 30 / 35

[0081] In another embodiment, the ISCOM matrix or ISCOM complex comprises from 40% to 99% by weight of one fraction, for example, fraction A, and from 1% to 60% by weight of another fraction, for example, a crude saponin fraction or fraction C. The weight is calculated as the total weight of the saponin fractions.

[0082] In yet another embodiment, the ISCOM matrix or ISCOM complex comprises 70% to 95% by weight of one fraction, for example, fraction A, and 30% to 5% by weight of another fraction, for example, a crude saponin fraction, or fraction C. The weight is calculated as the total weight of the saponin fractions. In other embodiments, the saponin fraction of quillaja (Quillaja saponaria) is selected from any of QA 1-21.

[0083] In addition to particles containing mixtures of saponin fractions, ISCOM matrix particles and ISCOM complex particles may be formed using only one saponin fraction. The compositions disclosed in this document may contain multiple particles in which each particle contains only one saponin fraction. That is, certain compositions may contain one or more different types of ISCOM matrix complex particles and / or one or more different types of ISCOM complex particles, wherein each individual particle contains a saponin fraction from Quillaja saponaria, wherein the saponin fraction in one complex is different from the saponin fraction in the other complex particles.

[0084] In particular aspects, one type of saponin fraction or a crude saponin fraction may be integrated into an ISCOM matrix complex or particle and another type of substantially pure saponin fraction, or a crude saponin fraction, may be integrated into another ISCOM matrix complex or particle. A composition or vaccine may comprise at least two types of complexes or particles, each type having a type of saponins integrated into physically different particles.

[0085] In the compositions, mixtures of complex particles of Petition 870260074126, dated 07 / 24 / 2026, pp. 40 / 51 31 / 35 ISCOM matrix and / or ISCOM complex particles can be used wherein a quillaja saponin fraction and another quillaja saponin fraction are incorporated separately into different ISCOM matrix complex particles and / or ISCOM complex particles.

[0086] The ISCOM matrix or ISCOM complex particles, each having a saponin fraction, may be present in the composition in any combination of % by weight. In particular aspects, a composition may contain 0.1% to 99.9% by weight, 5% to 95% by weight, 10% to 90% by weight, 15% to 85% by weight, 20% to 80% by weight, 25% to 75% by weight, 30% to 70% by weight, 35% to 65% by weight, 40% to 60% by weight, 45% to 55% by weight, 40% to 60% by weight or 50% by weight of an ISCOM matrix or complex containing a first saponin fraction with the remaining portion consisting of an ISCOM matrix or complex containing a different saponin fraction. In some respects, the remaining portion is one or more ISCOM matrices or complexes in which each matrix or complex particle contains only one saponin fraction. In other respects, the ISCOM matrix or complex particles may contain more than one saponin fraction.

[0087] In particular compositions, the saponin fraction in a first ISCOM matrix or ISCOM complex particle is Fraction A and the saponin fraction in a second ISCOM matrix or ISCOM complex particle is Fraction C.

[0088] Preferred compositions comprise a first ISCOM matrix containing Fraction A and a second ISCOM matrix containing Fraction C, wherein the ISCOM matrix of Fraction A constitutes about 70% by weight of the total saponin adjuvant, and the ISCOM matrix of Fraction C constitutes about 30% by weight of the total saponin adjuvant. In another preferred composition, the ISCOM matrix of Fraction A constitutes about 85% by weight of the total saponin adjuvant and the ISCOM matrix of Fraction C constitutes about 15% by weight of the total saponin adjuvant. Petition 870260074126, dated 07 / 24 / 2026, pp. 41 / 51 32 / 35 Thus, in certain compositions, the ISCOM matrix of Fraction A is present in a range of about 70% to about 85%, and the ISCOM matrix of Fraction C is present in a range of about 15% to about 30%, of the total weight amount of saponin adjuvant in the composition. Fractions QS-7 and QS-21, for example, their production and use are described in US Patent Nos. 5,057,540; 6,231,859; 6,352,697; 6,524,584; 6,846,489; 7,776,343 and 8,173,141, which are incorporated by reference for these disclosures. Other Supporting Agents

[0089] In some cases, compositions of other adjuvants may be used in addition to, or as an alternative to, 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 anticipated within the scope of this disclosure. Other adjuvants include Freund's complete adjuvant (a nonspecific immune response stimulator containing killed Mycobacterium tuberculosis), Freund's incomplete adjuvants, 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, which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2% squalene / Tween® 80 emulsion.In some embodiments, the adjuvant may be a paucilamellar lipid vesicle; for example, Novasomes®. Novasomes® are paucilamellar non-phospholipid vesicles ranging in size from about 100 nm to about 500 nm. They comprise Brij 72, cholesterol, oleic acid, and squalene. Novasomes have been shown to be an effective adjuvant (see U.S. Patents Nos. 5,629,021, 6,387,373, and 4,911,928). Administration and Dosage

[0090] The compositions disclosed in this document may be Petition 870260074126, dated 07 / 24 / 2026, pp. 42 / 51 33 / 35 administered via a systemic route or a mucosal route or a transdermal route or directly into a specific tissue. As used in this document, the term “systemic administration” includes parenteral routes of administration. In particular, parenteral administration includes subcutaneous, intraperitoneal, intravenous, intra-arterial, intramuscular or intrasternal, intravenous or renal dialysis infusion techniques. Typically, systemic parenteral administration is intramuscular injection. As used in this document, the term “mucosal administration” includes oral, intranasal, intravaginal, intrarectal, intratracheal, intestinal and ophthalmic administration. Preferably, administration is intramuscular.

[0091] 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 immunization schedule. In a multiple-dose schedule, the various doses can be administered by the same or different routes, for example, an initial parenteral dose and a mucosal booster, an initial mucosal dose and a parenteral booster, etc. In some cases, a subsequent booster dose is administered approximately 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 5 weeks, or approximately 6 weeks after the previous dose. Typically, however, the compositions disclosed in this document are administered only once, but still provide a protective immune response.

[0092] In some embodiments, the dose, measured in μg, may be the total weight of the dose including the solute, or the weight of the RSV F nanoparticles or the weight of the RSV F protein. The dose is measured using the A280 protein concentration assay or ELISA.

[0093] The antigen dose, including for pediatric administration, may be in the range of approximately 30 μg to approximately 300 μg, approximately 90 μg to approximately 270 μg, approximately 100 μg to approximately Petition 870260074126, dated 07 / 24 / 2026, pp. 43 / 51 34 / 35 160 μg, approximately 110 μg to approximately 150 μg, approximately 120 μg to approximately 140 μg, or approximately 140 μg to approximately 160 μg. In particular embodiments, the dose is approximately 120 μg, administered with alum. In some cases, a pediatric dose may be in the range of approximately 30 μg to approximately 90 μg. Certain populations may be administered with or without adjuvants. For example, when administered to the elderly, alum is preferably not used. In certain cases, the compositions may be free of added adjuvant. Under such circumstances, the dose may be increased by approximately 10%.

[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 is a typical amount.

[0095] In particular embodiments of an RSV vaccine, the dose may comprise an RSV F protein concentration of approximately 175 μg / mL to approximately 325 μg / mL, approximately 200 μg / mL to approximately 300 μg / mL, approximately 220 μg / mL to approximately 280 μg / mL, or approximately 240 μg / mL to approximately 260 μg / mL.

[0096] Compositions containing RSV F protein, such as vaccine compositions and nanoparticles, are further described in U.S. Application No. 16 / 009,257 and U.S. Application No. 15 / 819,962, both of which are incorporated herein by reference in their entirety for all purposes.

[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. EXAMPLES EXAMPLE 1 - Protection of infants against RSV lower respiratory tract infection (LRTI) through vaccination of pregnant mothers

[0098] A vaccine composition comprising a Petition 870260074126, dated 07 / 24 / 2026, pp. 44 / 51 35 / 35 recombinant RSV fusion protein (F) nanoparticle with aluminum adjuvant was administered to women at approximately 28 to 33 weeks of gestation. The results showed that the vaccine protected the children from serious consequences of RSV infection, including severe hypoxemia. The protective effect reduced hospitalization.

[0099] The vaccine efficacy rates against RSV LRTI hospitalization were 53 percent and against severe RSV hypoxemia were 70 percent during the first 90 days of life of the infants. In stark contrast, administering the vaccine to women more than 33 weeks pregnant showed that the vaccine efficacy rates were substantially reduced. Administration beyond 33 weeks results in efficacy rates of only 26 percent against LRTI hospitalization and 44% against severe RSV hypoxemia, measured during the first 90 days of life of their infants.

[00100] This study highlights the surprising finding that administering the vaccine to women during a narrow window of pregnancy can have significantly beneficial outcomes for babies after birth. These results represent the first time that an RSV vaccine composition has shown high efficacy rates against severe hypoxemia caused by RSV infection in a Phase III trial. Petition 870260074126, dated 07 / 24 / 2026, pages 45 / 51

Claims

1 / 2 CLAIMS 1. Use of a composition comprising an RSV F glycoprotein nanoparticle and a saponin-based adjuvant characterized in being in the preparation of a medicament for immunizing a pregnant woman carrying a gestational baby, wherein immunization induces an immune response against at least one symptom associated with RSV lower respiratory tract infection (LRTI) in the baby after birth and wherein the pregnant woman is about 28 weeks to about 33 weeks pregnant.

2. Use according to claim 1, characterized in that at least one symptom is hypoxemia.

3. Use, according to claim 1 or 2, characterized in that the saponin-based adjuvant comprises a saponin fraction of Quillaja saponaria Molina.

4. Use according to claim 3, characterized in that the saponin fraction comprises Fraction A and Fraction C.

5. Use according to claim 4, characterized in that the saponin fraction comprises a first ISCOM matrix containing Fraction A and a second ISCOM matrix containing Fraction C.

6. Use according to claim 5, characterized in that Fraction A constitutes about 70% to about 85% by weight of the total saponin adjuvant and Fraction C constitutes about 15% to about 30% by weight of the total saponin adjuvant.

7. Use, according to any one of claims 1 to 6, characterized in that the RSV F glycoprotein nanoparticle comprises a nonionic detergent core and an RSV F glycoprotein, wherein the RSV F glycoprotein is associated with the core and the detergent is present in about 0.03% to about 0.05%.

8. Use, according to claim 7, characterized in that the detergent is selected from the group consisting of Petition 870260074126, dated 07 / 24 / 2026, page 46 / 51 2 / 2 PS-20, PS-40, PS-60, PS-65 and PS-80.

9. Use, according to any one of claims 1 to 8, characterized in that the F glycoprotein of RSV 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 to 136 of SEQ ID NO: 2, wherein the primary furin cleavage site is inactivated by mutation.

10. Use, according to any one of claims 1 to 9, characterized in that the F glycoprotein of RSV is selected from the group consisting of SEQ ID NOs: 3-12.

11. Use, according to claim 10, characterized in that the F glycoprotein of RSV is encoded by SEQ ID NO:

8.

12. Use, according to any one of claims 1 to 11, characterized in that the F glycoprotein of RSV comprises SEQ ID NO:

19. Petition 870260074126, dated 07 / 24 / 2026, pp. 47 / 51