Conformationally stabilized RSV pre-fusion f proteins

Abstract

To provide an RSV F protein stabilized in its pre-fusion conformation, serving as a candidate of RSV vaccine immunogen.SOLUTION: The present invention provides mutant RSV F molecules, such as those that can be, or are stabilized, in a pre-fusion conformation by the introduction of one or more DT cross-links. The present invention also provides methods of making such mutant RSV F molecules, compositions comprising such mutant RSV F molecules, and methods of use of such mutant RSV F molecules, for example in vaccination methods, therapeutic methods, and antibody production methods.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 542,247, filed August 7, 2017, U.S. Provisional Patent Application No. 62 / 629,685, filed February 12, 2018, U.S. Provisional Patent Application No. 62 / 640,467, filed March 8, 2018, and U.S. Provisional Patent Application No. 62 / 674,791, filed May 22, 2018, the contents of each of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. Said ASCII copy, created on August 3, 2018, is named Avatar_009_WO1_SL.txt and is 452,739 bytes in size.

[0003] STATEMENT REGARDING FEDERALLY FUNDED RESEARCH This invention was made with Government support under Grant No. AI112124 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0004] Incorporation by Reference The contents of all references cited herein are hereby incorporated by reference in their entirety solely to the extent that the law permits incorporation by reference. [Background technology]

[0005] Respiratory syncytial virus (RSV) infects 4-5 million children annually in the United States, making it the leading cause of hospitalization in infants (approximately 150,000 hospitalizations). Globally, RSV accounts for 6.7% of deaths in infants under 1 year of age, second only to malaria. In addition, it poses a serious threat to other high-risk groups, including the elderly and immunocompromised, causing an estimated 180,000 additional hospitalizations and 12,000 deaths in the United States. There is no current frontline treatment for RSV, and the only currently approved preventive treatment for RSV is passive administration of the approved monoclonal antibody Synagis® (palivizumab), which recognizes the RSV fusion (F) protein and reduces the incidence of severe disease by approximately 50%. Due to the high cost of Synagis® prophylaxis, its use is limited to premature infants and infants under 24 months of age with congenital heart disease. For a review, see Costello et al., “Targeting RSV with Vaccines and Small Molecule Drugs, Infectious Disorders,” Drug Targets, 2012, vol. 12, no. 2. The development of a more effective, and ideally more cost-effective, RSV vaccine would be of enormous value.

[0006] The RSV F protein is an RSV envelope glycoprotein. In nature, the RSV F protein is translated as a single precursor polypeptide designated "F0". The F0 precursor polypeptide is generally 574 amino acids in length, of which amino acids 1-25 constitute a signal peptide. The precursor polypeptide F0 forms a precursor trimer, which is proteolytically cleaved by cellular proteases to produce a Pep 27 polypeptide, an F1 polypeptide, and an F2 polypeptide. The F2 polypeptide typically comprises amino acid residues 26-109 of the F0 precursor. The F1 polypeptide typically comprises amino acid residues 137-574 of the F0 precursor. The F1 and F2 polypeptides are linked by disulfide bonds to form a heterodimer called the RSV F "protomer". Three such protomers form the mature trimeric form of the RSV F molecule.

[0007] The RSV F protein is known to induce strong neutralizing antibodies that are involved in protection against RSV. In recent years, it has been shown that the pre-fusion conformation of the RSV F protein trimer (hereinafter sometimes referred to as "pre-fusion F" or "pre-F") is the most determinant of neutralizing activity in human serum. The most potent neutralizing antibodies (nAbs) isolated to date also specifically bind only to the pre-fusion conformation. However, soluble pre-F is highly unstable and easily transitions to the post-fusion conformation, limiting its usefulness as an immunogen for vaccines.

[0008] The RSV F protein stabilized in the prefusion conformation is a potentially highly valuable candidate RSV vaccine immunogen.

[0009] The crystal structure of the RSV F protein (bound to the potent neutralizing Ab D25) in its pre-fusion conformation is described in McLellan et al., 2013, Science, 340, p. 1113-1117, the contents of which are incorporated herein by reference in their entirety.

[0010] Several different approaches have been developed to stabilize RSV F in its pre-F conformation. For example, Marshall et al. have previously described the introduction of "tyrosine-to-point mutations and dityrosine (DT) bridges at certain positions in the RSV F protein. See International Patent Application PCT / US2014 / 048086. Other approaches include the introduction of "cysteine-to-point mutations, disulfide bonds, and / or cavity-filling amino acid substitutions at certain positions in the RSV F protein. See U.S. Patent No. 9,738,689, U.S. Patent No. 9,950,058, or McLellan et al. (2013) Science 342: 592-598. Summary of the Invention

[0011] The present invention provides certain new advances and improvements over the work previously described by Marshall et al. in International Patent Application PCT / US2014 / 048086, including, but not limited to, providing certain novel mutant RSV F molecules that are not described in PCT / US2014 / 048086 and have improved properties compared to the molecules described in PCT / US2014 / 048086, as well as compared to other RSV F variants and candidate RSV F immunogens. As shown in the examples herein, these novel mutant RSV F molecules are highly immunogenic when administered to animals in vivo using recognized preclinical animal models.

[0012] In some embodiments, the present invention provides a mutant RSV F molecule that includes a point mutation at amino acid position 428 to tyrosine (abbreviated as "Try" or "Y" according to standard convention), i.e., a "428Y mutation." As further described in the Examples herein, mutant RSV F molecules that include the 428Y mutation (referred to as "428Y mutants") have improved properties compared to identical mutant RSV F molecules that include the 427Y mutation instead of the 428Y mutation. These improved properties include (i) an approximately 5-fold increase in serum neutralization titers after vaccination, (ii) increased serum neutralization titers specific for antigenic sites IV-V and pre-fusion specific antigenic site "O" (translator's note: "O" is a

[0013]

number

[0014] The term "RSV F" refers to the letter "RSV F". This note will be omitted below. Also, "o" is a lowercase letter.) The antigen profile determined by binding to an antibody specific for RSV F is significantly improved, and (iii) the production of RSV F trimeric complexes is significantly increased compared to hexameric complexes. Based on these discoveries, the present invention provides various novel and improved mutant RSV F molecules, including, but not limited to, mature RSV F trimers, mature RSV F trimers conformationally locked in a pre-fusion conformation by one or more DT bridges, various precursors thereof, and various components thereof (including F1 and F2 polypeptides and promoters). The present invention also provides amino acid sequences of such mutant RSV F molecules, sequences encoding such mutant RSV F molecules, vectors containing such sequences, cells containing and / or expressing such sequences or vectors, methods for producing such mutant RSV F molecules, and methods of using such mutant RSV F molecules, including, but not limited to, in vivo use for vaccination against RSV and as an immunogen for antibody production.

[0015] Thus, in one embodiment, the present invention provides a mutant RSV F molecule comprising a point mutation to tyrosine at amino acid position 428 (i.e., a 428Y mutation), or a point mutation to tyrosine at an amino acid position corresponding to 428, e.g., as determined by alignment with SEQ ID NO: 1 and / or as determined using the amino acid numbering of SEQ ID NO: 1. In some embodiments, such mutant RSV F molecules are or include RSV F monomers or promoters each containing a 428Y mutation. In some embodiments, such mutant RSV F molecules are or include RSV F trimers (e.g., mature RSV F trimers) containing three 428Y mutations.

[0016] In some embodiments, such mutant RSV F molecules containing the 428Y mutation also contain a point mutation to tyrosine at amino acid position 226 (i.e., a 226Y mutation), or a point mutation to tyrosine at an amino acid position corresponding to 226, e.g., as determined by alignment with SEQ ID NO: 1 and / or as determined using the amino acid numbering of SEQ ID NO: 1. In some embodiments, such mutant RSV F molecules are or contain RSV F monomers or promoters each containing both a 428Y mutation and a 226Y mutation. In some embodiments, such mutant RSV F molecules are or contain RSV F trimers (e.g., mature RSV F trimers) containing three 428Y mutations and three 226Y mutations.

[0017] In some embodiments, such mutant RSV F molecules containing the 428Y mutation also contain a point mutation to tyrosine at amino acid position 185 (i.e., a 185Y mutation), or a point mutation to tyrosine at an amino acid position corresponding to 185, for example, as determined by alignment with SEQ ID NO: 1 and / or as determined using the amino acid numbering of SEQ ID NO: 1. In some embodiments, such mutant RSV F molecules are or contain RSV F monomers or promoters each containing both a 428Y mutation and a 185Y mutation. In some embodiments, such mutant RSV F molecules are or contain RSV F trimers (e.g., mature RSV F trimers) containing three 428Y mutations and three 185Y mutations.

[0018] In some embodiments, the mutant RSV F molecule also includes (a) a point mutation to tyrosine at amino acid position 226 (i.e., a 226Y mutation), or a point mutation to tyrosine at an amino acid position corresponding to 226, e.g., when aligned with SEQ ID NO: 1 and / or when determined using the amino acid numbering of SEQ ID NO: 1, and (b) a point mutation to tyrosine at amino acid position 185 (i.e., a 185Y mutation), or a point mutation to tyrosine at an amino acid position corresponding to 185, e.g., when aligned with SEQ ID NO: 1 and / or when determined using the amino acid numbering of SEQ ID NO: 1. In some embodiments, the mutant RSV F molecule is or includes a RSV F monomer or promoter, each of which includes a 428Y mutation, a 226Y mutation, and a 185Y mutation. In some embodiments, the mutant RSV F molecule is or includes an RSV F trimer (e.g., a mature RSV F trimer) that includes three 428Y mutations, three 226Y mutations, and three 185Y mutations.

[0019] Each of the various mutant RSV F molecules described herein can also contain various other mutations.

[0020] In some embodiments, the mutant RSV F molecules described herein also include one or more additional tyrosine mutations, such as one or more of the tyrosine mutations described in International Patent Application PCT / US2014 / 048086 (WO 2015 / 013551), the contents of which are incorporated herein by reference.

[0021] In some embodiments, the mutant RSV F molecules described herein also include one or more cysteine ​​mutations, such as one or more of those described in U.S. Patent No. 9,738,689, U.S. Patent No. 9,950,058, or McLellan et al. (2013) Science 342: 592-598, each of which is incorporated herein by reference. In some such embodiments, the RSV F molecule includes a so-called "DS" mutation described in U.S. Patent No. 9,738,689, U.S. Patent No. 9,950,058, or McLellan et al. (2013) Science 342: 592-598. For example, in some embodiments, the RSV F molecule includes a 155C mutation, a 290C mutation, or both a 155C mutation and a 290C mutation.

[0022] In some embodiments, the mutant RSV F molecules described herein also include one or more cavity-filling amino acid substitutions, such as one or more of those described in U.S. Patent No. 9,738,689, U.S. Patent No. 9,950,058, or McLellan et al. (2013) Science 342: 592-598, each of which is incorporated herein by reference. In some such embodiments, the RSV F molecule includes a so-called "Cav1" mutation described in U.S. Patent No. 9,738,689, U.S. Patent No. 9,950,058, or McLellan et al. (2013) Science 342: 592-598. For example, in some embodiments, the RSV F molecule includes an S190F cavity-filling mutation, a V207L ​​cavity-filling mutation, or both an S190F cavity-filling mutation and a V207L ​​cavity-filling mutation.

[0023] In some embodiments, the mutant RSV F molecules described herein further comprise a heterotrimerization domain. In some such embodiments, the trimerization domain is a foldon domain.

[0024] In some embodiments, the mutant RSV F molecules described herein further comprise one or more tags that can be useful for detecting and / or purifying the RSV F molecule. In some embodiments, the tag can be a cleavable tag. In some embodiments, the tag is a His tag, a Strep tag, or a StrepII tag.

[0025] Each of the mutant RSV F molecules described herein can be provided in either a membrane-bound or soluble (i.e., non-membrane-bound) form. In some embodiments, the membrane-bound form includes the transmembrane and cytoplasmic domains of the naturally occurring RSV F protein, the details of which (including sequences) are set forth in the detailed description herein. In some embodiments, the membrane-bound form may include other transmembrane and cytoplasmic domains that are not native to the RSV F protein. In some embodiments, a soluble (i.e., non-membrane-bound) form of the mutant RSV F molecule described herein is used. The soluble form may be particularly useful when the RSV F molecule is used as a component of a pharmaceutical composition, for example, when used as a vaccine for protection against RSV. Such a soluble form of the mutant RSV F molecule described herein can be produced, for example, by removing the transmembrane and cytoplasmic domains, as further described in the detailed description herein. In some such embodiments, heterologous sequences may be added to replace transmembrane and cytoplasmic domains, e.g., heterotrimerization domains, e.g., foldon domains, as further described in the detailed description herein.

[0026] Each of the mutant RSV F molecules described herein can be provided in the form of a mature RSV F trimer that contains one or more DT bridges and is conformationally locked in its pre-F conformation. Such conformationally locked RSV F molecules can be produced by performing a DT bridge reaction as described in the detailed description and examples of this specification and / or as described in International Patent Application PCT / US2014 / 048086 (WO 2015 / 013551), the contents of which are incorporated herein by reference.

[0027] In some embodiments, the present invention provides a mutant RSV F molecule as described herein that comprises at least one DT bridge. In some embodiments, the present invention provides a mutant RSV F molecule as described herein that comprises at least three DT bridges. In some embodiments, the present invention provides a mutant RSV F molecule as described herein that comprises at least six DT bridges.

[0028] In some embodiments, the present invention provides a mutant RSV F molecule as described herein, comprising at least one DT bridge, wherein at least one tyrosine of at least one DT bridge is introduced by a point mutation to tyrosine. In some embodiments, the present invention provides a trimeric mutant RSV F molecule as described herein, comprising at least three DT bridges, wherein at least one tyrosine of each of the at least three DT bridges is introduced by a point mutation to tyrosine.

[0029] For example, in some embodiments, a DT bridge is introduced between a tyrosine at residue 198 (generally a naturally occurring tyrosine) and an introduced tyrosine at residue 226 (i.e., resulting from a 226Y mutation) to form an intramolecular 198Y-226Y DT bridge, which creates an RSV F trimer containing three intramolecular DT bridges.

[0030] In some embodiments, a DT bridge is introduced between the introduced tyrosine at residue 185 (i.e., resulting from a 185Y mutation) and the introduced tyrosine at residue 428 (i.e., resulting from a 428Y mutation) to form a 185Y-428Y intermolecular DT bridge, thereby creating an RSV F trimer containing three intermolecular DT bridges.

[0031] In some embodiments, both a 198Y-226Y intramolecular DT bridge and a 185Y-428Y intermolecular DT bridge are introduced, creating an RSV F trimer containing six DT bridges, i.e., three intramolecular DT bridges and three intermolecular DT bridges.

[0032] In some embodiments, the present invention provides a composition comprising one or more of the mutant RSV F molecules described herein. In some such embodiments, the composition is a pharmaceutical composition, i.e., its components are suitable for administration to a living subject. In some such embodiments, the pharmaceutical composition comprises one or more adjuvants. Exemplary adjuvants include, but are not limited to, alum adjuvants. In some embodiments, the pharmaceutical composition comprises a carrier. In some embodiments, the pharmaceutical composition comprises an immunostimulant. In some embodiments, the composition is, or forms part of, a RSV vaccine, or can be used in a method of vaccination.

[0033] In some embodiments, the present invention provides nucleic acid molecules encoding the various different mutant RSV F molecules described herein. In some embodiments, the present invention provides vectors comprising such nucleic acid molecules. In some embodiments, the present invention provides cells comprising such nucleic acid molecules or vectors.

[0034] In some embodiments, the present invention provides a method for treating or preventing RSV in a subject. In some embodiments, the present invention provides a method for vaccinating a subject against RSV. In some embodiments, the present invention provides a method for inducing the production of RSV neutralizing antibodies in a subject. Each of such methods includes administering to a subject an effective amount of a mutant RSV F molecule described herein, or an effective amount of a pharmaceutical composition comprising such a mutant RSV F molecule. In some such embodiments, the subject is a non-human mammal. For example, in some embodiments, the subject may be a non-human mammal useful for conducting preclinical trials, such as a rodent (e.g., a mouse or cotton rat) or a non-human primate. In some embodiments, the subject may be a human. In embodiments in which the subject is a human, the human subject can be of any age. In some embodiments, the human subject is an infant. In some embodiments, the human subject is a child. In some embodiments, the human subject is less than 24 months of age. In some embodiments, the human subject is an adult. In some embodiments, the human subject is over 50 years of age. In some embodiments, the human subject is over 60 years of age.

[0035] In some embodiments, the present invention provides a method for producing anti-RSV F antibodies using the mutant RSV F molecules described herein as immunogens. Such antibodies can be useful for a variety of applications, including research and therapeutic applications. For example, in some embodiments, such methods include administering the mutant RSV F molecules described herein to an animal subject, such as a mouse or rabbit, that is useful for producing antibodies. In some embodiments, the subject may be an animal that has been genetically modified to produce fully human antibodies. In other embodiments, the mutant RSV F molecules described herein can be used as immunogens in other systems useful for producing antibodies, including, but not limited to, library screening methods, such as phage display screening methods.

[0036] These and other aspects of the invention are further described in this patent application's detailed description, examples, claims, drawings, and brief description of the drawings, all of which are intended to be read in combination with one another. Moreover, those skilled in the art will recognize that the various aspects of the invention described herein can be combined in a variety of different ways, and that such combinations are within the scope of the invention. [Brief description of the drawings]

[0037] [Figure 1] Exemplary nucleic acid vectors for expressing the RSV F molecules described herein in mammalian cells. [Diagram 2] Schematic diagram of mutant RSV F molecules containing a 198Y-226Y DT bridge ("Design 1") and a 185Y-428Y DT bridge (Design 2). The antigenic sites "O", IV, and V in the structure of the molecule are shown. [Diagram 3] Figure 3A: After performing the DT cross-linking reaction, the stability introduced by intramolecular cross-linking between 198Y and 226Y ("Design 1") was measured over 2 weeks at 4°C by ELISA using D25, a prefusion specific mAb that binds to site "O". Figure 3B: Intermolecular DT bond formation between 185Y and 428Y ("Design 2") was assayed by comparison to WT by Western blotting (Motavizumab is the primary Ab). The F1 protein migrates to the size of a trimer (TM). [Figure 4] Schematic diagram of an exemplary protocol for preparing DT cross-linked AVR02. [Diagram 5] Comparison of conformational integrity of DS-Cav1, uncrosslinked 428Y RSV F mutant (AVR02), and DT crosslinked 428Y mutant (DT-AVR02) by ELISA using motavizumab for normalization and antibodies binding to antigenic sites IV / V (Figure 5A) and site "O" (Figure 5B) to examine conformational integrity. [Figure 6]Antigenicity of site "O" after 100 hours of heat challenge at 37°C. Figure 6A: Comparison of retained binding by 5C4 (site "O") to 428Y RSV F mutant (AVR02) and DT crosslinked form of 428Y RSV F mutant (DT-AVR02) after incubation at 4°C and 37°C. Figure 6B: Retained binding of 5C4 and D25 to DS-Cav1 and DT-AVR02 after 100 hours of incubation at 37°C. The left bar of each pair is 5C4. The right bar of each pair is D25. Figure 6C: Retained binding by 5C4 (site "O") to 428Y RSV F mutant (AVR02) after 5 weeks at 4°C. [Figure 7] Efficacy of DT cross-linked 428Y RSV F mutant (DT-AVR02 or DT-preF) in vivo in mice. Neutralization titers. RSV-Renilla luciferase neutralization titers from animals vaccinated with either DSCAV1 or DT-AVR02 supplemented with alum. [Figure 8] Schematic diagram of an exemplary vaccination schedule. [Figure 9] In vivo efficacy of DT-crosslinked 428Y RSV F mutant (referred to as DT-AVR02 or DT-preF) in cotton rats. Effect of DT-crosslinked 428Y RSV F mutant adjuvanted with Alhydrogel® 2% on RSV / A / Tracy titers in nasal washes on day +39. RSV titers are shown for each of the control and treatment groups indicated. [Figure 10] In vivo efficacy of DT cross-linked 428Y RSV F mutant (DT-AVR02 / DT-preF) in cotton rats. DT cross-linked 428Y RSV F mutant adjuvanted with Alhydrogel® 2% on RSV / A / Tracy titers in lung lavage fluid on day +39. RSV titers are shown for each of the control and treatment groups indicated. [Figure 11]In vivo efficacy of DT cross-linked 428Y RSV F mutant (DT-AVR02 / DT-preF) in cotton rats. Effect of DT-preF adjuvanted with Alhydrogel® 2% on the production of RSV / A / Tracy serum neutralizing antibodies. [Figure 12] In vivo efficacy of DT cross-linked 428Y RSV F mutant (DT-AVR02 / DT-preF) in cotton rats. Effect of DT-preF adjuvanted with Alhydrogel® 2% on the generation of RSV / B / 1853 cross-reactive neutralizing antibodies. [Figure 13] The tyrosine substitution at position 428 (448Y) rather than position 427 (447Y) dramatically improves the antigenic profile of the RSV F mutant. Streptactin®-purified soluble 427Y (DT-Cav1) mutant (FIGS. 13A-B) or 428Y (DT-AVR02) mutant (FIGS. 13C-D) were subjected to DT cross-linking, purified, and analyzed for antigenicity using the indicated mAbs. ELISA was performed on Ni-NTA-coated plates, normalized for total protein using motavizumab. DSCav1 was used as a positive control. The prefusion structure was probed using two conformational antibodies, a mAb specific for site "O" and a prefusion-specific mAb that binds between antigenic sites IV / V. [Figure 14] Size exclusion chromatography (SEC) and SDS-PAGE analysis reveal significantly different profiles of the DT-crosslinked 427Y RSV F mutant (DT-Cav1) and the DT-crosslinked 428Y RSV F mutant (DT-AVR02). Size exclusion chromatography profiles of the indicated RSV F mutants (427Y / DT-Cav1 FIG. 14A top, left, and 428Y / DT-AVR02 FIG. 14B bottom, left) immediately after the dityrosine crosslinking reaction are shown. The 428Y mutation results in a dramatic reduction in higher order species after the crosslinking reaction. The final product after separation under reducing and denaturing conditions on an SDS-PAGE gel and Coomassie staining is shown in FIG. 14C (right). Arrows indicate the higher order species (427Y / DT-Cav1) and the crosslinked trimer species (428Y / DT-AVR02). [Figure 15] Serum neutralization titers of animals vaccinated with DT-bridged 428Y RSV F mutant (DT-AVR02) are dramatically higher than those of animals vaccinated with DT-bridged 427Y RSV F mutant (DT-Cav1). Animals were vaccinated with 10 μg of either the DS-Cav1 control (benchmark comparison standard) or the DT bridged molecules shown (427Y / DT-Cav1 in FIG. 15A (left) and 428Y / DT-AVR02 in FIG. 15B (right)) in a prime-boost regimen. Serum was collected after boosting, heat inactivated, and neutralization titers were obtained using the RSV-Renilla luciferase reporter virus. Neutralization titers shown are the average of 5 animals / group and are calculated as the reciprocal serum dilution resulting in 50% inhibition of luciferase activity. [Figure 16] Formulation with sucrose reduces the formation of RSV F aggregates. The experiment was performed with the DT-bridged 428Y mutant (DT-AVR02). After final concentration, analytical size exclusion chromatography (SEC) was performed. As shown in FIG. 16A, three peaks were identified, of which peak C is a trimer and peaks A and B are aggregates. Formulation of DT-AVR02 molecules with 10% sucrose during the elution of the purification step appears to eliminate the formation of aggregates. As shown in FIG. 16B, in the sample formulated with 10% sucrose, peak C (trimer) remained, but peaks A and B (aggregates) were not evident. Detailed Description of the Invention

[0038] The present invention provides, in part, mutant RSV F molecules that contain one or more tyrosine mutations and can be or are stabilized in a pre-fusion conformation by introducing one or more DT bridges. The present invention also provides nucleic acid molecules encoding such mutant RSV F molecules, methods for making such mutant RSV F molecules, compositions comprising such mutant RSV F molecules, and methods of using such mutant RSV F molecules, including, but not limited to, vaccination methods, treatment methods, and antibody production methods.

[0039] definition Scientific and technical terms used in this disclosure have meanings that are commonly understood by those of ordinary skill in the art and / or that are clear from the context in which the term is used, unless specifically defined otherwise herein. Some terms are defined below. Other terms are defined elsewhere in this specification.

[0040] As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The terms "a" (or "an") and "one or more" and "at least one" can be used interchangeably.

[0041] Furthermore, "and / or" shall be construed as specifically disclosing each of the two specified features or components with or without the other. Thus, the term "and / or" when used in a phrase such as "A and / or B" is intended to include A and B, A or B, A (alone), and B (alone). Similarly, the term "and / or" when used in a phrase such as "A, B, and / or C" is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).

[0042] Units, prefixes, and symbols are written in the form accepted by the International System of Units (SI). Numerical ranges given herein are inclusive of the numbers defining the range.

[0043] As used herein, the terms "about" and "approximately" when used in connection with a numerical value mean within a range of + or -20% of that value.

[0044] The word "exemplary" is used herein means serving as an example, instance, or illustration.

[0045] As used herein, the term "RSV F molecule" refers to any form of the RSV F protein, including the F1 and F2 polypeptides, i.e., the F0 precursor polypeptide, the trimer of the F0 precursor polypeptide, the RSV F promoter, and the mature RSV F trimer, which may be membrane-bound or soluble. As used herein, the term "mutated RSV F molecule" refers to a RSV F molecule that contains one or more artificially introduced / manufactured mutations.

[0046] As used herein, the term "F1 polypeptide" refers to a polypeptide comprising amino acid residues 137-513 of the RSV F F0 precursor sequence. Amino acid residues 137-513 do not include the RSV F transmembrane and cytoplasmic domains. In some embodiments, the F1 polypeptide may also include the RSV F transmembrane and cytoplasmic domains (located within residues 514-574 of the RSV F F0 precursor sequence).

[0047] As used herein, the term "F2 polypeptide" refers to a polypeptide comprising amino acid residues 26-109 of the RSV F F0 precursor sequence.

[0048] As used herein, the terms "protein" and "polypeptide" are used interchangeably unless otherwise specified. As used herein, the term "protein complex" is an assembly of two or more proteins or protein subunits, e.g., two or more monomers or protomers. Unless otherwise specified, all descriptions herein relating to proteins or polypeptides apply equally to protein complexes, and vice versa.

[0049] As used herein, the terms "nucleic acid molecule," "nucleic acid sequence," and "base sequence" are used interchangeably.

[0050] As used herein, the terms "stabilization" and "fixation" are used interchangeably, for example, in reference to the effect of cross-linking in stabilizing or fixing the RSV F protein in its pre-fusion conformation. These terms do not require 100% stability. Rather, they refer to some degree of improved or increased stability. For example, in some embodiments, when the term "stabilization" is used in reference to a RSV F protein that is cross-linked in a pre-fusion conformation, the term means that the pre-fusion conformation has greater stability than it would have before or in the absence of such cross-linking. Stability and relative stability may be measured in a variety of ways, for example, based on the half-life of the pre-fusion conformation of RSV, as described elsewhere in this application. The improvement or increase in stability may be any degree that is useful or important for the intended application. For example, in some embodiments, stability may be increased by about 10%, 25%, 50%, 100%, 200% (i.e., 2-fold), 300% (i.e., 3-fold), 400% (i.e., 4-fold), 500% (i.e., 5-fold), 1000% (i.e., 10-fold), or more.

[0051] The term "adjuvant" as used herein refers to a substance that can enhance, accelerate, or prolong the body's immune response to an immunogen or immunogenic composition, such as a vaccine.

[0052] As used herein, the term "DS-Cav1" refers to the mutant RSV F protein described in McLellan et al., Science, 342(6158), 592-598, 2013, which includes the S155C, S290C, S190F, and V207L ​​mutations.

[0053] As used herein, the term "pre-fusion conformation" refers to a conformation of the RSV F molecule that can be specifically bound by a pre-fusion specific antibody.

[0054] As used herein, the term "pre-fusion specific antibody" refers to an antibody that specifically binds to the RSV F molecule in a pre-fusion conformation but does not bind to the RSV F protein in a post-fusion conformation. Pre-fusion specific antibodies include, but are not limited to, D25, AM22, 5C4, and AM14 antibodies.

[0055] As used herein, the term "AM14" refers to the antibody described in WO 2008 / 147196 (A2).

[0056] As used herein, the term "AM22" refers to the antibody described in WO 2011 / 043643 (A1).

[0057] As used herein, the term "D25" refers to the antibody described in WO 2008 / 147196(A2).

[0058] As used herein, the term "5C4" refers to the antibody described in McLellan et al., 2010, Nat. Struct. Mol. Biol., Feb 17(2): 248-50. McLellan et al. (Science 340: 1113-1117 (2013)).

[0059] Mutant RSV F molecule The RSV fusion or "F" protein is an envelope glycoprotein of respiratory syncytial virus. In nature, the RSV F protein is translated as a single precursor polypeptide (designated F0). The F0 precursor polypeptide is generally 574 amino acids in length. Amino acids 1-25 of the F0 precursor generally constitute a signal peptide. The precursor polypeptide F0 forms a precursor trimer that is typically proteolytically cleaved by one or more cellular proteases at a conserved furin consensus cleavage site to produce a Pep 27 polypeptide, an F1 polypeptide, and an F2 polypeptide. The Pep 27 polypeptide (generally amino acids 110-136 of the F0 precursor) is removed and does not form part of the mature RSV F trimer. The F2 polypeptide (hereinafter sometimes referred to as "F2" or "F2 region") generally consists of amino acid residues 26-109 of the F0 precursor. The F1 polypeptide (hereinafter sometimes referred to as "F1" or "F1 region") generally consists of amino acid residues 137-574 of the F0 precursor and includes an extracellular domain (generally residues 137-524), a transmembrane domain (generally residues 525-550), and a cytoplasmic domain (generally residues 551-574). The F1 and F2 polypeptides are linked by disulfide bonds to form a heterodimer called an RSV F "protomer". Three such promoters form a mature RSV F trimer, i.e., a homotrimer of three promoters. In nature, the mature RSV F trimer is generally membrane-bound. However, a soluble form (i.e., non-membrane-bound) of the mature RSV F trimer can be produced by removing the transmembrane and cytoplasmic domains. For example, the RSV F protein can be converted to a soluble form by truncating it at amino acid 513 (i.e., by removing amino acids from 514 onward).

[0060] In nature, the mature RSV F trimer mediates the fusion of the viral membrane with the cellular membrane. The pre-fusion conformation of the mature RSV F trimer (hereinafter sometimes referred to as "pre-F") is very unstable (metastable). However, when the RSV virus combines with the cellular membrane, the RSV F protein trimer undergoes a series of conformational changes and transitions to a very stable post-fusion ("post-F") conformation. It is known that the mature RSV F protein induces potent neutralizing antibodies ("nAbs") that are involved in protection against RSV. For example, immunization with the RSV F protein induces protective nAbs in humans (e.g., Synagis®). The post-fusion form of the RSV F protein has several neutralizing epitopes (sites I, II, and IV). Recently, however, Magro et al. showed that incubation of the RSV F protein in a post-fusion conformation with human serum failed to deplete most of the neutralizing activity against the F protein, suggesting the presence of a neutralizing antigenic site unique to the pre-fusion conformation (Magro et al., 2012, PNAS 109(8): 3089). X-ray crystallography has mapped the epitopes recognized by palivizumab (Synagis®), motavizumab (Numax), and the more recently discovered 101F monoclonal antibody (McLellan et al., 2010, J. Virol., 84(23): 12236-44; and McLellan et al., 2010, Nat. Struct. Mol. Biol., Feb 17(2): 248-50). McLellan et al. (Science 340: 1113-1117 (2013)) solved the structure of the F protein in the pre-fusion conformation, revealing a novel neutralizing epitope, site "o", which is only present in the pre-fusion conformation and is bound by a series of antibodies, e.g., 5C4, that are up to 50-fold more potent at neutralizing than Synagis® and Numax.Thus, there is growing evidence that RSV vaccine immunogens that present site "o" in this pre-fusion conformation can induce effective protection. However, the highly unstable (metastable) nature of the pre-fusion conformation of the RSV F protein has proven to be a major obstacle to the development of such a vaccine. Comparing the pre-fusion and post-fusion RSV F structures of McLellan et al., there appear to be two regions of the F protein that undergo large conformational changes (>5 Å). These regions are located at the N- and C-termini of the F1 subunit (residues 137-216 and 461-513, respectively). In the crystal structure of the RSV F protein held in the pre-fusion conformation by the D25 antibody bound to the site "o" epitope, the C-terminal F1 residues can be stabilized in the pre-fusion conformation by adding a foldon trimerization domain. To stabilize the N-terminal region of F1, McLellan et al. found that the binding of antibody D25 was sufficient for crystal structure studies. However, another stabilization strategy is needed to produce immunogens for vaccines, for example, one that does not require the RSV F protein to be bound to a large antibody molecule. One alternative approach that has been attempted so far involves the introduction of paired cysteine ​​mutations (for disulfide bond formation) and cavity-filling mutations near the F1 N-terminus (see DS-Cav1 RSV F protein variants described in McLellan et al, (2013) Science 342: 592-598, which is incorporated herein by reference in its entirety). However, crystallographic analysis of such variants revealed that the structure is only partially present in the pre-fusion conformation. Therefore, further manipulation of the RSV F protein is required to obtain an immunogen for clinical vaccine development.

[0061] The present invention provides several alternative approaches to stabilizing the RSV F protein in its pre-fusion conformation, which are based on introducing one or more "to tyrosine" mutations and one or more DT bridges at specific positions in the RSV F molecule.

[0062] The amino acid sequences of some exemplary RSV F and mutant RSV F molecules are shown in Table 1 and the sequence listing. Many of the sequences presented in Table 1 are represented as "F0" sequences, i.e., these sequences include a signal peptide and a pep27 peptide that is present in the F0 precursor but not in the mature RSV F protein. Similarly, some of the sequences presented in Table 1 include native transmembrane and cytoplasmic domains that can be removed in some embodiments to form a soluble form of the mature RSV trimer. Thus, the final / mature forms of these exemplary mutant RSV molecules (i.e., forms that may be DT-bridged) typically do not include all of the amino acids shown in the sequences in Table 1. However, the final / mature forms of these exemplary mutant RSV molecules include the F2 region and at least a portion of the F1 region of these sequences. Typically, the final / mature forms of these exemplary mutant RSV molecules include amino acid residues 26-109 and amino acid residues 137-513 of these sequences. It should be noted that in all embodiments herein referring to RSV F molecules having specific exemplary amino acid sequences, either the complete F0 sequence or amino acid residues 26-109 and 137-513 therein are contemplated.

[0063] Throughout this specification, when a specific amino acid position / residue of the RSV F molecule is referred to by an amino acid residue number (e.g., amino acid residue at position 428), unless otherwise stated, the amino acid numbering is the numbering used for the RSV F amino acid sequence presented in the sequence listing and Table 1 of this application (see, for example, SEQ ID NOs: 1-91). This is the same numbering system as is commonly used in the art when describing RSV F sequences. However, it should be noted that different numbering systems can be used, and those skilled in the art will understand this. For example, when there are additional amino acid residues added or deleted compared to any of SEQ ID NOs: 1-91. As such, it should be understood that when a particular amino acid residue is referred to using its number, the recitation is not limited to the amino acid located exactly at the numbered position counting from the start of a given amino acid sequence, but rather, the "corresponding" amino acid residue is intended in any and all RSV F sequences, even if the residue is not at the exact same numbered position in a given molecule, for example, even if the RSV sequence is shorter or longer than SEQ ID NO: 1, or has an insertion or deletion compared to SEQ ID NO: 1. One of skill in the art can readily determine which "corresponding" amino acid positions are for any of the specific numbered residues described herein, for example, by aligning a given RSV F sequence with SEQ ID NO: 1 or any of the other RSV F amino acid sequences set forth herein (i.e., SEQ ID NOs: 1-91). Such alignments can be readily performed, either by computer or by eye, given the highly conservative nature of the RSV F sequence between RSV subtypes and RSV strains. Numerous WT / native RSV F molecule amino acid sequences from different RSV subtypes and strains, as well as nucleic acid sequences encoding such RSV F molecules, are known in the art. Some exemplary WT / native RSV F molecule amino acid sequences are set forth in SEQ ID NOs: 1-20 and 82-85. Other such sequences can be found in public sequence databases.Other such sequences are described in International Patent Application PCT / US2014 / 048086, U.S. Pat. No. 9,738,689, and U.S. Pat. No. 9,950,058, the contents of each of which are incorporated herein by reference.

[0064] The WT / native RSV F molecule shows a surprisingly high level of sequence conservation both between RSV subtypes and between RSV strains. See, for example, WO 2014 / 160463. For example, RSV subtypes A and B have 90% sequence identity in the F0 precursor molecule. Within a given RSV subtype (e.g., subtype A or B), the sequence identity between strains is about 98%. Furthermore, nearly all RSV F F0 precursors identified to date consist of 574 amino acids. Although there may be some minor differences in length, such differences generally occur in the cytoplasmic domain.

[0065] The specific mutations to tyrosine described herein can be introduced into any suitable RSV F "background" sequence.

[0066] In some embodiments, such a "background" sequence is a sequence of a WT / native RSV F molecule (i.e., a RSV F molecule that occurs in nature). Numerous amino acid and nucleotide sequences of WT / native RSV F molecules from different RSV subtypes and strains are known in the art. Some exemplary amino acid sequences of WT / native RSV F molecules are shown in SEQ ID NOs: 1-20 and 82-85 (see Table 1 and Sequence Listing). Other such sequences are described in International Patent Application PCT / US2014 / 048086, U.S. Patent No. 9,738,689, and U.S. Patent No. 9,950,058, the contents of each of which are incorporated herein by reference.

[0067] In some embodiments, such a "background" sequence is a sequence of a mutant RSV F molecule, i.e., a sequence that contains one or more artificially introduced mutations when compared to a WT / native RSV F molecule. For example, in some embodiments, the "background" RSV F sequence may contain a "Cav1" mutation, a "DS" mutation, or a combination thereof. Non-limiting examples of suitable mutant background RSV molecules into which specific mutations described herein may be introduced include those having the amino acid sequence of any of SEQ ID NOs: 86-91, or those containing amino acids 26-109 (i.e., F2) and amino acids 137-513 (i.e., F1) of any of SEQ ID NOs: 86-91 (see Table 1 and the sequence listing). Other suitable mutant background RSV molecules into which specific mutations described herein may be introduced include those described in International Patent Application PCT / US2014 / 048086, U.S. Patent No. 9,738,689, and U.S. Patent No. 9,950,058.

[0068] In some embodiments, the "background" RSV F molecule into which the specific mutations described herein may be introduced may be a "full-length" RSV F molecule, i.e., an RSV F molecule including a transmembrane domain and a cytoplasmic domain. Non-limiting examples of suitable "full-length" background RSV molecules into which the specific mutations and bridges described herein may be introduced include those having any of the amino acid sequences of SEQ ID NOs: 1-9, 11-29, 31-49, 51-80, 83, 85, 87, or 89, or those including amino acids 26-109 (F2) and amino acids 137-513 (F1) of any of SEQ ID NOs: 1-9, 11-29, 31-49, 51-80, 83, 85, 87, or 89 (see Table 1 and the Sequence Listing).

[0069] In some embodiments, the "background" RSV F molecule into which the specific mutations and bridges described herein may be introduced may be a "soluble" RSV F molecule, i.e., an RSV F molecule that does not include a transmembrane domain and a cytoplasmic domain. Non-limiting examples of suitable "soluble" background RSV molecules into which the specific mutations and bridges described herein may be introduced include those having the amino acid sequence of any of SEQ ID NOs: 10, 30, 50, 81, 82, 84, 86, 88, or 90, or those including amino acids 26-109 (i.e., F2) and amino acids 137-513 (i.e., F1) of any of SEQ ID NOs: 10, 30, 50, 81, 82, 84, 86, 88, or 90.

[0070] Similarly, in some embodiments, a soluble "background" RSV F molecule, into which certain mutations and cross-links described herein may be introduced, can be created by removing the transmembrane and cytoplasmic domains from a "full-length" RSV F molecule, for example, by removing amino acids from position 514 onwards from a "full-length" RSV F molecule, for example, one of those described above.

[0071] In some embodiments, the present invention provides a mutant RSV F molecule that includes a point mutation to tyrosine at amino acid residue 428 (sometimes referred to as a 428Y point mutation), or a point mutation to tyrosine at the amino acid corresponding to position 428, for example, when aligned with SEQ ID NO:1 and / or when determined using the amino acid numbering of SEQ ID NO:1.

[0072] In some embodiments, such mutant RSV F molecules containing a 428Y mutation further include a point mutation to tyrosine at amino acid residue 185 (sometimes referred to as a 185Y point mutation), or a point mutation to tyrosine at the amino acid corresponding to position 185, e.g., as determined by alignment with SEQ ID NO: 1 and / or as determined using the amino acid numbering of SEQ ID NO: 1. Thus, such mutants include both the 428Y mutation and the 185Y mutation.

[0073] In some embodiments, such mutant RSV F molecules containing a 428Y mutation further include a point mutation to tyrosine at amino acid residue 226 (sometimes referred to as a 226Y point mutation), or a point mutation to tyrosine at the amino acid corresponding to position 226, e.g., as determined by alignment with SEQ ID NO: 1 and / or as determined using the amino acid numbering of SEQ ID NO: 1. Thus, such mutants include both the 428Y mutation and the 226Y mutation.

[0074] In some embodiments, such mutant RSV F molecules comprising a 428Y mutation further comprise: (a) a point mutation to tyrosine at amino acid residue 226 (also referred to as a 226Y point mutation), or a point mutation to tyrosine at the amino acid corresponding to position 226, e.g., when aligned to SEQ ID NO: 1 and / or when determined using the amino acid numbering of SEQ ID NO: 1; and (b) a point mutation to tyrosine at amino acid residue 185 (also referred to as a 185Y point mutation), or a point mutation to tyrosine at the amino acid corresponding to position 185, e.g., when aligned to SEQ ID NO: 1 and / or when determined using the amino acid numbering of SEQ ID NO: 1. Thus, such mutants include a 428Y mutation, a 185Y mutation, and a 226Y mutation.

[0075] In some embodiments, the mutant RSV F molecule comprises a transmembrane domain and a cytoplasmic domain. In some embodiments, the mutant RSV F molecule does not comprise a transmembrane domain and / or a cytoplasmic domain, i.e., is a soluble (non-membrane-bound) RSV F molecule.

[0076] In some embodiments, the mutant RSV F molecule is a RSV type A or a RSV type B molecule.

[0077] In some embodiments, the mutant RSV F molecule can bind to a pre-fusion specific antibody. In some embodiments, the mutant RSV F molecule can bind to an antibody that recognizes the antigenic site "o", such as one of those described in U.S. Patent No. 9,738,689, U.S. Patent No. 9,950,058, or McLellan et al. (2013) Science 342: 592-598, each of which is incorporated by reference in its entirety for this purpose. Non-limiting examples of such antibodies include D25, 5C4, and AM22. Other antibodies that recognize the antigenic site "o" are disclosed herein or known in the art.

[0078] In some embodiments, the mutant RSV F molecule comprises an F2 polypeptide and an F1 polypeptide, wherein the C-terminus of the F2 polypeptide is linked to the N-terminus of the F1 polypeptide by a disulfide bond. In some embodiments, the mutant RSV F molecule comprises an F2 polypeptide and an F1 polypeptide, wherein the C-terminus of the F2 polypeptide is linked to the N-terminus of the F1 polypeptide by an artificially introduced peptide linker.

[0079] In some embodiments, the mutant RSV F molecule comprises (a) an F2 polypeptide comprising or consisting of approximately 84 amino acid residues, and (b) an F1 polypeptide comprising or consisting of approximately 375 amino acid residues.

[0080] In some embodiments, the mutant RSV F molecule comprises (a) an F2 polypeptide comprising or consisting of approximately 74-84 amino acid residues; and (b) an F1 polypeptide comprising or consisting of approximately 365-375 amino acid residues.

[0081] In some embodiments, the mutant RSV F molecule comprises (a) an F2 polypeptide comprising or consisting of amino acid residues 26-109 of any of SEQ ID NOs: 21-81, and (b) an F1 polypeptide comprising or consisting of amino acid residues 137-513 of any of SEQ ID NOs: 21-81.

[0082] In some embodiments, the mutant RSV F molecule comprises (a) an F2 polypeptide comprising or consisting of approximately 74-84 amino acids among amino acid residues 26-109 of any of SEQ ID NOs: 21-81; and (b) an F1 polypeptide comprising or consisting of approximately 365-375 amino acids among amino acid residues 137-513 of any of SEQ ID NOs: 21-81.

[0083] In some embodiments, the mutant RSV F molecule comprises (a) an F2 polypeptide comprising, or consisting of, amino acid residues 26-109 of SEQ ID NO: 81; and (b) an F1 polypeptide comprising, or consisting of, amino acid residues 137-513 of SEQ ID NO: 81.

[0084] In some embodiments, the mutant RSV F molecule comprises (a) an F2 polypeptide comprising or consisting of approximately 74-84 amino acids among amino acid residues 26-109 of SEQ ID NO: 81; and (b) an F1 polypeptide comprising or consisting of approximately 365-375 amino acids among amino acid residues 137-513 of SEQ ID NO: 81.

[0085] In some embodiments, the mutant RSV F molecule is stabilized in a pre-fusion conformation by one or more dityrosine bridges.

[0086] In some embodiments, the mutant RSV F molecule is a mature RSV F trimer. In some such embodiments, the RSV F molecule is a mature RSV F trimer stabilized in a pre-fusion conformation by three or more dityrosine bridges.

[0087] In some embodiments, the mutant RSV F molecule comprises a tyrosine bridge between the tyrosine at amino acid position 428 (or a corresponding amino acid position, e.g., as determined by alignment with SEQ ID NO:1 and / or as determined using the amino acid numbering of SEQ ID NO:1) and the tyrosine at amino acid position 185 (or a corresponding amino acid position, e.g., as determined by alignment with SEQ ID NO:1 and / or as determined using the amino acid numbering of SEQ ID NO:1).

[0088] In some embodiments, the mutant RSV F molecule comprises a tyrosine bridge between the tyrosine at amino acid position 198 (or a corresponding amino acid position, e.g., when aligned to SEQ ID NO:1 and / or determined using the amino acid numbering of SEQ ID NO:1) and the tyrosine at amino acid position 226 (or a corresponding amino acid position, e.g., when aligned to SEQ ID NO:1 and / or determined using the amino acid numbering of SEQ ID NO:1).

[0089] In some embodiments, the mutant RSV F molecule comprises (a) a dityrosine bridge between a tyrosine at amino acid position 198 (or a corresponding amino acid position, e.g., when aligned to SEQ ID NO:1 and / or determined using the amino acid numbering of SEQ ID NO:1) and a tyrosine at amino acid position 226 (or a corresponding amino acid position, e.g., when aligned to SEQ ID NO:1 and / or determined using the amino acid numbering of SEQ ID NO:1); and (b) a dityrosine bridge between a tyrosine at amino acid position 428 (or a corresponding amino acid position, e.g., when aligned to SEQ ID NO:1 and / or determined using the amino acid numbering of SEQ ID NO:1) and a tyrosine at amino acid position 185 (or a corresponding amino acid position, e.g., when aligned to SEQ ID NO:1 and / or determined using the amino acid numbering of SEQ ID NO:1).

[0090] In some embodiments, the mutant RSV F molecule is a mature RSV F trimer containing three dityrosine bridges, each of which is between a tyrosine at amino acid position 428 (or a corresponding amino acid position, e.g., when aligned with SEQ ID NO:1 and / or when determined using the amino acid numbering of SEQ ID NO:1) and a tyrosine at amino acid position 185 (or a corresponding amino acid position, e.g., when aligned with SEQ ID NO:1 and / or when determined using the amino acid numbering of SEQ ID NO:1).

[0091] In some embodiments, the mutant RSV F molecule is a mature RSV F trimer containing three dityrosine bridges, each of which is between a tyrosine at amino acid position 198 (or a corresponding amino acid position, e.g., when aligned with SEQ ID NO:1 and / or when determined using the amino acid numbering of SEQ ID NO:1) and a tyrosine at amino acid position 226 (or a corresponding amino acid position, e.g., when aligned with SEQ ID NO:1 and / or when determined using the amino acid numbering of SEQ ID NO:1).

[0092] In some embodiments, the mutant RSV F molecule is a mature RSV F trimer containing six dityrosine bridges, three of which are between the tyrosine at amino acid position 198 (or a corresponding amino acid position, e.g., when aligned with SEQ ID NO:1 and / or determined using the amino acid numbering of SEQ ID NO:1) and the tyrosine at amino acid position 226 (or a corresponding amino acid position, e.g., when aligned with SEQ ID NO:1 and / or determined using the amino acid numbering of SEQ ID NO:1), and three of which are between the tyrosine at amino acid position 428 (or a corresponding amino acid position, e.g., when aligned with SEQ ID NO:1 and / or determined using the amino acid numbering of SEQ ID NO:1) and the tyrosine at amino acid position 185 (or a corresponding amino acid position, e.g., when aligned with SEQ ID NO:1 and / or determined using the amino acid numbering of SEQ ID NO:1).

[0093] In some embodiments, the mutant RSV F molecule further comprises one or more artificially introduced non-DT bridges. In some embodiments, the non-DT bridges are disulfide bonds. In some embodiments, the RSV F molecule comprises one or more point mutations to cysteine. In some embodiments, one or more of the disulfide bonds are formed between two cysteines, one or both of which are introduced by point mutation. In some embodiments, the RSV F molecule comprises a point mutation to cysteine ​​at amino acid residue 155 (i.e., 155C mutation). In some embodiments, the RSV F molecule comprises a point mutation to cysteine ​​at amino acid residue 290 (i.e., 290C mutation). In some embodiments, the RSV F molecule comprises both a point mutation to cysteine ​​at amino acid residue 155 (i.e., 155C mutation) and a point mutation to cysteine ​​at amino acid residue 290 (i.e., 290C mutation). In some such embodiments, the RSV F molecule includes one or more of the point mutations to cysteines disclosed in U.S. Pat. No. 9,738,689, U.S. Pat. No. 9,950,058, or McLellan et al. (2013) Science 342: 592-598, each of which is incorporated herein by reference in its entirety for this purpose.

[0094] In some embodiments, the mutant RSV F molecule further comprises one or more artificially introduced cavity-filling mutations (e.g., substitutions). In some such embodiments, the RSV F molecule comprises a mutation at amino acid residue 190 to F (190F mutation). In some such embodiments, the RSV F molecule comprises a point mutation at amino acid residue 207 to L (207L mutation). In some such embodiments, the RSV F molecule comprises both a mutation at amino acid residue 190 to F (190F mutation) and a point mutation at amino acid residue 207 to L (207L mutation). In some such embodiments, the RSV F molecule comprises one or more cavity-filling amino acid substitutions selected from the group consisting of 58W, 83W, 87F, 90L, 153W, 190F, 203W, 207L, 220L, 260W, 296F, and 298L. In some such embodiments, the RSV F molecule comprises one or more of the cavity-filling amino acid substitutions disclosed in U.S. Pat. No. 9,738,689, U.S. Pat. No. 9,950,058, or McLellan et al. (2013) Science 342: 592-598, each of which is incorporated herein by reference in its entirety for this purpose.

[0095] In some embodiments, the mutant RSV F molecule comprises an oligomerization domain, such as a trimerization domain. In one embodiment, the trimerization domain is a T4 foldon domain, a GCN4 domain, or a T4 fibrintin domain. Examples of other trimerization domains that can be used include those described in Habazettl et al., 2009 (Habazettl et al., 2009. NMR Structure of a Monomeric Intermediate on the Evolutionarily Optimized Assembly Pathway of a Small Trimerization Domain. J.Mol.Biol. pp. null); Kammerer et al., 2005 (Kammerer et al., 2005. A conserved trimerization motif controls the topology of short coiled coils. Proc Natl Acad Sci USA 102 (39): 13891-13896); Innamorati et al., 2006 (Innamorati et al., 2006. An intracellular role for the C1q-globular domain. Cell signal 18(6): 761-770); Schelling et al., 2007 (Schelling et al., 2007. The reovirus σ-1 aspartic acid sandwich: A trimerization motif poised for conformational change.Biol Chem 282(15): 11582-11589; Pancera et al., 2005 (Soluble Mimetics of Human Immunodeficiency Virus Type 1 Viral Spikes Produced by Replacement of the Native Trimerization Domain with a Heterologous Trimerization Motif: Characterization and Ligand Binding Analysis. J Virol 79 (15): 9954-9969); Guthe et al., 2004 (Very fast folding and association of a trimerization domain from bacteriophage T4 fibritin. J.Mol.Biol. v337 pp. 905-15); and Papanikolopoulou et al., 2008 (Creation of hybrid nanorods from sequences of natural trimeric fibrous proteins using the fibritin trimerization motif. Methods Mol Biol 474: 15-33).

[0096] In some embodiments, the mutated RSV F molecule comprises a foldon domain of SEQ ID NO:93.

[0097] In some embodiments, the mutant RSV F molecule further comprises one or more tags (e.g., C-terminal tags) useful for detection and / or purification of the RSV F molecule. Exemplary tags include, but are not limited to, a Strep tag, a Strep II tag, a FLAG tag, a glutathione S-transferase (GST) tag, a green fluorescent protein (GFP) tag, a hemagglutinin A (HA) tag, a histidine (His) tag, a luciferase tag, a maltose binding protein (MBP) tag, a c-Myc tag, a protein A tag, and a protein G tag. In some such embodiments, the RSV F molecule comprises a His tag of SEQ ID NO: 95. In some such embodiments, the RSV F molecule comprises a Strep II tag of SEQ ID NO: 96. In some such embodiments, the tag is a cleavable tag, i.e., it can be cleaved / removed from the mutant RSV F molecule if desired. In some such embodiments, the tag is located adjacent to (e.g., C-terminally) a proteolytic cleavage site that allows the tag to be removed using a protease. In some such embodiments, the RSV F molecule comprises a thrombin cleavage site of SEQ ID NO:94.

[0098] In some embodiments, the mutant RSV F molecule further comprises one or more peptide "linker" sequences. Suitable linker sequences include, but are not limited to, G, GG, GGG, GS, and SAIG ​​(amino acids 1-4 of SEQ ID NO: 92) linker sequences. Such linkers may be provided between the C-terminus of F2 and the N-terminus of F1, between the C-terminus of F1 and the N-terminus of any artificial trimerization domain, and / or between any other artificially introduced sequences, such as trimerization domains, proteolytic cleavage domains, and tags useful for detection and / or purification.

[0099] In some embodiments, the mutant RSV F molecule comprises one or more leader sequences, precursor polypeptide sequences, secretion signals, and / or localization signals.

[0100] In the above embodiments of the present invention relating to certain exemplary amino acid sequences of mutant RSV F molecules (e.g., amino acid sequences of SEQ ID NOs: 21-81) or "background" RSV F molecules (e.g., amino acid sequences of SEQ ID NOs: 1-20 or 82-91), or to certain regions of such sequences (e.g., the F1 and / or F2 regions thereof), equivalent variant forms of such amino acid sequences can also be used. For example, in some embodiments, an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the RSV F amino acid sequences described herein across its F1 and F2 regions (specifically across amino acid residues 26-109 (F2) and 137-513 (a portion of F1 excluding the transmembrane and cytoplasmic domains)) can be used. In some embodiments, an amino acid sequence having at least about 80% or at least 85% sequence identity to such amino acid sequences across its F1 and F2 regions can be used. Such variant forms may have additional, deleted or substituted amino acids when compared to one or more of the specific amino acid sequences set forth herein, and thus may be longer or shorter than the specified sequences.

[0101] Similarly, the amino acid residues from position 514 onwards of each of the exemplary RSV F amino acid sequences set forth herein may be deleted or altered. For example, in some of the amino acid sequences set forth herein, the amino acid residues from position 514 onwards comprise the native RSV F transmembrane and cytoplasmic domains. In some embodiments, these native RSV F transmembrane and cytoplasmic domains can be removed to generate a soluble form (i.e., a non-membrane-bound form) of the RSV F molecule, or can be replaced with a different transmembrane and / or cytoplasmic domain. Similarly, in some of the amino acid sequences set forth herein, the amino acid residues from position 514 onwards comprise a variety of artificially added C-terminal sequences, such as a combination of the artificial C-terminal sequences set forth in SEQ ID NO: 92, including a foldon domain (SEQ ID NO: 93), a thrombin cleavage site (SEQ ID NO: 94), a histidine tag (SEQ ID NO: 95), a Strep II tag (SEQ ID NO: 96), and various linkers. In some embodiments, such artificial C-terminal sequences can be deleted, modified, rearranged or substituted as necessary. For example, in some embodiments, different trimerization domains may be used, and / or different cleavage sites may be used, and / or different epitope tags may be used.

[0102] In some embodiments, one or more amino acid residues in a particular mutant RSV F molecule described herein can be replaced with another amino acid. In some embodiments, one or more amino acid residues can be replaced with another amino acid of similar polarity that can act as a functional equivalent, resulting in a silent change. In some embodiments, substitutions for an amino acid in a sequence can be selected from other members of the class to which the amino acid belongs, for example, to produce a conservative substitution. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. In general, such substitutions are understood to be conservative substitutions.

[0103] In some embodiments, artificial, synthetic, or non-classical amino acids or chemical amino acid analogs can be used to generate the mutant RSV F molecules described herein. Non-classical amino acids include, but are not limited to, D isomers of common amino acids, fluoroamino acids, and "designer" amino acids, such as β-methyl amino acids, Cγ-methyl amino acids, Nγ-methyl amino acids, and amino acid analogs in general. Further non-limiting examples of non-classical amino acids include α-aminocaprylic acid, Acpa; (S)-2-aminoethyl-L-cysteine / HCl, Aecys; aminophenylacetate, Afa; 6-aminohexanoic acid, Ahx; γ-aminoisobutyric acid and α-aminoisobutyric acid, Aiba; alloisoleucine, Aile; L-allylglycine, Alg; 2-aminobutyric acid, 4-aminobutyric acid, and α-aminobutyric acid, Aba; p-amino Phenylalanine, Aphe; b-alanine, Bal; p-bromophenylalanine, Brphe; cyclohexylalanine, Cha; citrulline, Cit; beta-chloroalanine, Clala; cycloleucine, Cle; p-chlorophenylalanine, Clphe; cysteic acid, Cya; 2,4-diaminobutyric acid, Dab; ​​3-aminopropionic acid and 2,3-diaminopropionic acid, Dap; 3,4-dehydroproline, Dhp; 3,4 -Dihydroxylphenylalanine, Dhphe;p-fluorophenylalanine, Fphe;D-glucosaminic acid, Gaa;Homoarginine, Hag;δ-hydroxylysine / HCl, Hlys;DL-β-hydroxynorvaline, Hnvl;Homoglutamine, Hog;Homophenylalanine, Hoph;Homoserine, Hos;Hydroxyproline, Hpr;p-iodophenylalanine, Iphe;Isoserine, Ise;α-methylroly Syn, Mle; DL-methionine-S-methylsulfonium chloride, Msmet; 3-(1-naphthyl)alanine, 1Nala; 3-(2-naphthyl)alanine, 2Nala; Norleucine, Nle; N-methylalanine, Nmala; Norvaline, Nva; O-benzylserine, Obser; O-benzyltyrosine, Obtyr; O-ethyltyrosine, Oetyr; O-methylserine, Omser; O-methylthreonine, Omthr;O-Methyltyrosine, Omtyr;Ornithine, Orn;Phenylglycine;Penicillamine, Pen;Pyroglutamic acid, Pga;Pipecolic acid, Pip;Sarcosine, Sar;t-Butylglycine;t-Butylalanine;3,3,3-Trifluoroalanine, Tfa;6-Hydroxydopa, Thphe;L-Vinylglycine, Vig;(-)-(2R)-2-Amino-3-(2-aminoethylsulfonyl)sulfonyl (-)-(2R)-2-amino-3-(2-hydroxyethylsulfonyl)propanoic acid dihydroxochloride, Aaspa;(2S)-2-amino-9-hydroxy-4,7-dioxanoic acid, Ahdna;(2S)-2-amino-6-hydroxy-4-oxahexanoic acid, Ahoha;(-)-(2R)-2-amino-3-(2-hydroxyethylsulfonyl)propanoic acid, Ahsopa;(-)-(2R)-2-amino-3-(2-hydroxyethylsulfanyl)propanoic acid Panic acid, Ahspa;(2S)-2-amino-12-hydroxy-4,7,10-trioxadodecanoic acid, Ahtda;(2S)-2,9-diamino-4,7-dioxanonoic acid, Dadna;(2S)-2,12-diamino-4,7,10-trioxadodecanoic acid, Datda;(S)-5,5-difluoronorleucine, Dfnl;(S)-4,4-difluoronorvaline, Dfnv;(3R)-1- These include, but are not limited to, 1-dioxo-[1,4]thiadiane-3-carboxylic acid, Dtca; (S)-4,4,5,5,6,6,6-heptafluoronorleucine, Hfnl; (S)-5,5,6,6,6-pentafluoronorleucine, Pfnl; (S)-4,4,5,5,5-pentafluoronorvaline, Pfnv; and (3R)-1,4-thiazinane-3-carboxylic acid, Tca. Furthermore, the amino acid may be D (dextrorotatory) or L (levorotatory). For a review of classical and non-classical amino acids, see Sandberg et al., 1998. New chemical descriptors relevant for the design of biologically active peptides. A multivariate characterization of 87 amino acids. J Med Chem 41(14): pp. 2481-91.

[0104] Characterization of mutant RSV F molecules The mutant RSV F molecules of the present invention include mutant RSV F molecules having an amino acid sequence of one of the exemplary amino acid sequences set forth herein (e.g., SEQ ID NOs: 21-81), mutant RSV F molecules having residues 26-109 and / or 137-513 of one of the exemplary amino acid sequences, F1 and / or F2 polypeptides of one of the exemplary amino acid sequences, and variant forms of certain amino acid sequences, but the mutant RSV F molecules of the present invention (a) include an F1 polypeptide and an F2 polypeptide, and (b) are capable of forming a mature RSV F trimer capable of adopting a pre-fusion conformation (as described / defined herein) or are capable of being processed to form such a trimer (e.g., a precursor such as F0). The mutant RSV F molecules of the present invention may also have one or more of the following properties in some embodiments: (1) bind to a pre-F specific antibody, (2) bind to an antibody that binds to the "o" site, (3) bind to a neutralizing antibody, (4) bind to a broadly neutralizing antibody, (5) bind to an antibody selected from the group consisting of D25, AM22, 5C4, 101F, (6) bind to palivizumab (Synagis®), (7) bind to and / or activate a B cell receptor, (8) induce an anti-RSV antibody response in an animal, (9) induce a protective anti-RSV antibody response in an animal, (10) induce the production of anti-RSV neutralizing antibodies in an animal, (11) induce the production of anti-RSV broadly neutralizing antibodies in an animal, (12) induce the production of anti-RSV antibodies that recognize the quaternary neutralizing epitope (QNE) in an animal, and / or (13) induce a protective anti-RSV immune response in an animal.

[0105] DT crosslinking In some embodiments, the present invention provides a mutant RSV F molecule comprising at least one artificially introduced DT bridge, which serves to stabilize the mutant RSV F molecule described herein in its pre-fusion conformation.

[0106] In some embodiments, such a DT bridge may be introduced between two endogenous tyrosine residues, between two artificially introduced tyrosine residues (i.e., originating from a "to tyrosine" mutation), or between an artificially introduced tyrosine residue and an endogenous tyrosine residue.

[0107] In some embodiments, at least one tyrosine of at least one DT bridge is derived from a point mutation to tyrosine (i.e., introduced by a point mutation to tyrosine). In some embodiments, the present invention provides a mutant trimeric RSV F molecule comprising at least three DT bridges, wherein at least one tyrosine of each of the at least three DT bridges is derived from a point mutation to tyrosine (i.e., introduced by a point mutation to tyrosine).

[0108] For example, in some embodiments, the present invention provides a mutant RSV F molecule that includes a DT bridge between a tyrosine at residue 198 (typically a naturally occurring tyrosine) and an introduced tyrosine at residue 226 (i.e., resulting from a 226Y mutation), i.e., an intramolecular 198Y-226Y DT bridge. Thus, in embodiments where the mutant RSV F molecule is a trimer, the trimer includes three intramolecular 198Y-226Y DT bridges.

[0109] In some embodiments, the present invention provides a mutant RSV F molecule comprising a DT bridge between an introduced tyrosine at residue 185 (i.e., resulting from a 185Y mutation) and an introduced tyrosine at residue 428 (i.e., resulting from a 428Y mutation), i.e., a 185Y-428Y intermolecular DT bridge. Thus, in embodiments where the mutant RSV F molecule is a trimer, the trimer comprises three intermolecular 185Y-428Y DT bridges.

[0110] And in some embodiments, the present invention provides mutant RSV F molecules that contain both a 198Y-226Y intramolecular DT bridge and a 185Y-428Y intermolecular DT bridge. Thus, in embodiments in which the mutant RSV F molecule is a trimer, the trimer contains six DT bridges, i.e., three intramolecular 198Y-226Y DT bridges and three intermolecular 185Y-428Y DT bridges.

[0111] As mentioned above, each promoter of the mature RSV F trimer contains two separate polypeptides, designated F1 and F2, which are non-covalently associated to form a promoter. The bond between the F1 and F2 polypeptides in the same promoter is an example of an intermolecular bond and an intraprotomer bond. The 185Y-428YDT bridge is designed to hold the two promoters of the trimer together, i.e., an intermolecular interprotomer bond. The tyrosine at position 185 on one promoter forms a dityrosine bond with the tyrosine at position 428 on the other promoter.

[0112] Exemplary methods for carrying out DT cross-linking reactions are shown in the Examples herein. Additionally, methods for carrying out DT cross-linking are known in the art, and are described, for example, in U.S. Patent Nos. 7,037,894 and 7,445,912 to Marshall et al., the contents of which are incorporated herein by reference. Dityrosine cross-linking introduces a carbon-carbon covalent bond that is minimally altered and zero length. DT cross-links are not hydrolyzed under physiological conditions. Dityrosine cross-links form naturally in vivo and are known to be safe because they are abundant in common foods. For example, DT bonds form the structure of wheat gluten. Dityrosine bonds do not form spontaneously in vitro. Rather, an enzymatic cross-linking reaction must be carried out in which a protein with a tyrosyl side chain is subjected to reaction conditions that result in the formation of a DT bond. Such conditions are or become oxidative reaction conditions, since the DT bond formation reaction is an oxidative cross-linking reaction. In some embodiments, the DT cross-linking reaction conditions result in a protein that is otherwise unmodified or not detectably modified. Such conditions include, but are not limited to, H2 O 2 This may be accomplished by using an enzyme, e.g., peroxidase, that catalyzes the formation of DT bond formation. DT bond formation may be monitored by spectrophotometry using an excitation wavelength near 320 nm and fluorescence measured at a wavelength near 400 nm (see, e.g., FIG. 34), and the disappearance of tyrosyl fluorescence may be monitored by standard procedures. When the disappearance of tyrosyl fluorescence is no longer stoichiometrically consistent with DT bond formation, the reaction may be stopped by any method known to those skilled in the art, e.g., by adding a reducing agent followed by cooling (on ice) or freezing the sample. Further details of suitable DT crosslinking methods are described in U.S. Pat. Nos. 7,037,894 and 7,445,912, and in the Examples herein.

[0113] nucleic acid molecule In some embodiments, the present invention provides nucleic acid molecules encoding the mutant RSV F molecules described herein, and vectors comprising such nucleic acid molecules. Given the nature of the genetic code, which is commonly known and understood by those skilled in the art, one of ordinary skill in the art can readily determine the nucleic acid sequence of a nucleic acid molecule encoding any one of the mutant RSV F molecules described herein.

[0114] In some embodiments, the nucleic acid molecule encodes a precursor F0 polypeptide that is properly processed to generate a mutant RSV F molecule when expressed in an appropriate cell. For example, in some embodiments, the nucleic acid molecule may encode one of the precursor (F0) polypeptides of the amino acid sequence set forth in Table 1 and in the sequence listing. In some embodiments, the nucleic acid molecule may encode only an F2 polypeptide or only an F1 polypeptide.

[0115] Nucleic acid molecules encoding mutant RSV F molecules described herein can be obtained or produced by any suitable method known in the art. For example, nucleic acid molecules encoding mutant RSV F molecules can be obtained from cloned DNA or produced by chemical synthesis. In some embodiments, nucleic acid molecules can be obtained by reverse transcribed RNA prepared by methods known to those of skill in the art. Point mutations or other modifications described herein (e.g., removal of C-terminal sequences, replacement of C-terminal sequences, etc.) can be made by standard recombinant DNA techniques well known and understood by those of skill in the art. For example, one of skill in the art can easily create the "to tyrosine" mutations described herein by identifying a nucleotide codon that encodes a particular amino acid residue to be mutated to tyrosine (e.g., encoding amino acid residues at positions 185, 226, or 428) and mutating the nucleotides of that codon as necessary to generate a codon encoding tyrosine.

[0116] Whatever the source, the nucleic acid molecule encoding the mutant RSV F molecule of the present invention can be cloned into any suitable vector, such as a vector used to propagate the nucleic acid molecule or a vector used to express the nucleic acid molecule. In embodiments requiring expression, the nucleic acid can be operably linked to a promoter suitable for expression in the desired cell type, such as a mammalian cell or an insect cell, and can be incorporated into any suitable expression vector, such as a mammalian or insect expression vector.

[0117] In some embodiments, the nucleic acid molecule encoding the mutant RSV F molecule of the present invention can be codon-optimized for expression in cells of a particular organism or species. For example, International Patent Application PCT / US2014 / 048086 provides RSV F molecule sequences that are codon-optimized for expression in human, hamster, mouse, and insect cells. Such codon-optimized sequences encoding the RSV F protein can be used as "background" sequences for introducing any of the specific mutations described herein.

[0118] Manufacturing method The mutant RSV F molecule of the present invention can be produced by any suitable means known in the art. In general, the mutant RSV F molecule is produced using standard methods used for the production of recombinant proteins. For example, the nucleic acid molecule encoding the mutant RSV F molecule of the present invention can be expressed in any suitable cell type, including, but not limited to, mammalian cells and insect cells (e.g., SF9 or Hi5 cells, e.g., using a baculovirus expression system). Methods for expressing proteins from nucleic acid molecules are routine and well known in the art, and suitable methods, vectors, systems, and cell types known in the art can be used. For example, typically, the nucleic acid molecule encoding the mutant RSV F molecule of the present invention will be inserted into a suitable expression construct containing a suitable promoter, which will then be sent to a cell for expression.

[0119] In some embodiments, the mutant RSV F molecules of the present invention are mature RSV F trimers stabilized in a pre-F conformation. In such embodiments, nucleic acid molecules encoding mutant RSV F molecules are typically expressed in a soluble form in cells and then subjected to an enzymatic DT cross-linking reaction after assembling into a normal trimeric pre-F conformation. In some embodiments, mutant RSV F molecules in (and / or maintained in) a pre-F conformation may be obtained before and / or during the enzymatic cross-linking reaction, e.g., while cross-linking. In some embodiments, mutant RSV F molecules may be produced and / or isolated such that most or substantially all mutant RSV F molecules are in a pre-F conformation. In some embodiments, mutant RSV F molecules in a pre-F conformation may be separated from a mixed population of RSV F protein molecules, including some in a pre-F conformation and some in other conformations. In some embodiments, the RSV F protein is expressed intracellularly (e.g., in its membrane-bound or soluble form) and spontaneously assembles into its normal pre-F conformation. In some embodiments, no further stabilization is required to maintain the mutant RSV F molecule in its pre-F form prior to DT cross-linking. In some embodiments, the mutant RSV F molecule may be maintained under certain conditions or in a certain composition suitable for the formation and / or maintenance of the pre-F conformation. For example, in some embodiments, the mutant RSV F molecule in the pre-F conformation is maintained in the absence of cells, since contact with cells may cause a change to the post-F conformation. The mutant RSV F molecule may be obtained and / or isolated and / or maintained in the pre-F conformation by any suitable method known in the art, including, but not limited to, standard protein purification methods such as ion exchange chromatography, size exclusion chromatography, and / or affinity chromatography.In some embodiments, the mutant RSV F molecule may be expressed in the presence of, co-expressed with, or contacted with a molecule that binds to the RSV F protein and stabilizes the RSV F protein in its pre-F conformation, including, but not limited to, an antibody, a small molecule, a peptide, and / or a peptidomimetic. Non-limiting examples of antibodies that bind to pre-fusion RSV F proteins include the 5C4, AM22, and D25 antibodies (see McLellan et al. (2013) Science 342: 592-598, which is incorporated herein by reference in its entirety). In some embodiments, the mutant RSV F molecule may be obtained, isolated, or maintained in its pre-F conformation by controlling the ionic strength of the medium / buffer in which the protein is present (e.g., by using a medium with high or low ionic strength). In some embodiments, the mutant RSV F molecule may be obtained, isolated, or maintained at one or more temperatures suitable for preserving the pre-F conformation. In some embodiments, mutant RSV F molecules may be obtained, isolated or maintained for a period of time during which the pre-F conformation is less likely to be lost.

[0120] In some embodiments, analysis may be performed to confirm that a desired conformation, such as a pre-F conformation, is formed and / or maintained in the mutant RSV F molecule. Such analysis may be performed before cross-linking, during the cross-linking process, after the cross-linking process, or any combination of these steps. Such analysis may include any suitable method known in the art for examining the three-dimensional structure of a protein or protein complex, including functional analysis and crystallographic analysis. In some embodiments, such analysis may include examining the binding of the mutant RSV F molecule to an antibody, such as an antibody specific for the pre-F conformation and / or an antibody known to bind to the "o" site, as described herein, including, but not limited to, the 5C4, AM22, and D25 antibodies.

[0121] In some embodiments, the mutant RSV F molecules of the present invention may be purified before, during, or after one or more steps during the manufacturing process. For example, in some embodiments, the mutant RSV F molecules may be purified after all of the manufacturing steps are completed. In some embodiments, the mutant RSV F molecules may be purified before the cross-linking process is started or after one or more intermediate process steps during the process, such as after expressing the mutant RSV F molecule, after assembling the mutant RSV F molecule into a mature trimer, after obtaining a mutant RSV F molecule in a pre-F conformation, or during or after performing a DT cross-linking reaction. The mutant RSV F molecules of the present invention may be isolated or purified using a suitable method known in the art. Such methods include, but are not limited to, chromatography (e.g., ion exchange, affinity, and / or size exclusion chromatography), ammonium sulfate precipitation, centrifugation, fractional lysis, or other techniques known to those skilled in the art for purifying proteins. In certain embodiments, it may be necessary to separate DT bridged mature trimeric mutant RSV F molecules from those that are not fully bridged or are not fully stabilized in the pre-F conformation. This can be done using a suitable system known in the art. For example, mutant RSV F molecules in the pre-F conformation can be separated from those that are not in the pre-F conformation using an antibody-based separation method using a pre-F or post-F specific antibody. The mutant RSV F molecules of the present invention may be purified from any source used to produce them. Although the purity may vary, in various embodiments, the purified mutant RSV F molecules of the present invention are provided in a form in which the molecule constitutes about 10%, 20%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.9% of the total protein in the final composition.In some embodiments, the mutant RSV F molecules of the present invention may be isolated or purified from other proteins or any other undesired products (e.g., non-crosslinked or non-pre-F RSV F) by standard methods, including, but not limited to, chromatography, glycerol gradients, affinity chromatography, centrifugation, ion exchange chromatography, size exclusion chromatography, and affinity chromatography, or other standard techniques known in the art for purifying proteins. The mutant RSV F molecules to be isolated may be expressed in high or low ionic media and isolated in high or low ionic buffers or solutions. The mutant RSV F molecules of the present invention may also be isolated at one or more temperatures suitable for preserving the desired conformation. The mutant RSV F molecules of the present invention may also be isolated for a period of time that reduces the extent to which the preparation loses the desired conformation. The extent to which the protein preparation maintains one or more desired conformations (e.g., pre-F conformation) may be assayed using suitable methods known in the art, including, but not limited to, biochemical, biophysical, immunological, and virological analyses. Such assays include, but are not limited to, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA) or enzyme-linked immunosorbent spot (ELISPOT) assays, crystallography (including co-crystallization with antibodies), sedimentation, analytical ultracentrifugation, dynamic light scattering (DLS), electron microscopy (EM), cryo-EM tomography, calorimetry, surface plasmon resonance (SPR), fluorescence resonance energy transfer (FRET), circular dichroism analysis and small angle X-ray scattering, neutralization assays, antibody-dependent cellular cytotoxicity assays, and / or in vivo viral infection tests.

[0122] The yield of the mutant RSV F molecules of the present invention can be determined by means known in the art, for example, by comparing the amount of the final engineered protein (e.g., cross-linked pre-F RSV) to the amount of starting material, or to the amount of material present in any preceding step of the manufacturing process. Protein concentration can be determined by standard procedures, for example, the Bradford or Lowry protein assay. The Bradford assay is compatible with reducing agents and denaturants (Bradford, M, 1976. Anal. Biochem. 72: 248). The Lowry assay is more compatible with detergents, and its response is more linear with respect to protein concentration and readout (Lowry, OJ, 1951. Biol. Chem. 193: 265).

[0123] Characterization assays In some embodiments, the mutant RSV F molecules of the present invention or any intermediates in their production may be analyzed to confirm that they have desirable properties, such as one or more of the properties described above or identified elsewhere in this patent specification. For example, in some embodiments, in vitro or in vivo assays may be performed to determine the conformation, stability (e.g., thermostability), half-life (e.g., in a subject's body), aggregation in solution, binding to antibodies (e.g., neutralizing antibodies, broadly neutralizing antibodies; pre-F specific antibodies; antibodies that recognize the site "o", conformation specific antibodies, antibodies that recognize metastable epitopes, D25, AM22, 5C4, 101F, or palivizumab), binding to B cell receptors, activation of B cell receptors, antigenicity, immunogenicity, ability to induce an antibody response, ability to induce a protective antibody / immune response, ability to induce the production of neutralizing antibodies, or ability to induce the production of broadly neutralizing antibodies of the RSV F protein. In embodiments in which the mutant RSV F molecules of the invention are tested in vivo in animals, the animal may be any suitable animal species, including, but not limited to, mammals (e.g., rodent species (e.g., mice or rats), rabbits, ferrets, porcine species, bovine species, equine species, ovine species, or primate species (e.g., humans or non-human primates)), or birds (e.g., chickens).

[0124] Assays for examining protein three-dimensional structure are well known in the art and any suitable assay can be used, including, but not limited to, crystallography (e.g., X-ray crystallography or electron crystallography), sedimentation analysis, analytical ultracentrifugation, electron microscopy (EM), cryo-electron microscopy (cryo-EM), cryo-EM tomography, nuclear magnetic resonance (NMR), small angle X-ray scattering, and fluorescence resonance energy transfer (FRET) assays.

[0125] Assays for determining protein stability are well known in the art and any suitable assay can be used, including, but not limited to, denaturing and non-denaturing electrophoresis, isothermal titration calorimetry, and time-course experiments in which proteins are incubated and analyzed over time under varying protein concentrations, temperatures, pH, or redox conditions.Proteins may also be analyzed for susceptibility to proteolysis.

[0126] Assays for determining binding of a protein to an antibody are well known in the art and any suitable assay can be used, including, but not limited to, immunoprecipitation assays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunosorbent spot assays (ELISPOT), crystallographic assays (including co-crystallization with an antibody), surface plasmon resonance (SPR) assays, and fluorescence resonance energy transfer (FRET) assays.

[0127] Assays for determining neutralizing activity are well known in the art, and any suitable assay can be used. For example, an assay can be performed to determine the neutralizing activity of antibodies or antisera generated by vaccination / immunization of animals with the RSV F polypeptides, proteins and / or protein complexes of the present invention. Neutralization assays known in the art include, but are not limited to, those described by Dey et al., 2007 (Dey et al., 2007, Characterization of Human Immunodeficiency Virus Type 1 Monomeric and Trimeric gp120 Glycoproteins Stabilized in the CD4-Bound State: Antigenicity, Biophysics, and Immunogenicity. J Virol 81(11): 5579-5593) and Beddows et al., 2006 (Beddows et al., 2007, A comparative immunogenicity study in rabbits of disulfide-stabilized proteolytically cleaved, soluble trimeric human immunodeficiency virus type 1 gp140, trimeric cleavage-defective gp140 and momomeric gp120. Virol 360: 329-340).

[0128] Assays for determining whether a vaccine immunogen can induce an immune response and / or demonstrate protective immunity are well known in the art, and any suitable assay can be used. For example, an assay can be performed to determine whether vaccination / immunization of an animal with a RSV F polypeptide, protein, and / or protein complex of the present invention results in an immune response and / or protective immunity against RSV infection. In some embodiments, a comparison can be made between placebo and vaccinated test groups for infection or seroconversion rates, or viral load.

[0129] Assays for determining the pharmacokinetics and biodistribution of proteins are also well known in the art, and any suitable assay can be used to determine the above properties of the RSV F polypeptides, proteins and / or protein complexes of the present invention.

[0130] composition In some embodiments, the present invention provides compositions comprising one or more of the mutant RSV F molecules described herein. In some embodiments, such compositions may be pharmaceutical compositions, i.e., may contain components suitable for administration to a living subject.

[0131] In some embodiments, the mutant RSV F molecule of the present invention may be provided in a composition that includes one or more additional active ingredients, such as one or more additional vaccine immunogens or therapeutic agents. In some embodiments, the mutant RSV F molecule of the present invention may be provided in a composition, such as a pharmaceutical composition, that includes one or more other components, including, but not limited to, a pharma- ceutically acceptable carrier, an adjuvant, an immunostimulant, a wetting or emulsifying agent, a pH buffer, a preservative, and / or any other component suitable for the intended use of the composition. Such compositions may take the form of a solution, a suspension, an emulsion, and the like. The term "pharmaceutically acceptable carrier" includes various diluents, excipients, and / or vehicles in which or with which the mutant RSV F molecules of the present invention can be provided, including, but not limited to, carriers that are known to be safe for delivery to humans and / or other animals, and / or approved by federal or state government regulatory agencies, and / or listed in the United States Pharmacopeia and / or other generally recognized pharmacopoeias, and / or have received explicit or specific approval for use in humans and / or other animals from one or more generally recognized regulatory agencies. Such pharmaceutically acceptable carriers include, but are not limited to, water, aqueous solutions (such as saline solutions and buffer solutions), and organic solvents (such as certain alcohols and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like). In some embodiments, the compositions of the present invention also include one or more adjuvants.Exemplary adjuvants include, but are not limited to, alum adjuvants, inorganic or organic adjuvants, oil-based adjuvants, virosomes, liposomes, lipopolysaccharides (LPS), molecular cages for antigens (e.g., immune stimulating complexes ("ISCOMs"), Ag-modified saponin / cholesterol micelles that form stable cage-like structures that are transported to draining lymph nodes, components of bacterial cell walls, endocytosed nucleic acids (e.g., double-stranded RNA (dsRNA), single-stranded DNA (ssDNA), and demethylated CpG dinucleotide-containing DNA), AUM, aluminum phosphate, aluminum hydroxide, and squalene. In some embodiments, virosomes are used as adjuvants. Additional commercially available adjuvants that can be used in accordance with the present invention include the Ribi Adjuvant System (RAS, an oil-in-water emulsion (Sigma) containing detoxified endotoxin (MPL) and mycobacterial cell wall components in 2% squalene. M6536), TiterMax (a stable, metabolizable water-in-oil adjuvant (CytRx Corporation 150 Technology Parkway Technology Park / Atlanta Norcross, Georgia 30092)), Syntex Adjuvant Formulation (SAF, an oil-in-water emulsion stabilized with Tween® 80 and Pluronic® polyoxyethylene / polyoxypropylene block copolymer L121 (Chiron Corporation, Emeryville, CA)), Freund's Complete Adjuvant, Freund's Incomplete Adjuvant, ALUM-Aluminum Hydroxide, Al(OH). 3(available as Alhydrogel®, Accurate Chemical & Scientific Co, Westbury, NY), SuperCarrier (Syntex Research 3401 Hillview Ave. PO Box 10850 Palo Alto, CA 94303), Elvax 40W1,2 (ethylene-vinyl acetate copolymer ((DuPont Chemical Co. Wilmington, DE)), L-tyrosine co-precipitated with antigen (available from a number of chemical companies); Montanide (manilide oleate, ISA Seppic Fairfield, NJ)), AdjuPrime (a carbohydrate polymer), protein absorbed to nitrocellulose, and Gerbu adjuvant (CC Biotech, Poway, CA).

[0132] In some embodiments, the mutant RSV F molecules of the present invention may be provided in a composition comprising sugar. In some embodiments, the RSV F polypeptides, proteins and / or protein complexes of the present invention may be provided in a composition comprising sucrose. In some embodiments, the RSV F polypeptides, proteins and / or protein complexes of the present invention may be provided in a composition comprising about 5% sucrose. In some embodiments, the RSV F polypeptides, proteins and / or protein complexes of the present invention may be provided in a composition comprising about 10% sucrose. In some embodiments, the RSV F polypeptides, proteins and / or protein complexes of the present invention may be provided in a composition comprising about 15% sucrose. In some embodiments, the RSV F polypeptides, proteins and / or protein complexes of the present invention may be provided in a composition comprising about 20% sucrose. In some embodiments, the RSV F polypeptides, proteins and / or protein complexes of the present invention may be provided in a composition comprising about 25% sucrose. Importantly, it has been found that the inclusion of sucrose reduces the formation of aggregates in the RSV F polypeptide, protein and / or protein complex compositions of the present invention.

[0133] In some embodiments, the present invention provides compositions comprising an "effective amount" of the mutant RSV F molecule of the present invention. Similarly, in some embodiments, the present invention provides methods involving administering to a subject an "effective amount" of a mutant RSV F molecule or a composition comprising a mutant RSV F. An "effective amount" is an amount required to achieve a desired end result. Examples of desired end results include, but are not limited to, the generation of a humoral immune response to RSV, the generation of a neutralizing antibody response to RSV, the generation of a broad neutralizing antibody response to RSV, the generation of protective immunity to RSV, inhibition or RSV viral replication, and the amelioration of one or more symptoms of RSV infection. The amount of the mutant RSV F molecule of the present invention or a composition comprising such a molecule that is effective to achieve a desired end result may depend on a variety of factors, including, but not limited to, the type, subtype, and strain of the RSV virus, the species of the subject (e.g., human or any other animal species), the age of the subject, the sex of the subject, the weight of the subject, the intended route of administration, the intended dosing regimen, and the severity of any ongoing RSV infection (e.g., in the case of therapeutic use). Effective amount may be a range of effective amount, and can be determined by standard techniques without undue experimentation, for example, using in vitro and / or in vivo assays in the intended target species or any suitable animal model species.Suitable assays include, but are not limited to, those involving extrapolation from dose-response curves and / or other data derived from in vitro and / or in vivo model systems.In some embodiments, effective amount can be determined according to the judgment of a physician or veterinarian based on the specific circumstances.

[0134] Use of mutant RSV F molecules In some embodiments, the mutant RSV F molecules of the present invention can be useful as research tools, diagnostic tools, therapeutic agents, targets for the production of antibody reagents or therapeutic antibodies, and / or components of vaccines or vaccine compositions.For example, in some embodiments, the mutant RSV F molecules of the present invention are useful as immunogens for vaccines in animal subjects, such as mammalian subjects, including humans.These and other uses of the mutant RSV F molecules of the present invention are described more fully below.Those skilled in the art should understand that the mutant RSV F molecules of the present invention can also be useful for a variety of other applications, and all such applications and uses are intended to be within the scope of the present invention.

[0135] Tools for studying RSV F antibodies In one embodiment, the mutant RSV F molecules of the present invention can be useful as analytes for assaying and / or measuring the binding and / or potency of anti-RSV F antibodies, for example, in ELISA assays, Biacore / SPR binding assays, and / or other assays known in the art for antibody binding. For example, the mutant RSV F molecules of the present invention can be used to analyze and / or compare the effectiveness of anti-RSV F antibodies.

[0136] Tools for antibody generation The mutant RSV F molecules of the present invention can also be useful for generating antibodies that can be used as therapeutic antibodies and / or research tools, or for any other desired use. For example, the mutant RSV F molecules of the present invention can be used for immunization to obtain antibodies against the RSV F protein for use as a research tool and / or therapeutic agent. In some embodiments, the mutant RSV F molecules of the present invention can be used to immunize non-human animals, such as, but not limited to, vertebrates, including mice, rats, guinea pigs, rabbits, goats, non-human primates, etc., to generate antibodies. Such antibodies, which may be monoclonal or polyclonal, and / or cells that produce such antibodies can then be obtained from the animals. For example, in some embodiments, the mutant RSV F molecules of the present invention can be used to immunize mice to generate and obtain monoclonal antibodies and / or hybridomas that produce such monoclonal antibodies. Such methods can be performed using standard methods for producing mouse monoclonal antibodies known in the art, including standard methods for hybridoma production. In some embodiments, the mutant RSV F molecules of the present invention may be used to produce chimeric, humanized, or fully human antibodies, for example, using any of the methods currently known in the art for producing chimeric (e.g., partially human), humanized, and fully human antibodies, including, but not limited to, CDR grafting, phage display, transgenic mouse methods (e.g., mice genetically modified to produce fully human antibodies, e.g., Xenomouse), and / or other suitable methods known in the art. Antibodies against the mutant RSV F molecules of the present invention produced using such systems can be characterized for antigenicity, preferably using one or several sets of antigens, including the mutant RSV F molecules of the present invention themselves.Further characterization of such antibodies may be performed by standard methods known to those of skill in the art, including, but not limited to, ELISA-based methods, SPR-based methods, biochemical methods (such as, but not limited to, isoelectric point measurement), and methods known in the art for studying the biodistribution, safety, and efficacy of antibodies, for example, in preclinical and clinical studies.

[0137] Administration to subjects In some embodiments, the present invention provides methods comprising administering to a subject a mutant RSV F molecule of the present invention or a composition comprising such a mutant RSV F molecule. Such methods may include methods for treating an individual with RSV (i.e., therapeutic methods) and / or methods for protecting an individual from future RSV infection (i.e., prophylactic methods).

[0138] The subject to which the mutant RSV F molecule of the present invention or a composition comprising such a RSV F polypeptide, protein and / or protein complex can be administered (e.g., during a method of treatment or vaccination) includes any animal species, particularly those that are susceptible to RSV infection or can be a model animal system for the study of RSV infection. In some embodiments, the subject is a mammalian species. In some embodiments, the subject is an avian species. Mammalian subjects include, but are not limited to, humans, non-human primates, rodents, rabbits, and ferrets. Avian species include, but are not limited to, chickens, such as chickens in poultry farms. In some embodiments, the subject to which the mutant RSV F molecule of the present invention or a composition comprising such a mutant RSV F molecule is administered either has RSV or is at risk of RSV infection. In some embodiments, the subject is immunocompromised. In some embodiments, the subject has a heart disease or disorder. In some embodiments, the subject is a human who is greater than about 50 years of age, or greater than about 55 years of age, or greater than about 60 years of age, or greater than about 65 years of age, or greater than about 70 years of age, or greater than about 75 years of age, or greater than about 80 years of age, or greater than about 85 years of age, or greater than about 90 years of age. In some embodiments, the subject is a human who is less than about 1 month old, or less than about 2 months old, or less than about 3 months old, or less than about 4 months old, or less than about 5 months old, or less than about 6 months old, or less than about 7 months old, or less than about 8 months old, or less than about 9 months old, or less than about 10 months old, or less than about 11 months old, or less than about 12 months old, or less than about 13 months old, or less than about 14 months old, or less than about 15 months old, or less than about 16 months old, or less than about 17 months old, or less than about 18 months old, or less than about 19 months old, or less than about 20 months old, or less than about 21 months old, or less than about 22 months old, or less than about 23 months old, or less than about 24 months old.

[0139] A variety of delivery systems are known in the art, and any suitable delivery system can be used to administer the composition of the present invention to a subject. Such delivery systems include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral delivery systems. The composition of the present invention can be administered by any convenient route, for example, by infusion or bolus administration, by absorption through epithelium or mucosa (for example, oral mucosa, rectal and intestinal mucosa, etc.), or together with other biologically active agents. Administration can be systemic or local. Pulmonary administration can also be employed, for example, using an inhaler or nebulizer and a formulation containing an aerosol agent.

[0140] In some embodiments, it may be desirable to administer the pharmaceutical compositions of the present invention locally to tissues where the mutant RSV F molecule may be most effective in producing the desired therapeutic outcome. This may be accomplished, for example, by local infusion, injection, catheter-based delivery, or by using an implant, such as a porous, non-porous, or gelatinous implant, or an implant that includes one or more membranes (e.g., sialastic membranes) or fibers that can locally release the protein or protein complex therethrough or through. In some embodiments, sustained release systems may be used. In some embodiments, pumps may be used (see Langer, supra; Sefton, 1987. CRC Crit. Ref. Biomed. Eng. 14: 201; Buchwald et al., 1980. Surgery 88: 507; Saudek et al., 1989. N. Engl. J. Med. 321: 574). In some embodiments, polymeric materials may be used to facilitate and / or control the release of the mutant RSV F molecules of the invention (Medical Applications of Controlled Release, Langer and Wise (eds.), 1974. CRC Pres., Boca Raton, Florida; Controlled Drug Bioavailability, 1984. Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York; Ranger & Peppas, 1983 Macromol. Sci. Rev. Macromol. Chem. 23: 61; see also Levy et al., 1985. Science 228: 190; During et al., 1989. Ann. Neurol. 25: 351; Howard et al., 1989. J. Neurosurg 71: 105).In some embodiments, a sustained release system can be placed near the tissue / organ to which the RSV pre-fusion F protein or polypeptide is delivered (see, e.g., Goodson, 1984. Medical Applications of Controlled Release, supra, vol. 2: 115 138). Some suitable sustained release systems that may be used in conjunction with the present invention are described in Langer, 1990, Science; vol. 249: pp. 527-1533.

[0141] In some embodiments, the administration of the compositions of the present invention can be performed in combination with the administration of one or more immunostimulants. Non-limiting examples of such immunostimulants include various cytokines, lymphokines and chemokines with immunostimulatory, immunopotentiating and proinflammatory activities, such as interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-12, IL-13); growth factors (e.g., granulocyte macrophage (GM) colony stimulating factor (CSF)); and other immunostimulants, such as macrophage inflammatory factor, Flt3 ligand, B7.1; B7.2. The immunostimulants can be administered in the same formulation as the mutant RSV F molecule or can be administered separately.

[0142] In some embodiments, the mutant RSV F molecule of the present invention or a composition comprising the same can be administered to a subject in a variety of different RSV vaccination methods or regimens. In some such embodiments, administration of a single dose is performed. However, in other embodiments, additional doses can be administered by the same or different routes to achieve the desired protective effect. For example, in newborns and infants, multiple doses may be required to induce a sufficient level of immunity. If necessary, administration can be continued at intervals throughout childhood to maintain a sufficient level of protection against RSV infection. Similarly, adults who are particularly susceptible to RSV infection, such as elderly and immunocompromised individuals, may require multiple immunizations to establish and / or maintain a protective immune response. To induce and maintain a desired level of protection, the level of induced immunity can be monitored, for example, by measuring the amount of secreted and serum neutralizing antibodies, and dosages can be adjusted or vaccinations can be repeated as necessary.

[0143] In some embodiments, the dosing regimen may include a single administration / immunization. In other embodiments, the dosing regimen may include multiple administrations / immunizations. For example, the vaccine may be given as a primary immunization followed by one or more boosts. In some embodiments of the present invention, such a "prime-boost" vaccination regimen may be used. For example, in some such prime-boost regimens, a composition comprising a mutant RSV F molecule as described herein may be administered to an individual multiple times (e.g., two, three, or even more times) at separate times, where the first administration is a "priming" administration and the subsequent administrations are "boosting" administrations. In other such prime-boost regimens, a composition comprising a virus or DNA vector encoding a RSV polypeptide, protein, or protein complex may be administered to an individual first as a "priming" administration, followed by one or more "boosting" administrations of a composition comprising a RSV F polypeptide, protein, or protein complex as described herein. The booster may be delivered by the same and / or different route as the priming immunization. The booster is generally administered a certain time after the priming immunization or a previously administered booster. For example, the booster may be administered about 2 weeks or more after the priming immunization and / or a second booster may be administered about 2 weeks or more after the first booster. The booster may be administered repeatedly throughout the life of the subject, for example, at intervals of about 2 weeks or more. The booster may be spaced, for example, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 3.5 years, about 4 years, about 4.5 years, about 5 years, or longer, after the priming immunization or after a previous booster.

[0144] In some embodiments, the pharmaceutical compositions of the present invention may conveniently be provided in unit dosage form. A unit dosage form contains a single dose or unit (e.g., an effective amount), or an appropriate fraction thereof, of a mutant RSV F molecule of the present invention. The unit dosage form may be provided in single-dose or multi-dose containers.

[0145] In some embodiments, compositions of the invention may be supplied in sealed ampoules or vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier (e.g., water) immediately prior to use.

[0146] kit The present invention further provides kits comprising the mutant RSV F molecules of the present invention or compositions containing such mutant RSV F molecules. To facilitate the use of the methods and compositions of the present invention, any of the components and / or compositions described herein and additional components useful for experimental or therapeutic or vaccine purposes can be packaged in the form of a kit. Typically, the kit contains the above components as well as additional components, which can include, for example, instructions for using the components, packaging materials, containers, and / or delivery devices.

[0147] [Table 1-1]

[0148] [Table 1-2]

[0149] [Table 1-3]

[0150] [Table 1-4]

[0151]

Table 1-5

[0152]

Table 1-6

[0153]

Table 1-7

[0154]

Table 1-8

[0155]

Table 1-9

[0156]

Table 1-10

[0157]

Table 1-11

[0158]

Table 1-12

[0159]

Table 1-13

[0160]

Table 1-14

[0161]

Table 1-15

[0162] [Table 1-16]

[0163] [Table 1-17]

[0164] [Table 1-18]

[0165] In Table 1, SEQ ID NOs: 1-91 are exemplary WT and mutant RSV F amino acid sequences, and SEQ ID NOs: 92-96 are exemplary artificial sequences that can be introduced into the C-terminus of the RSV F molecule. In SEQ ID NOs: 1-91, the underlined residues are F1 and F2 polypeptides, the bolded residues are introduced mutations, the italicized residues are residues that may be deleted or substituted in some embodiments, and the double underlined residues are artificially introduced C-terminal sequences.

[0166] [Table 2]

[0167] Various aspects of the present invention are also further illustrated by the following non-limiting examples:

[0168] Example 1. Selected materials and methods The mutant RSV F protein was expressed in human cells and modified by the introduction of dityrosine bonds as described below.

[0169] Using standard methods, cDNAs encoding mutant RSV F molecules were cloned into the pCDNA3.1 / zeo+ expression vector (Invitrogen) via the 5'BamHI and 3'XhoI restriction endonuclease sites (see FIG. 1).

[0170] HEK 293 cells (ATCC) were grown in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum and 50 μg / ml gentamicin. Cells were seeded into 6-well tissue culture plates (Corning) and grown to 80% confluency (approximately 24 hours). Cells were transfected with 2 μg of each RSV F expression plasmid per well using a 1:4 (M / V) ratio of DNA to polyethyleneimine (25 kDa, linear). 16 hours after transfection, medium was removed and replaced with 2 ml / well of serum-free Freestyle-293 expression medium (Invitrogen). Cells were incubated at 37°C, 5% CO prior to harvesting and analysis. 2 The mixture was then cultured for an additional 48 to 72 hours.

[0171] Detection of RSV-F in cell supernatants by ELISA After harvesting, total RSV F protein was captured directly from cell supernatants in EIA / RIA high binding 96-well plates (Corning) for 1 hour at room temperature. Protein-containing and control wells were then blocked with 4% nonfat milk in PBS-tween® 20 (0.05%) for 1 hour at room temperature. Plates were washed 3 times with PBS-T (400 μl / well). Total RSV F was detected for 1 hour using a high affinity human anti-hRSV antibody (100 ng / ml in PBS) that recognizes both pre-fusion and post-fusion forms of RSV F. Pre-fusion F was detected using a pre-F specific human monoclonal antibody (2 μg / ml in PBS) that recognizes site "o". Wells were washed again 3 times in PBS-T and then incubated for 1 hour at room temperature with HRP-conjugated goat anti-human F(ab)2 (Jackson Immunoresearch) diluted 1:5000 in PBS. Wells were washed six times with PBS-T and total RSV-F was detected and quantified using 100 μl of 3,3',5,5'-tetramethylbenzidine (TMB), which produces a chromogenic signal. The chromogenic reaction was stopped by adding an equal volume of 4N sulfuric acid. Final optical density readings were taken at 450 nm using a BioRad Benchmark Plus microplate absorbance spectrophotometer. A two-fold serial dilution series of each supernatant was used to determine the linear range of detectable signal for each sample, allowing accurate comparison of the relative amounts of RSV-F between samples.

[0172] Dityrosine crosslinks in cell supernatants Immediately after harvesting, 100 μl of transfected and control cell supernatants were transferred to wells of a black flat-bottom non-binding 96-well FIA plate (Greiner bio-one). 300 ng of Arthromyces ramosus peroxidase was added to each sample for cross-linking. Then, 1 μl of 1.2 mM H 2 O 2 was added to both control and DT reactions, and 120 μM H 2 O 2The final reaction concentration was 100 mM NaCl. The reaction was allowed to proceed at room temperature for 20 min, after which the reaction was alkalized by adding an equal volume of pH 10 sodium phosphate buffer. Dityrosine-specific fluorescence was read using a Thermo Scientific Fluoroskan Ascent FL at an excitation wavelength of 320 nm and an emission wavelength of 405 nm.

[0173] Seventy-two hours after transfection, supernatants were either crosslinked (DT) or left uncrosslinked, and total protein was measured by ELISA using a high-affinity human anti-hRSV antibody (100 ng / ml in PBS) that recognizes both pre- and post-fusion forms of RSV-F. In some experiments, after 16 days of storage at 4°C, presentation of the "o" site was measured by ELISA using a preF-specific human monoclonal antibody (2 μg / ml in PBS) that recognizes the "o" site.

[0174] Example 2: Production and in vitro characterization of 428Y, 185Y, and 226Y mutant RSV F molecules 2 is a schematic diagram of an exemplary mutant RSV F molecule containing three tyrosine mutations, namely, the 428Y mutation, the 185Y mutation, and the 226Y mutation. This exemplary mutant RSV F molecule is referred to as "AVR02". The F0 precursor form of AVR02 has the amino acid sequence of SEQ ID NO: 81. The mature AVR02 molecule (formed after transfection and expression in a cell of a nucleic acid molecule encoding SEQ ID NO: 81) contains amino acid residues 26-109 and 137-568 of SEQ ID NO: 81, where amino acid residues 26-109 are RSV F F2 residues, amino acid residues 137-513 are RSV F F1 residues, and amino acid residues 514-568 are a heterologous sequence, including a foldon domain, a His tag, a StrepII tag, and a thrombin cleavage site. The signal peptide from amino acids 1-25 and the pep27 peptide from amino acids 100-136 are not present in the final mature trimeric AVR02 molecule.

[0175] Figure 4 is a schematic diagram of the method for producing DT cross-linked AVR02 (referred to as "DT-AVR02"). Uncross-linked AVR02 is transiently transfected into 293 Freestyle cells (Invitrogen) by standard methods using PEI and expressed as a secreted / soluble form for 5 days. On day 5, cells are harvested, pelleted by low speed centrifugation in a swinging bucket rotor (1000xg x 10 min), decanted into a second tube, clarified by spinning at a higher speed (3000xg x 15 min), and then passed through a 70uM cell strainer prior to purification. AVR02 is purified using Streptactin® resin (IBA Lifesciences) according to the manufacturer's instructions. AVR02 is eluted using a PBS-based elution buffer containing 1 mM EDTA and 2.5 mM desthiobiotin, concentrated using tangential flow filtration (Pall Corporation) and clarified by a final centrifugation step (4° C., >20,000×g×5 min).

[0176] The crosslinking reaction is carried out in a phosphate-containing buffer containing 150 mM NaCl, 300-600 uM HO, using 20-40 ug / mL peroxidase enzyme from Arthromyces ramosus and 10-20 ug / mL AVR02. 2 O 2The reaction is started by the addition of 20 min and stopped by the addition of ascorbic acid (80ng / mL-50ug / mL) with imidazole added to 20mM. The AVR02 protein is then purified using FPLC through an IMAC chromatography column (Ni-NTA) to remove the peroxidase, followed by size exclusion chromatography (Superdex 200 pg) with PBS as eluent. Fractions containing AVR02 are then pooled, concentrated to 500ug / mL, and cleaved overnight (20 hours) at 4°C with 2 units / mL biotinylated thrombin (Millipore). The thrombin is then removed according to the manufacturer's instructions, and any uncleaved AVR02 protein is removed by a 30 minute batch incubation with Streptactin® beads at RT. The protein is passed through a spin filter and finally spun at 4°C to remove the beads before a final assay for protein quantification is performed. The final protein assay is a ninhydrin protein assay according to the method of Starcher, B. Analytical Biochemistry 292: 125-129 (2001). Proteins are flash frozen in liquid nitrogen and stored at -80°C until use.

[0177] Figure 3 shows data showing that each of the two "designs" incorporated into AVR02 forms dityrosine bonds. Figure 3A: After applying the cross-linking reaction, the stability introduced by intramolecular cross-linking between 198Y and 226Y (design 1) was measured over 2 weeks at 4°C by ELISA using D25, a prefusion specific mAb that binds to site "O". Figure 3B: Intermolecular DT bond formation between 185Y and 428Y (design 2) was assayed by Western blotting in comparison to WT (Motavizumab is the primary Ab). F 1 The protein migrates to a trimer (TM) size.

[0178] Figures 5A-B show the conformational integrity of AVR02 and DT-crosslinked AVR02 as determined by site IV / V antibody (Figure 5A) and site "O" antibody (Figure 5B) binding, as compared to the RSV mutant called DS-Cav1 (described in McLellan et al. (2013) Science 342:592-598, which contains the S155C, S290C, S190F, and V207L ​​mutations).

[0179] Figures 6A-B provide data showing the temperature stability of DT-AVR02. Figure 6C provides data showing that AVR02 did not lose binding to 0-specific mAb (5C4) after 5 weeks of storage at 4°C.

[0180] Example 3 In vivo testing of 428Y, 185Y, and 226Y mutant RSV F molecules in mice The immunogenicity of DT-crosslinked AVR02 (DT-AVR02) compared to DS-Cav1 was tested. Details of the AVR02 and DT-AVR02 molecules are described above. The DS-Cav1 mutants are described in McLellan et al. (2013) Science 342: 592-598 and include S155C, S290C, S190F, and V207L ​​mutations. Two groups of 10 6-8 week old female pathogen-free BALB / c mice (Jackson Labs) were immunized intramuscularly in a prime (day 0)-boost (day 21) regimen. Group 1 received DT-AVR02 supplemented with 10 μg of alum adjuvant per animal per dose, and group 2 received DS-Cav1 supplemented with 10 μg of alum adjuvant per animal per dose. Serum was collected on day 35, two weeks after the second immunization (booster).

[0181] Heat-inactivated serum was diluted 1:800 in phenol-free MEM supplemented with glutamax, 5% FBS, and penicillin / streptomycin and serially diluted 4-fold (final volume of 50 uL). RSV-Renilla luciferase virus was then diluted in the same medium as above to 2 × 10 4pfu / mL and 50 uL was added to each well (10,000 pfu / well) along with virus alone or motavizumab control. Virus and serum were incubated at 37°C, 5% CO 2 Hep-2 cells (ATCC) were then trypsinized, counted, and cultured at 1 × 10 6 The cells were diluted to 2.5 × 10 4 The cells were then incubated at 37°C, 5% CO 2 The cells were incubated for 60-72 h at 4°C and luciferase activity was quantified using the Renilla-Glow Luciferase Assay System (Promega) according to the manufacturer's instructions. The IC of each serum dilution was calculated using the following formula: 50 To calculate concentrations, data was analyzed using nonlinear regression (Graphpad Prism).

[0182] FIG. 7 presents the results of this study and shows RSV-Renilla luciferase neutralization titers from animals vaccinated with either DS-Cav1 or DT-AVR02 supplemented with alum.

[0183] Example 4 In vivo testing of 428Y, 185Y, and 226Y mutant RSV F molecules in cotton rats The term "DT-preF" as used in this example and in the figures refers to the molecule called "DT-AVR02" in the above examples, and the term DT-preF can be used interchangeably with the term DT-AVR02. Details of the structure of the DT-AVR02 molecule and its preparation are described above.

[0184] Overview / Overview The goal of this study was to evaluate the efficacy and safety of DT-AVR02 in protecting against RSV in cotton rats (CR). The effect of adding an adjuvant (4 mg / CR of 2% Alhydrogel® (ALH)) was also evaluated. A prime-boost strategy was used and the vaccine was injected intramuscularly. On day +39 after RSV / A / Tracy infection (day +35), lung lavage fluids from two (2) large lobes of the right lung and nasal washes were obtained and RSV titers were determined by plaque assay. One (1) lobe of the right lung was snap frozen in liquid nitrogen and stored at -80°C. Serum samples were obtained throughout the study and used to measure neutralizing antibody activity against RSV / A / Tracy and RSV / B / 18537.

[0185] Both 2 and 10 μg doses of DT-AVR02 protein / cotton rats increased lung activity by approximately 4 log compared to saline controls. 10 PFU / g lung, approximately 1.5 log in nose 10 Total PFU alone was highly effective in reducing RSV / A / Tracy. In these naive cotton rats, all vaccines generated RSV / A / Tracy-specific neutralizing antibody responses after the second (booster) vaccination that were greater than those generated by infection with live RSV / A / Tracy on day 0.

[0186] DT-AVR02 (DT-preF) adjuvanted boost vaccines administered at either 2 μg or 10 μg doses using a two-dose regimen produced robust immune responses in RSV-naïve cotton rats. Both the 2 μg and 10 μg doses provided excellent protection against RSV / A / Tracy replication in the lungs and a significant but moderate reduction in the nose, and induced RSV / A / Tracy- and RSV / B / 18537-specific serum neutralizing antibodies. The data suggest that these vaccines may be particularly effective in RSV pre-immune populations, such as the elderly adult population.

[0187] Experimental design details, results and conclusions The present inventors have designed and tested a series of RSV F mutants with tyrosine substitutions that can introduce targeted dityrosine bridges (DTs) to lock the RSV F protein in its pre-fusion conformation. One such molecule is referred to herein as DT-AVR02 (also referred to herein as DT-preF). The structure and production details of this molecule are described in the Examples above. Preliminary studies in mice (see Examples above) have shown that DT-AVR02 is at least immunogenic equivalent to DS-Cav1, one of the two currently "best" preF designs (DS-Cav1 and SC-DM), which is partially stabilized and at the same time has dramatically improved thermal stability. Although DS-Cav1 and SC-DM have shown promise as preF antigens, these proteins are still relatively unstable and lose antigenicity after only a few weeks at 4 ° C. Our in vitro stability assays showed that DT-AVR02 is more thermally stable than DS-Cav1 and SC-DM, retaining preF antigenicity for at least 100 hours at 37°C, indicating that DT-AVR02 is likely stable enough for clinical development and that its improved stability will also improve vaccine exposure and efficacy in vivo.

[0188] The goal of the experiments described in this example was to evaluate the DT-AVR02 / DT-preF RSV pre-fusion F protein vaccine in vivo in cotton rats at two doses (2 or 10 μg / CR).

[0189] The effect of adding the adjuvant 2% Alhydrogel® (ALH) to the formulation was also evaluated. A prime-boost strategy was used and the vaccine was injected intramuscularly. On day +39 after RSV / A / Tracy infection (day +35), lung lavage fluids from two (2) large lobes of the right lung and nasal washes were obtained and RSV titers were determined by plaque assay. Serum samples were obtained throughout the experiment and used to measure neutralizing antibody activity against RSV / A / Tracy and RSV / B / 18537.

[0190] Vaccination (intramuscular) was performed according to the vaccination schedule shown in FIG. 8 - vaccination on day 0, boost on day 21, and virus challenge 14 days later.

[0191] All animals were infected with live RSV / A / Tracy on day +35. Group 1 consisted of five cotton rats (CR) that were injected intramuscularly with saline (positive virus-infected control). Group 2 consisted of five CR that were infected with live RSV / A / Tracy on day 0 (the "gold standard"). Group 3 consisted of five CR that were injected intramuscularly with 2 μg of DT-preF and adjuvanted with 100 μg of 2% ALH / CR. Groups 4-5 consisted of CR that were injected intramuscularly with 10 μg of DT-preF and adjuvanted with 100 μg of 2% ALH / CR.

[0192] Cotton rats used had a body weight ranging from approximately 75 to 150 g (depending on age at the start of the experiment). Body weights were also measured at the end of the experiment. At the start, the weight, age, and sex distribution of the animals was similar among all groups. Experiments were performed using the NIH and USDA guidelines, The Public Health Service Policy on Humane Care and Use of Laboratory Animals, and the experimental protocol was approved by the Investigational Animal Care and Use Committee (IACUC).

[0193] Cotton rats were lightly anesthetized with isoflurane and were then injected with 1.21 × 10 5 PFU of RSV / A / Tracy (RSV / A / Tracy) (P3 wp 3 / 13 / 2015) was administered intranasally (100 μL). After inoculation on day 0 (group 2) and day 35 (all groups), the virus inoculum was back-titrated to determine the initial concentration (log 10 TCID 50 / mL).

[0194] Alhydrogel® (aluminum hydroxide gel; ALH) was stored at RT [21° C.]. The protein concentration of DT-AVR02 stock solution was 160 μg / mL in PBS (stored at −80° C.). All steps were performed under a sterile hood. The Alhydrogel® 2% formulation was used seven times (total of 700 μL) at a dose of 100 μL per animal.

[0195] Protein samples were thawed on wet ice. For the 10 μg dose, protein was diluted to a concentration of 105.26 μg / mL in PBS (total 665 μL, 437 μL DT-PreF, 228 μL PBS). For the 2 μg dose, protein was diluted to a concentration of 21.05 μg / mL in PBS (total 665, 87.4 μL DT-PreF, 577.6 μL PBS). Protein samples were mixed by inversion. Alhydrogel® (2%, Brenntag) was mixed thoroughly by inversion. The bottle of Alhydrogel® was opened and 35 μL was pipetted into each dose and mixed thoroughly by hand to obtain a homogenous suspension. Prior to vaccination, the compounded solution was mixed several times and mixed again before each injection to ensure a homogenous solution was maintained.

[0196] DT-AVR02 and adjuvant (100 μL of DT-AVR02 and ALH (Groups 3, 4)) were injected intramuscularly (Tuberculin syringe) into the left tibialis anterior (TA). The contralateral leg was used for the second vaccination (booster on day +21).

[0197] Blood was collected from the orbital plexus of one cotton rat in each group on day 0. Blood was collected from all cotton rats (5CR / group; four groups) on all other days indicated.

[0198] CO 2 After euthanasia with acetaminophen, the right lung was ligated. Two lobes were excised from the right lung, rinsed with sterile water to remove external blood stains, and weighed. The same two right lobes were lavaged transpleurally with 3 mL of Iscove's medium (1:1, v / v) containing 15% glycerol and 2% FBS-MEM in a 3 mL syringe with a 26G3 / 8 needle, injected at multiple sites to inflate the lobes as a whole. The lavage fluid was collected by gently pressing flat on one of the inflated lobes and used to lavage the other lobe transpleurally following the same technique. The lavage fluid was collected and kept on ice until titration. Nasal lavage of the upper airways was obtained by dislocating the jaw. The head was excised and 1 mL of Iscove's medium (1:1, v / v) containing 15% glycerol and 2% FBS-MEM was flushed into each nostril (total of 2 mL). The effluent was collected from the rear opening of the pallet and kept on ice until titration. Samples were not frozen prior to titration, which was performed at the end of sampling. An aliquot of the serum sample was set aside for antibody analysis.

[0199] RSV Tracy lung lavage titers (PFU / g lung) and nasal lavage titers (total PFU) were determined as follows: 24 h prior to the start of the assay, cells were cultured to near confluence (approximately 2 × 10 5 Plaque assays were performed in 24-well tissue culture plates containing monolayers of HEp-2 cells (cells / well). At the start of each assay, dilutions (usually log 10 ) was prepared. 0.2 mL of each sample was then added to the wells in duplicate and allowed to adsorb for 90 minutes with occasional gentle agitation. After removal of the inoculum, the monolayer was then overlaid with 0.75% methylcellulose in MEM containing antibiotics, vitamins, and other nutrients. Each assay included tissue culture and positive virus controls. Plates were incubated at 36°C, 5% CO 2Plates were stained with 0.01% crystal violet / 10% formalin solution (approximately 1.5 mL / well) and left at room temperature for 24-48 hours. Wells were rinsed with water. When present, plaques were readily visible (distinct circles on a very dark blue background). All plaques in wells containing approximately 20-100 plaques were counted and averaged, and viral titers were calculated as log 1 for nasal washes and log 2 for total nasal washes. 10 As PFU, log for lung or other organs 10 The lower limit of detection by this method was 0.70 log 10 Total PFU, or approximately 1.4 logs 10 PFUg lung tissue.

[0200] Anti-RSV neutralizing antibody tests on days 0, 21, 35, and 39 were performed as follows: Serum neutralizing antibody tests against RSV / A / Tracy and RSV / B / 18537 were performed in 96-well microtiter plates containing HEp-2 cells. Plaque-purified RSV virus was used in the microneutralization (Nt) assay. Samples were heat-inactivated at 56°C for 30 min. A 3 log 2 Neutralizing antibody (Ab) titers were determined for each sample by performing repeated two-fold serial dilutions starting at 0.01. Neutralizing antibody titers were defined as the serum dilution at which a 50% or greater reduction in viral cytopathic effect (CPE) was observed. CPE was defined as the amount of tissue destruction and was determined visually after fixing wells with 10% neutral buffered formalin and staining with crystal violet. Neutralizing antibody (NtAb) titers are presented as categorical log numbers, not continuous values. The minimum detectable NtAb titer was 2.5 log 2 Samples with undetectable NtAb titers had a 2 log 2 The NtAb titer was 0.5 log 2 The samples were reported as multiples of the upper limit of the test (e.g., 2.5, 3.0, 3.5, 4.0, etc.). 2If the patient had an NtAb titer of 26log or more, the dilution was increased to 26log 2 The samples were retested so that NtAb titers could be determined by extending the incubation time to 40 μg / mL Palivizumab was included as an internal standard.

[0201] RSV-specific antibody titers were determined by ELISA. Serum samples were stored at -20°C before performing the ELISA assay. First, log 10 Transformed viral titer or log 2 Viral infection or RSV-specific neutralizing antibody concentrations were analyzed between control and vaccine groups by conversion antibody titers. Further comparisons can be analyzed by ANOVA using InStat3 (GraphPad) with post hoc Teukese comparisons.

[0202] Further details of the experimental design are provided in Table A. The results are discussed above. Further details of the results are provided below, including Tables B-L.

[0203] [Table 3]

[0204] [Table 4]

[0205] [Table 5]

[0206] [Table 6]

[0207] [Table 7]

[0208] [Table 8]

[0209] [Table 9]

[0210] [Table 10]

[0211] [Table 11]

[0212] [Table 12]

[0213] [Table 13]

[0214] [Table 14]

[0215] Cotton rat 2B was found dead in its cage on day 9. Cause of death is unknown.

[0216] Effect of DT-preF / DT-AVR02 adjuvanted with Alhydrogel® 2% on RSV / A / Tracy titers in nasal washes: Live RSV / A / Tracy infection on day 0 (Group 2) reduced titers by 4.42 log compared to saline control (Group 1). 10 The total PFU (P<0.00001; two-tailed Student's t test) was reduced (Figure 9, Table B). Both vaccine combinations reduced viral titers by 1.37 to 1.77 log 10There was a decrease in total PFU (P≦0.00024; two-tailed Student's t test). There were no statistical differences between the vaccine combinations.

[0217] Effect of DT-preF / DT-AVR02 adjuvanted with Alhydrogel® 2% on RSV / A / Tracy titers in lung lavage fluid: Infection with live RSV / A / Tracy on day 0 (Group 2) reduced titers below the limit of detection (4.03 log 10 PFU / g lung; P<0.00001; two-tailed Student's t test) (Figure 10, Table C). The vaccine combination for groups 3 and 4 reduced RSV titers below the limit of detection (4.04 to 4.13 log 10 PFU / g lung; P<0.00001; two-tailed Student's t test). There were no statistical differences between the vaccine combinations.

[0218] Effect of DT-preF / DT-AVR02 adjuvanted with Alhydrogel® 2% on the generation of RSV / A / Tracy or RSV / B / 18537-specific neutralizing antibodies: For RSV / A / Tracy-specific neutralizing antibodies (NtAb), natural infection with live RSV / A / Tracy (Group 2) resulted in NtAb by day 21 (approximately 5.5 log 2 / 0.05 mL) and remained at that concentration through day 39 (Figure 11; Tables D-G). At day 21 this was statistically different from either DT-AVR02 (P < 0.0036; two-tailed Student's t-test). However, after the second vaccination on day 21, there was a robust NtAb response with either vaccine that was greater than infection with live RSV / A / Tracy.

[0219] Infection with live RSV / A / Tracy and the two vaccines generated RSV / B / 1853 cross-linking NtAbs (Fig. 12; Tables H-L). The pattern of NtAb responses to the two vaccines was similar to that of RSV / A / Tracy, but was 1-2 log 2 It was at a low level.

[0220] Example 5 Comparison of 428Y and 427Y mutant RSV F molecules in vitro and in vivo Using the methods described in the Examples above, two DT bridge mutant RSV F molecules stabilized in the pre-F conformation were generated. The two mutants differed only in that one had a point mutation to tyrosine at amino acid position 428 (428Y mutation) and the other had a point mutation to tyrosine at amino acid position 427 (427Y mutation). Both molecules contained S190F and V207L ​​mutations (i.e., "Cav1" mutations) and tyrosine mutations at amino acid residues 185 and 226 (i.e., 185Y and 226Y mutations).

[0221] The 428Y mutant (including the V185Y, K428Y, K226Y, S190F, and V207L ​​mutations) is referred to herein interchangeably as either the "428Y mutant" or "DT-AVR02." The F0 precursor form of the 428Y mutant has the amino acid sequence of SEQ ID NO: 81. The mature 428Y mutant molecule (formed after transfection and expression in a cell of a nucleic acid molecule encoding SEQ ID NO: 81) comprises amino acid residues 26-109 and 137-568 of SEQ ID NO: 81, where amino acid residues 26-109 are RSV F F2 residues, amino acid residues 137-513 are RSV F F1 residues, and amino acid residues 514-568 are a heterologous sequence, including a foldon domain, a His tag, a Strep II tag, and a thrombin cleavage site. The signal peptide from amino acids 1-25 and the pep27 peptide from amino acids 100-136 are not present in the final mature trimeric 428Y mutant molecule.

[0222] The 427Y mutant (including the V185Y, K427Y, K226Y, S190F, and V207L ​​mutations) is referred to herein interchangeably as either the "427Y mutant" or "DT-CAV1." The F0 precursor form of the 428Y mutant has the amino acid sequence of SEQ ID NO:81, except that it contains a lysine (K) residue at amino acid position 428 and a tyrosine at amino acid position 427. The mature 427Y mutant molecule (formed after transfection and expression in a cell of a nucleic acid molecule encoding this modified version of SEQ ID NO:81) comprises amino acid residues 26-109 and 137-568 of this modified sequence, where amino acid residues 26-109 are RSV F F2 residues, amino acid residues 137-513 are RSV F F1 residues, and amino acid residues 514-568 are a heterologous sequence, including a foldon domain, a His tag, a Strep II tag, and a thrombin cleavage site. The signal peptide from amino acids 1-25 and the pep27 peptide from amino acids 100-136 are not present in the final mature trimeric 427Y mutant molecule.

[0223] The 427Y and 428Y mutants were expressed and purified according to the methods described above and analyzed for conformational integrity by ELISA using motavizumab to normalize total protein, as well as the conformational 5C4 (site "O", prefusion specific) antibody and a prefusion specific monoclonal antibody that binds to the region between antigenic site IV and antigenic site V. The assay was developed with one-step TMB reagents (Pierce) using Ni-NTA coated plates (Qiagen). As shown in Figure 13, the N428Y substitution instead of K427Y significantly improves binding to site IV-V antibodies (Figure 13B and D) and also improves binding to site "O" antibodies (Figure 13A and C).

[0224] Substitution of N428Y instead of K427Y also affects the aggregation state of the molecule after the cross-linking reaction. The proteins were purified according to the methods described herein, and both proteins were cross-linked under identical conditions and processed as described herein. During size-exclusion chromatography using a Superdex 200 26 / 60 pg column with PBS as eluent, the chromatograms of the cross-linked material are significantly different. As shown in Figure 14A, after the cross-linking reaction, the 427Y mutant (DT-CAV1) produces mainly higher aggregate species, as shown in the Coomassie-stained SDS-PAGE analysis of the corresponding final product in Figure 14 (Figure 14C, two gels on the left). When the 428Y mutant (DT-AVR02) was produced by the same treatment, the majority of the protein was present in the trimer fraction, as evident from the chromatogram (Figure 14B) and the Coomassie-stained SDS-PAGE gel of the corresponding final product (Figure 14C, right gel).

[0225] We also tested the immunogenicity of the 427Y mutant (DT-CAV1), the 427Y mutant (DT-AVR02), and the DS-Cav1 (containing S155C, S290C, S190F, and V207L ​​mutations) described in McLellan et al. (2013) Science 342:592-598. Two groups of 10 6-8 week-old pathogen-free female CB6 / F1J mice (Jackson Labs) were immunized with a prime (day 0)-boost (day 21) regimen. In both experiments, group 1 was vaccinated with 10 μg of DS-Cav1 adjuvanted with alum, whereas group 2 was vaccinated with 10 μg per animal per dose of 427Y mutant (DT-CAV1) adjuvanted with alum in the first study, and 10 μg per animal per dose of 428Y mutant (DT-AVR02) adjuvanted with alum in the second study. Serum was collected on day 35, two weeks after the second vaccination (booster). The results are shown in Figures 15A-B. The results show that the serum neutralization titers of animals vaccinated with the 428Y mutant (DT-AVR02) (Figure 15B) are dramatically higher than those vaccinated with the 427Y mutant (DT-Cav1) (Figure 15A).

[0226] Example 6. Formulation with sucrose reduces the formation of RSV F aggregates It has been found that the inclusion of sucrose in the formulation of the mutant RSV F molecule of the present invention significantly reduces the formation of undesirable aggregates. The experiment was carried out using the exemplary DT-bridged 428Y mutant (DT-AVR02) described above. After final concentration, analytical size exclusion chromatography (SEC) demonstrated the presence of the desired DT-AVR02 trimer (peak C in FIG. 16A) and also aggregates (peaks A and B in FIG. 16A). Formulation of the DT-AVR02 molecule with 10% sucrose during the elution of the purification step appeared to prevent the formation of aggregates. As shown in FIG. 16B, in the sample formulated with sucrose, peak C (trimer) remained, but peaks A and B (aggregates) were not visible.

[0227] Example 7 In vivo testing of AVR02 in non-human primates Six groups of five non-human primates are immunized by intramuscular injection with a prime (day 0)-boost (day 28) regimen. Groups 1 and 2 are vaccinated with a high dose (50 mg / animal / injection) of DT-AVR02 with (group 1) or without (group 2) adjuvant; groups 3 and 4 are vaccinated with a low dose (10 mg / animal / injection) of DT-AVR02 with (group 3) or without (group 4) adjuvant; groups 5 and 6 are vaccinated with a high dose (50 mg / animal / injection) of DS-Cav1, the control of the present invention, with (group 1) or without (group 2) adjuvant.

[0228] Animals in all groups are injected twice, on days 0 and 28. Blood is taken from all animals prior to the first immunization on day 0 and the second immunization on day 28. Blood is again taken / harvested on days 42 and 49. Peripheral blood mononuclear cells are isolated by standard methods for characterization of the frequency and phenotype (Th1 / Th2) of RSV-specific T cell responses; serum is analyzed to quantify NHP anti-F responses (IgM, total IgG, IgG1, IgG2, IgG3), epitope analysis is performed by competitive analysis by standard methods (site "O" and site II), and neutralization titers are quantified, also by standard methods.

[0229] Example 8. Phase I human clinical trial of AVR02 A Randomized, Placebo-Controlled, Blinded, Dose-Escalation Study to Investigate the Safety, Tolerability, and Immunogenicity of the Stabilized Pre-fusion RSV F Subunit Protein Vaccine DT-AVR02, Alone or Adjuvanted with Alum, in Healthy (Non-Pregnant) Adults (Age 18-50) 150 subjects will be distributed into 7 cohorts to test 6 formulations and a placebo. Subjects will be randomly assigned to vaccine treatment or saline placebo in a 4:1 ratio, with each cohort containing 20 subjects receiving active vaccine (groups A, B, C, D, E, and F) and 5 subjects receiving placebo (group G). Six groups of 20 (active vaccine) and 5 (placebo) subjects will be tested. 6 x 25 = 150 subjects. Low, medium, and high doses (50 mcg, 150 mcg, and 500 mcg per injection), with each dose formulated with or without alum as an adjuvant.

[0230] Subjects will receive two intramuscular injections on days 0 and 90 (± 5 days). Subjects will be followed for any serious adverse events (SAEs) and non-serious AEs from the time of each vaccination (days 0 and 90) through 30 days post-vaccination. Additionally, subjects will be contacted by telephone on days 60, 150, 180, 210, and 310) to inquire about the occurrence of SAEs and new notable medical conditions.

[0231] The primary objective is to evaluate the safety, reactogenicity, and tolerability of three doses of DT-AVR02, alone or with alum, when injected intramuscularly in healthy adults monitored over 44 weeks. The secondary objective is to evaluate antibody responses in study subjects to three doses of DT-AVR02, without or with adjuvant addition, as measured by ELISA (including Ab subtype analysis) to assess total Ab responses, competitive ELISA to assess epitope-specific responses at 2 and 4 weeks after each injection, and neutralization assays at 2 and 4 weeks after each injection.

[0232] Although the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be apparent to those skilled in the art upon reading this disclosure that various changes in form and detail may be made therein without departing from the true scope of the invention. The invention may also be further defined in the terms of the following claims.

Claims

1. A mutant RSV F molecule comprising a point mutation to tyrosine at amino acid residue 428, or the amino acid corresponding to residue 428 as determined by alignment with SEQ ID NO:

1.

2. 2. The mutant RSV F molecule of claim 1, further comprising a point mutation to tyrosine at amino acid residue 185, or the amino acid corresponding to residue 185 as determined by alignment with SEQ ID NO:

1.

3. 2. The mutant RSV F molecule of claim 1, further comprising a point mutation to tyrosine at amino acid residue 226, or the amino acid corresponding to residue 226 as determined by alignment with SEQ ID NO:

1.

4. 2. The mutant RSV F molecule of claim 1, further comprising: (a) a point mutation to tyrosine at amino acid residue 185, or the amino acid corresponding to residue 185 as determined by alignment to SEQ ID NO:1; and (b) a point mutation to tyrosine at amino acid residue 226, or the amino acid corresponding to residue 226 as determined by alignment to SEQ ID NO:

1.

5. The mutant RSV F molecule of any one of claims 1 to 4, comprising a transmembrane domain and a cytoplasmic domain.

6. The mutant RSV F molecule of any one of claims 1 to 4, wherein the molecule does not contain a transmembrane domain or a cytoplasmic domain.

7. The mutant RSV F molecule of any one of claims 1 to 6, wherein the molecule is an RSV type A or an RSV type B molecule.

8. The mutant RSV F molecule of any one of claims 1 to 7, wherein the molecule binds to a prefusion specific antibody.

9. The molecule is an antigenic site [0010] The mutant RSV F molecule of any one of claims 1 to 8, which binds to an antibody that recognizes

10. The mutant RSV F molecule of any one of claims 1 to 9, wherein the molecule binds to an antibody selected from the group consisting of D25, 5C4, AM22, and AM14.

11. The mutant RSV F molecule of any one of claims 1 to 10, wherein the molecule comprises an F2 polypeptide and an F1 polypeptide, and the C-terminus of the F2 polypeptide is linked to the N-terminus of the F1 polypeptide by a disulfide bond.

12. The mutant RSV F molecule of any one of claims 1 to 11, wherein the molecule comprises an F2 polypeptide and an F1 polypeptide, and the C-terminus of the F2 polypeptide is linked to the N-terminus of the F1 polypeptide by an artificially introduced peptide linker.

13. The mutant RSV F molecule of any one of claims 1 to 12, comprising an F2 polypeptide having approximately 84 amino acid residues and an F1 polypeptide having approximately 375 amino acid residues.

14. The mutant RSV F molecule of any one of claims 1 to 13, comprising an F2 polypeptide having approximately 74-84 amino acid residues and an F1 polypeptide having approximately 365-375 amino acid residues.

15. 15. The mutant RSV F molecule of any one of claims 1 to 14, comprising: (a) an F2 polypeptide comprising, or consisting of, amino acid residues 26 to 109 of any one of SEQ ID NOs:21 to 81; and (b) an F1 polypeptide comprising, or consisting of, amino acid residues 137 to 513 of any one of SEQ ID NOs:21 to 81.

16. 16. The mutant RSV F molecule of any one of claims 1 to 15, comprising: (a) an F2 polypeptide comprising, or consisting of, approximately 74-84 amino acids from amino acid residues 26 to 109 of any of SEQ ID NOs:21 to 81; and (b) an F1 polypeptide comprising, or consisting of, approximately 365-375 amino acids from amino acid residues 137 to 513 of any of SEQ ID NOs:21 to 81.

17. 17. The mutant RSV F molecule of any one of claims 1 to 16, comprising: (a) an F2 polypeptide comprising, or consisting of, amino acid residues 26 to 109 of SEQ ID NO:81; and (b) an F1 polypeptide comprising, or consisting of, amino acid residues 137 to 513 of SEQ ID NO:

81.

18. 18. The mutant RSV F molecule of any one of claims 1 to 17, comprising: (a) an F2 polypeptide comprising, or consisting of, approximately 74-84 amino acids from amino acid residues 26 to 109 of SEQ ID NO:81; and (b) an F1 polypeptide comprising, or consisting of, approximately 365-375 amino acids from amino acid residues 137 to 513 of SEQ ID NO:

81.

19. The mutated RSV F molecule of any one of claims 1 to 18, wherein the molecule is stabilized in a pre-fusion conformation by one or more dityrosine bridges.

20. The mutant RSV F molecule of any one of claims 1 to 19, wherein the molecule is a mature RSV F trimer.

21. 21. The mutant RSV F molecule of any one of claims 1 to 20, wherein the molecule is a mature RSV F trimer stabilized in a pre-fusion conformation by three or more dityrosine bridges.

22. 22. The mutant RSV F molecule of any one of claims 1 to 21, wherein the molecule comprises a dityrosine bridge between (a) a tyrosine at amino acid residue 428, or a corresponding amino acid residue as determined by alignment with SEQ ID NO:1, and (b) a tyrosine at amino acid residue 185, or a corresponding amino acid residue as determined by alignment with SEQ ID NO:

1.

23. 23. The mutant RSV F molecule of any one of claims 1-22, wherein the molecule comprises a dityrosine bridge between (a) a tyrosine at amino acid residue 198, or a corresponding amino acid residue as determined by alignment with SEQ ID NO:1, and (b) a tyrosine at amino acid residue 226, or a corresponding amino acid residue as determined by alignment with SEQ ID NO:

1.

24. 24. The mutant RSV F molecule of any one of claims 1-23, wherein the molecule comprises both (i) a dityrosine bridge between the tyrosine at amino acid residue 198, or a corresponding amino acid residue as determined by alignment to SEQ ID NO:1, and the tyrosine at amino acid residue 226, or a corresponding amino acid residue as determined by alignment to SEQ ID NO:1, and (ii) a dityrosine bridge between the tyrosine at amino acid residue 428, or a corresponding amino acid residue as determined by alignment to SEQ ID NO:1, and the tyrosine at amino acid residue 185, or a corresponding amino acid residue as determined by alignment to SEQ ID NO:

1.

25. 25. The mutant RSV F molecule of any one of claims 1 to 24, wherein the molecule is a mature RSV F trimer comprising three dityrosine bridges, each of the bridges being between (a) a tyrosine at amino acid residue 428, or a corresponding amino acid residue as determined by alignment with SEQ ID NO:1, and (b) a tyrosine at amino acid residue 185, or a corresponding amino acid residue as determined by alignment with SEQ ID NO:

1.

26. 26. The mutant RSV F molecule of any one of claims 1 to 25, wherein the molecule is a mature RSV F trimer comprising three dityrosine bridges, each of the bridges being between (a) a tyrosine at amino acid residue 198, or a corresponding amino acid residue as determined by alignment with SEQ ID NO:1, and (b) a tyrosine at amino acid residue 226, or a corresponding amino acid residue as determined by alignment with SEQ ID NO:

1.

27. 27. The mutant RSV F molecule of any one of claims 1-26, wherein the molecule is a mature RSV F trimer comprising six dityrosine bridges, wherein (i) three of the dityrosine bridges are between the tyrosine at amino acid residue 198, or a corresponding amino acid residue as determined by alignment to SEQ ID NO:1, and the tyrosine at amino acid residue 226, or a corresponding amino acid residue as determined by alignment to SEQ ID NO:1, and (ii) three of the dityrosine bridges are between the tyrosine at amino acid residue 428, or a corresponding amino acid residue as determined by alignment to SEQ ID NO:1, and the tyrosine at amino acid residue 185, or a corresponding amino acid residue as determined by alignment to SEQ ID NO:

1.

28. The mutant RSV F molecule of any one of claims 1 to 27, wherein the molecule further comprises one or more artificially introduced non-DT bridges.

29. The mutated RSV F molecule of any one of claims 1 to 28, wherein the molecule further comprises one or more artificially introduced disulfide bonds.

30. The mutated RSV F molecule of any one of claims 1 to 29, wherein the molecule further comprises one or more point mutations to cysteine.

31. 30. The mutant RSV F molecule of claim 29, wherein the disulfide bond is formed between two cysteines, one or both of which are introduced by a point mutation.

32. 32. The mutant RSV F molecule of any one of claims 1 to 31, wherein the molecule comprises a point mutation to cysteine ​​at amino acid residue 155, or a corresponding amino acid residue thereto as determined by alignment with SEQ ID NO:

1.

33. 33. The mutant RSV F molecule of any one of claims 1 to 32, wherein the molecule comprises a point mutation to cysteine ​​at amino acid residue 290, or a corresponding amino acid residue thereto, as determined by alignment with SEQ ID NO:

1.

34. 34. The mutant RSV F molecule of any one of claims 1 to 33, wherein the molecule comprises both: (a) a point mutation to cysteine ​​at amino acid residue 155, or a corresponding amino acid residue as determined by alignment to SEQ ID NO:1; and (b) a point mutation to cysteine ​​at amino acid residue 290, or a corresponding amino acid residue as determined by alignment to SEQ ID NO:

1.

35. The mutant RSV F molecule of any one of claims 1 to 34, wherein the molecule comprises one or more artificially introduced cavity-filling mutations.

36. 36. The mutant RSV F molecule of claim 35, wherein the molecule comprises a point mutation to phenylalanine at amino acid residue 190, or a corresponding amino acid residue thereto, as determined by alignment with SEQ ID NO:

1.

37. 36. The mutant RSV F molecule of claim 35, wherein the molecule comprises a point mutation to leucine at amino acid residue 207, or a corresponding amino acid residue thereto, as determined by alignment with SEQ ID NO:

1.

38. 36. The mutant RSV F molecule of claim 35, wherein the molecule comprises both (a) a point mutation to phenylalanine at amino acid residue 190, or a corresponding amino acid residue as determined by alignment with SEQ ID NO:1, and (b) a point mutation to leucine at amino acid residue 207, or a corresponding amino acid residue as determined by alignment with SEQ ID NO:

1.

39. 36. The mutant RSV F molecule of claim 35, wherein the molecule comprises one or more cavity-filling mutations selected from the group consisting of 58W, 83W, 87F, 90L, 153W, 190F, 203W, 207L, 220L, 260W, 296F, and 298L.

40. The mutant RSV F molecule of any one of claims 1 to 39, wherein the molecule comprises a heterologous oligomerization domain.

41. 41. The mutant RSV F molecule of claim 40, wherein the heterologous oligomerization domain is a trimerization domain.

42. 42. The mutant RSV F molecule of claim 41, wherein the trimerization domain is a foldon domain, a GCN4 domain, or a T4 fibrintin domain.

43. 42. The mutant RSV F molecule of claim 41, wherein the trimerization domain is a foldon domain comprising SEQ ID NO:

93.

44. The mutant RSV F molecule of any one of claims 1 to 43, wherein the molecule comprises one or more tags useful for detection of the RSV F molecule.

45. The mutant RSV F molecule of any one of claims 1 to 44, wherein the molecule comprises one or more tags useful for purification of the RSV F molecule.

46. 46. ​​The mutant RSV F molecule of claim 44 or 45, wherein the tag is selected from the group consisting of a Strep tag, a StrepII tag, a FLAG tag, a glutathione S-transferase (GST) tag, a green fluorescent protein (GFP) tag, a hemagglutinin A (HA) tag, a histidine (His) tag, a luciferase tag, a maltose binding protein (MBP) tag, a c-Myc tag, a protein A tag, and a protein G tag.

47. The mutant RSV F molecule of claim 44 or 45, wherein the tag comprises SEQ ID NO:

95.

48. The mutant RSV F molecule of claim 44 or 45, wherein the tag comprises SEQ ID NO:

96.

49. The mutant RSV F molecule of any one of claims 44 to 48, wherein the tag is a proteolytically cleavable tag.

50. A mutant RSV F molecule comprising a point mutation to tyrosine at each of: (a) the amino acid residue at position 428, or a corresponding amino acid when determined by alignment to SEQ ID NO:1; (b) the amino acid residue at position 226, or a corresponding amino acid when determined by alignment to SEQ ID NO:1; and (c) the amino acid residue at position 185, or a corresponding amino acid when determined by alignment to SEQ ID NO:

1.

51. 51. The mutant RSV F molecule of claim 50, comprising amino acid residues 26-109 and 137-513 of SEQ ID NO:

81.

52. (i) a mutant RSV F molecule comprising: (a) a point mutation to tyrosine at each of the amino acid residue at position 428, or the amino acid corresponding thereto, as determined by alignment to SEQ ID NO:1; (b) the amino acid residue at position 226, or the amino acid corresponding thereto, as determined by alignment to SEQ ID NO:1; and (c) the amino acid residue at position 185, or the amino acid corresponding thereto, as determined by alignment to SEQ ID NO:1; (ii) a point mutation to phenylalanine at the amino acid residue at position 190, or the amino acid residue corresponding thereto, as determined by alignment to SEQ ID NO:1; and (iii) a point mutation to leucine at the amino acid residue at position 207, or the amino acid residue corresponding thereto, as determined by alignment to SEQ ID NO:

1.

53. 53. The mutant RSV F molecule of claim 52, comprising amino acid residues 26-109 and 137-513 of SEQ ID NO:

81.

54. A soluble mature trimeric RSV F molecule comprising a point mutation to tyrosine at each of: (a) amino acid residue at position 428, or a corresponding amino acid when determined by alignment to SEQ ID NO:1; (b) amino acid residue at position 226, or a corresponding amino acid when determined by alignment to SEQ ID NO:1; and (c) amino acid residue at position 185, or a corresponding amino acid when determined by alignment to SEQ ID NO:

1.

55. 55. The soluble mature trimeric RSV F molecule of claim 54, comprising amino acid residues 26-109 and 137-513 of SEQ ID NO:

81.

56. (i) a soluble mature trimeric RSV F molecule comprising: (a) a point mutation to tyrosine at each of the amino acid residue at position 428, or the amino acid corresponding thereto, as determined by alignment to SEQ ID NO:1; (b) the amino acid residue at position 226, or the amino acid corresponding thereto, as determined by alignment to SEQ ID NO:1; and (c) the amino acid residue at position 185, or the amino acid corresponding thereto, as determined by alignment to SEQ ID NO:1; (ii) a point mutation to phenylalanine at the amino acid residue at position 190, or the amino acid residue corresponding thereto, as determined by alignment to SEQ ID NO:1; and (iii) a point mutation to leucine at the amino acid residue at position 207, or the amino acid residue corresponding thereto, as determined by alignment to SEQ ID NO:

1.

57. 57. The soluble mature trimeric RSV F molecule of claim 56, comprising amino acid residues 26-109 and 137-513 of SEQ ID NO:

81.

58. 58. The RSV F molecule of any one of claims 50 to 57, wherein the molecule comprises one or more DT bridges and is stabilized in a pre-F conformation.

59. A nucleic acid encoding a mutant RSV F molecule of any one of claims 1 to 58.

60. A vector comprising the nucleic acid molecule of claim 59.

61. 60. A cell comprising the nucleic acid molecule of claim 59.

62. A cell comprising the vector of claim 60.

63. A pharmaceutical composition comprising a mutant RSV molecule according to any one of claims 1 to 58.

64. 64. The pharmaceutical composition of claim 63, further comprising an adjuvant.

65. 65. A method of vaccinating against RSV, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition of claim 64.

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