Conformationally stable RSV pre-fusion F proteins

Abstract

The present invention provides mutant RSV F molecules, such as those cross-linked by the introduction of one or more DTs, which may be or be stabilized in a pre-fusion conformation. The invention also provides methods of making the mutant RSV F molecules, compositions comprising the mutant RSV F molecules, and methods of using the mutant RSV F molecules, e.g., in methods of vaccination, methods of treatment, and methods of antibody preparation.

Description

[0001] Divisional application

[0002] This application is a divisional application of application number 201880066038.5, filing date August 7, 2018, and titled “Conformational Stabilized RSV Prefusion F Protein”.

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to U.S. Provisional Patent Application No. 62 / 542,247 filed on August 7, 2017, U.S. Provisional Patent Application No. 62 / 629,685 filed on February 12, 2018, U.S. Provisional Patent Application No. 62 / 640,467 filed on March 8, 2018, and U.S. Provisional Patent Application No. 62 / 674,791 filed on May 22, 2018, the contents of each of which are incorporated herein by reference in their entirety.

[0005] Sequence Listing

[0006] This application contains a sequence listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on August 3, 2018, named Avatar_009_WO1_SL.txt, and is 452739 bytes in size.

[0007] Statement Regarding Federally Funded Research

[0008] 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.

[0009] Incorporated by Reference

[0010] The contents of all documents cited herein are incorporated by reference in their entirety only to the extent such incorporation by reference is permitted. Background Art

[0011] In the United States, respiratory syncytial virus (RSV) infects 4–5 million children annually and is the leading cause of infant hospitalization (approximately 150,000 hospitalizations). Globally, it accounts for 6.7% of infant deaths under the age of 1 year, second only to malaria. It also poses a serious threat to other high-risk populations, including the elderly and immunocompromised, causing approximately 180,000 additional hospitalizations and 12,000 deaths in the United States. There is currently no first-line treatment for FSV, and the only approved preventive treatment for RSV is passive administration of the licensed monoclonal antibody Synagis (palivizumab), which recognizes the RSV fusion (F) protein and reduces the incidence of severe disease by only approximately 50%. The high cost of preventive treatment with Synagis limits its use to premature infants and infants under 24 months of age with congenital heart disease. See the review by Costello et al., “Targeting RSV with Vaccines and Small Molecule Drugs, Infectious Disorders,” Drug Targets, 2012, Vol. 12, No. 2. Developing a more effective, ideal, and cost-effective RSV vaccine would be of great value.

[0012] RSV F protein is an RSV envelope glycoprotein. In nature, RSV F protein is translated into a single precursor polypeptide, called "F0". The length of the F0 precursor polypeptide is generally 574 amino acids, of which the 1st to 25th amino acids contain a signal peptide. The precursor polypeptide F0 forms a precursor trimer, which is hydrolyzed and cleaved by cellular proteases to produce Pep 27 polypeptide, F1 polypeptide and F2 polypeptide. The F2 polypeptide usually contains amino acid residues 26 to 109 of the F0 precursor. The F1 polypeptide usually contains amino acid residues 137 to 574 of the F0 precursor. The F1 and F2 polypeptides are linked by disulfide bonds to form heterodimers, which are called RSV F "protomers". Three such protomers form the mature trimer form of the RSV F molecule.

[0013] RSV F protein is known to induce potent neutralizing antibodies, which are associated with protection against RSV. It has recently been shown that the prefusion conformation of the RSV F protein trimer (referred to herein as "pre-fusion F" or "pre-F") is the major determinant of neutralizing activity in human serum. In addition, the most potent neutralizing antibodies (nAbs) isolated to date bind specifically only to the prefusion conformation. However, soluble pre-F is highly unstable and easily converts to a post-fusion conformation, limiting its use as a vaccine immunogen.

[0014] RSV F protein stabilized in its prefusion conformation may be of great value, providing a candidate RSV vaccine immunogen.

[0015] The crystal structure of the RSV F protein in a prefusion conformation (bound to the potent neutralizing Ab-D25) is described in McLellan et al., 2013, Science, 340, p. 1113-1117, which is incorporated herein by reference in its entirety.

[0016] In order to try to stabilize the pre-F conformation of RSV F, people have developed several different methods. For example, Marshall et al. introduced point mutations "to tyrosine" and dityrosine (DT) cross-linking at certain specific positions of RSV F protein. See international patent application PCT / US2014 / 048086. Other methods include introducing mutations "to cysteine", disulfide bonds and / or cavity filling amino acid replacements at certain specific positions of 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

[0017] The present invention provides some new developments and improvements, surpassing the work of Marshall et al. in patent application PCT / US2014 / 048086, including but not limited to providing some new mutant RSV F molecules with improved properties that are not described in PCT / US2014 / 048086, and compared to the molecules described in PCT / US2014 / 048086, and compared to other RSV F mutants and RSV F candidate immunogens. As shown in the Examples section of this patent disclosure, when these new mutant RSV F molecules are administered to animals using recognized preclinical animal models, they are highly immunogenic.

[0018] In some embodiments, the present invention provides a mutant RSV F molecule comprising a point mutation at position 428 that mutates to tyrosine (abbreviated as "Tyr" or "Y" according to standard convention), i.e., a "428Y mutation". As further described in the Examples section of this patent disclosure, a mutant RSV F molecule comprising this 428Y mutation (referred to as a "428Y mutant") has improved properties compared to other identical mutant RSV F molecules comprising a 427Y mutation instead of a 428Y mutation. These improved properties include: (i) after vaccination, serum neutralization titers increased by about 5 times; (ii) the antigenic profile was significantly improved, which was achieved by specific antibodies to antigenic sites IV-V and to pre-fusion specific antigenic sites. The binding assay of specific antibodies; and, (iii) the yield of RSV-F trimer complexes is significantly increased compared to hexamer complexes. On the basis of these findings, the present invention provides various new and improved mutant RSV F molecules, including but not limited to, mature RSV F trimers, mature RSV F trimers that are conformationally locked as pre-fusion conformations by one or more DT cross-links, its various precursors, and its various components (including F1 and F2 polypeptides and protomers). The present invention also provides amino acid sequences of the mutant RSV F molecules, nucleotide sequences encoding the mutant RSV F molecules, vectors comprising the nucleotide sequences, cells comprising and / or expressing the nucleotide sequences or vectors, methods for preparing the mutant RSV F molecules, and methods using the mutant RSV F molecules - including but not limited to, in vivo vaccinations against RSV and immunogens for antibody production.

[0019] Therefore, in one embodiment, the invention provides a mutation RSV F molecule, which is included in the point mutation (i.e., 428Y mutation) that mutates to tyrosine at the 428th amino acid, or mutates to the point mutation of tyrosine at the amino acid position corresponding to the 428th (for example, by comparison and / or using the amino acid numbering of SEQ ID NO.1 to determine). In some embodiments, the mutation RSV F molecule is or includes RSV F monomer or protomer, and each monomer or protomer includes 428Y mutation. In some embodiments, the mutation RSV F molecule is or includes RSV F trimer (for example, mature RSV F trimer), and each trimer includes three 428Y mutations.

[0020] In some embodiments, the above-mentioned mutation RSV F molecule comprising 428Y mutation is also included in the point mutation (i.e. 226Y mutation) that mutates to tyrosine at the 226th amino acid, or mutates to the point mutation of tyrosine at the amino acid position corresponding to the 226th (for example, by comparison and / or using the amino acid numbering of SEQ ID NO.1 to determine).In some embodiments, the mutation RSV F molecule is or includes RSV F monomer or protomer, and each monomer or protomer includes 428Y mutation and 226Y mutation.In some embodiments, the mutation RSV F molecule is or includes RSV F trimer (for example, mature RSV F trimer), and each trimer includes three 428Y mutations and three 226Y mutations.

[0021] In some embodiments, the above-mentioned mutation RSV F molecule comprising 428Y mutation is also included in the point mutation (i.e. 185Y mutation) that mutates to tyrosine at the 185th amino acid, or mutates to the point mutation of tyrosine at the amino acid position corresponding to the 185th (for example, by comparison and / or using the amino acid numbering of SEQ ID NO.1 to determine).In some embodiments, the mutation RSV F molecule is or includes RSV F monomer or protomer, and each monomer or protomer includes 428Y mutation and 185Y mutation.In some embodiments, the mutation RSV F molecule is or includes RSV F trimer (for example, mature RSV F trimer), and each trimer includes three 428Y mutations and three 185Y mutations.

[0022] In some embodiments, the mutant RSV F molecule comprising the 428Y mutation further comprises: (a) a point mutation (i.e., a 226Y mutation) at the 226th amino acid position to tyrosine, or a point mutation (e.g., determined by comparison and / or using the amino acid numbering of SEQ ID NO.1) corresponding to the 226th amino acid position to tyrosine; and, (b) a point mutation (i.e., a 185Y mutation) at the 185th amino acid position to tyrosine, or a point mutation (e.g., determined by comparison and / or using the amino acid numbering of SEQ ID NO.1) corresponding to the 185th amino acid position to tyrosine. In some embodiments, the mutant RSV F molecule is or comprises an RSV F monomer or protomer, each monomer or protomer comprising a 428Y mutation, a 226Y mutation, and a 185Y mutation. In some embodiments, the mutant RSV F molecule is or comprises an RSV F trimer (eg, a mature RSV F trimer), each trimer comprising three 428Y mutations, three 226Y mutations, and three 185Y mutations.

[0023] Each of the various mutant RSV F molecules described above and elsewhere herein can also contain various other mutations.

[0024] In some embodiments, the mutant RSV F molecules described herein further include one or more additional mutations to tyrosine—for example, one or more mutations to tyrosine described in International Patent Application PCT / US2014 / 048086 (Publication No. WO / 2015 / 013551), the contents of which are incorporated herein by reference.

[0025] In some embodiments, the mutation RSV F molecule described herein also includes one or more mutations that are cysteine, such as one or more or U.S. Patent No. 9,738,689, U.S. Patent No. 9,950,058 or McLellan et al. (2013) Science 342: Those described in 592-598, each of which is incorporated herein by reference. In some such embodiments, RSV F molecules include so-called "DS" mutations--such as U.S. Patent No. 9,738,689, U.S. Patent No. 9,950,058 or McLellan et al. (2013) Science 342: described in 592-598. For example, in some embodiments, RSV F molecules include 155C mutations, 290C mutations, or both 155C and 290C mutations.

[0026] In some embodiments, the mutant RSV F molecule described herein also includes one or more cavity filling amino acid replacements, such as one or more or U.S. Patent No. 9,738,689, U.S. Patent No. 9,950,058 or McLellan et al. (2013) Science 342: Those described in 592-598, each of which is incorporated herein by reference. In some such embodiments, the RSV F molecule includes so-called "Cav1" mutations--such as U.S. Patent No. 9,738,689, U.S. Patent No. 9,950,058 or McLellan et al. (2013) Science 342: described in 592-598. For example, in some embodiments, the RSV F molecule includes S190F cavity filling mutations, V207L ​​cavity filling mutations, or both S190F and V207L ​​cavity filling mutations.

[0027] In some embodiments, the mutant RSV F molecules described herein further comprise a heterologous trimerization domain. In some such embodiments, the trimerization domain is a foldon domain.

[0028] In some embodiments, mutation RSV F molecule as herein described also includes one or more labels, which can be used for detecting and / or purifying RSV F molecule. In some embodiments, these labels can be cleavable labels. In some such embodiments, the label is a His label, a Strep label or a StrepII label.

[0029] Each mutant RSV F molecule described above and elsewhere herein can be provided in 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 naturally occurring RSV F protein--its details (including sequence) are provided in the specific embodiments disclosed in this patent. In some embodiments, the membrane-bound form may include other transmembrane and cytoplasmic domains--that is, it is not inherent in the natural nature of RSV F protein. In some embodiments, the soluble (i.e., non-membrane-bound) form of the mutant RSV F molecule described herein is used. When RSV F molecules are used as components of pharmaceutical compositions--for example, as vaccines for preventing RSV, soluble forms may be particularly useful. As further described in the specific embodiments disclosed in this patent, this soluble form of the mutant RSV F molecule described herein can be produced by, for example, removing transmembrane and cytoplasmic domains. As further described in the specific embodiments disclosed in this patent, in some such embodiments, heterologous sequences can be added to replace transmembrane and cytoplasmic domains, such as heterologous trimerization domains, such as foldon domains.

[0030] Each mutation RSV F molecule described above and elsewhere herein can be provided in the form of a mature RSV F trimer, wherein the mature RSV F trimer comprises one or more DT cross-links and is conformationally locked to its pre-F conformation. This conformationally locked RSV F molecule is prepared by carrying out a DT cross-linking reaction, as described in the specific embodiments and examples of the application, and / or as described in international patent application PCT / US2014 / 048086 (publication number WO / 2015 / 013551), the contents of which are incorporated herein by reference.

[0031] In some embodiments, the invention provides a mutant RSV F molecule as described herein, comprising at least one DT cross-linked. In some embodiments, the invention provides a trimer mutant RSV F molecule as described herein, comprising at least three DT cross-linked. In some embodiments, the invention provides a trimer mutant RSV F molecule as described herein, comprising at least six DT cross-linked.

[0032] In some embodiments, the invention provides a mutant RSV F molecule as described herein, comprising at least one DT cross-link, wherein at least one tyrosine of at least one DT cross-link is introduced by a point mutation that mutates to tyrosine. In some embodiments, the invention provides a mutant trimeric RSV F molecule as described herein, comprising at least three DT cross-links, wherein at least one tyrosine in each of the at least three DT cross-links is introduced by a point mutation that mutates to tyrosine.

[0033] For example, in some embodiments, a DT crosslink is introduced between the tyrosine at residue 198 (typically a naturally occurring tyrosine) and the tyrosine introduced at residue 226 (i.e., resulting from a 226Y mutation) - forming an intramolecular 198Y-226Y DT crosslink. This results in an RSV F trimer containing three intramolecular DT crosslinks.

[0034] In some embodiments, a DT cross-link is introduced between the tyrosine introduced at residue 185 (i.e., resulting from the 185Y mutation) and the tyrosine introduced at residue 428 (i.e., resulting from the 428Y mutation), forming a 185Y-428Y intermolecular DT cross-link. This results in an RSV-F trimer containing three intermolecular DT cross-links.

[0035] In some embodiments, both 198Y-226Y intramolecular DT crosslinks and 185Y-428Y intermolecular DT crosslinks are introduced. This results in an RSV-F trimer containing six DT crosslinks—three intramolecular DT crosslinks and three intermolecular DT crosslinks.

[0036] In some embodiments, the invention provides compositions comprising one or more mutant RSV F molecules described above or elsewhere herein. In some such embodiments, the composition is a pharmaceutical composition, that is, its components are suitable for administration to living subjects. In some such embodiments, the pharmaceutical composition comprises one or more adjuvants. Exemplary adjuvants include but are not limited to alum (alum) adjuvant. In some embodiments, the pharmaceutical composition comprises a carrier. In some embodiments, the pharmaceutical composition comprises an immunostimulant. In some embodiments, the composition is a part of an RSV vaccine or constitutes an RSV vaccine, or can be used for a vaccination method.

[0037] In some embodiments, the invention provides nucleic acid molecules encoding various different mutation RSV F molecules described in other places herein. In some embodiments, the invention provides vectors comprising the nucleic acid molecules. In some embodiments, the invention provides cells comprising the nucleic acid molecules or the vectors.

[0038] 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 inoculating a subject with a RSV vaccine. In some embodiments, the present invention provides a method for inducing the production of RSV neutralizing antibodies in a subject. These methods all include administering an effective amount of a mutant RSV F molecule as described herein to a subject, or an effective amount of a pharmaceutical composition comprising the mutant RSV F molecule. In some such embodiments, the subject is a non-human mammal. For example, in some embodiments, the subject is a non-human mammal for preclinical studies, such as a rodent (e.g., mouse or cotton rat) or a non-human primate. In some embodiments, the subject is a human. In embodiments where the subject is a human, the human subject can be any age. In some embodiments, the human subject is an infant. In some embodiments, the human subject is a child. In some embodiments, the age of the human subject is less than 24 months. In some embodiments, the human subject is an adult. In some embodiments, the age of the human subject is greater than 50 years old. In some embodiments, the age of the human subject is greater than 60 years old.

[0039] In some embodiments, the invention provides methods for preparing anti-RSV F antibodies using mutant RSV F molecules as described herein as immunogens. The antibodies can be used for a variety of applications, including research applications and therapeutic applications. For example, in some embodiments, the method includes applying mutant RSV F molecules as described herein to animal subjects, such as mice or rabbits, for preparing antibodies. In some embodiments, the subject is an animal that has been genetically modified to allow the production of fully human antibodies. In other embodiments, mutant RSV F molecules as described herein can be used as immunogens in other systems for antibody preparation, wherein other systems for antibody preparation include but are not limited to library screening methods, such as phage display screening methods.

[0040] These and other aspects of the present invention are further described in the detailed description, examples, claims, drawings / figures and figure description sections of this patent application - all of which are intended to be read in conjunction with each other. In addition, those skilled in the art will appreciate that the various embodiments described above and elsewhere in this patent disclosure may be combined in a variety of different ways, and such combinations are within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Exemplary nucleic acid vectors for expressing RSV F molecules described herein in mammalian cells.

[0042] Figure 2Schematic representation of mutant RSV F molecules containing 198Y-226Y DT crosslinks ("Design 1") and 185Y-428Y DT crosslinks (Design 2). Antigenic sites are indicated IV and V are related to the molecular structure.

[0043] Figure 3A-B Figure 3A - After DT cross-linking, the D25 (binding site Figure 3B - The formation of intermolecular DT bonds between 185Y and 428Y ("Design 2") was analyzed by Western blotting and compared with WT (Movizumab was the primary antibody). The F1 protein shifted towards the size of the trimer (TM).

[0044] Figure 4 Schematic representation of an exemplary protocol for preparing DT-crosslinked AVR02.

[0045] Figure 5A-B The conformational integrity of DS-Cav1, the uncrosslinked form of the 428Y RSV F mutant (AVR02), and the DT-crosslinked form of the 428Y mutant (DT-AVR02) were compared by ELISA with normalization using motavizumab, using antibodies binding to antigenic sites IV / V ( FIG5A ) and binding sites 5B) to assess conformational integrity.

[0046] Figure 6A-C After 100 h of heat shock at 37°C, Figure 6A After incubation at 4°C and 37°C, 5C4 (site ) with 428Y RSV F mutant (AVR02) and DT cross-linked version of 428Y RSV F mutant (DT-AVR02). FIG6B Retained binding of 5C4 and D25 to DS-Cav1 and DT-AVR02 after incubation at 37°C for 100 hours. The left bar in each pair is 5C4. The right bar in each pair is D25. FIG6C Retained binding of 5C4 (site 5C4) after 5 weeks at 4°C ) retained binding to the 428YRSV F mutant (AVR02).

[0047] Figure 7Effect of DT-cross-linked 428Y RSV F mutant (DT-AVR02 or DT-preF) in mice. Neutralization titers. RSV-Renoluciferase (RenillaLuc) neutralization titers in animals vaccinated with alum-loaded DSCAV1 or DT-AVR02.

[0048] Figure 8 Schematic representation of an exemplary vaccination procedure.

[0049] Fig. 9 Effect of DT cross-linked 428Y RSV F mutant (referred to as DT-AVR02 or DT-preF) in cotton rats. Effect of DT cross-linked 428Y RSV F mutant with 2% alum adjuvant on RSV / A / Tracy titers in nasal washes at day +39. RSV titers for each indicated control and treatment group are shown.

[0050] Fig.10 Effect of DT-cross-linked 428Y RSV F mutant (DT-AVR02 / DT-preF) in cotton rats. Effect of DT-cross-linked 428Y RSV F mutant with 2% alum adjuvant on RSV / A / Tracy titers in lung lavage fluid at day +39. RSV titers for each indicated control and treatment group are shown.

[0051] Fig.11 Effect of DT-cross-linked 428Y RSV F mutant (DT-AVR02 / DT-preF) in cotton rats. Effect of DT-preF with 2% alum adjuvant on the production of neutralizing antibodies in RSV / A / Tracy serum.

[0052] Fig.12 Effect of DT-cross-linked 428Y RSV F mutant (DT-AVR02 / DT-preF) in cotton rats. Effect of DT-preF with 2% alum adjuvant on the production of cross-reactive neutralizing antibodies against RSV / B / 1853.

[0053] Fig. 13A-D Tyrosine substitution at position 428 (428Y) instead of position 427 (447Y) significantly improved the antigenic profile of RSV F mutants. After purification with streptavidin, soluble 427Y (DT-Cav1) mutants (Figures 13A-B) or 428Y (DT-AVR02) mutants (Figures 13C-D) were DT cross-linked, purified, and analyzed for antigenicity using the indicated mAbs. ELISA was normalized to total protein using motavizumab and performed on Ni-NTA coated plates. DSCav1 was used as a positive control. Prefusion structures were probed with two conformational antibodies; for site Specific mAb, pre-fusion specific mAb that binds between antigenic sites IV / V.

[0054] Figure 14A-C Size exclusion chromatography (SEC) and SDS-PAGE analysis show that the overview of DT cross-linked 427Y RSV F mutant (DT-Cav1) and DT cross-linked 428Y RSV F mutant (DT-AVR02) is significantly different.Show, after dityrosine cross-linking reaction, the size exclusion chromatogram of RSV F mutant obtained immediately (427Y / DT-Cav1 Figure 14A upper left, 428Y / DT-AVR02 Figure 14B lower left).428Y mutation significantly reduces high-order species after cross-linking reaction.Under reduction and denaturation conditions, the final product is separated with SDS-PAGE gel, and then Coomassie staining is performed, as shown in Figure 14 C (right).Arrows point out high-order species (427Y / DT-Cav1) and cross-linked trimer species (428Y / DT-AVR02).

[0055] Figure 15A-B Serum neutralization titers of animals vaccinated with DT cross-linked 428Y RSV F mutant (DT-AVR02) were significantly higher than those of animals vaccinated with DT cross-linked 427Y RSV F mutant (DT-Cav1). Animals were vaccinated with 10 μg (micrograms) of DS-Cav1 control (benchmark comparator) or the DT cross-linked molecules shown (427Y / DT-Cav1 in Figure 15A (left) and 428Y / DT-AVR02 in Figure 15B (right)) according to the initial immunization: boost immunization schedule. Serum was obtained after boost immunization, heat inactivated, and then neutralization titers were obtained using RSV-Renilla luciferase reporter virus. The neutralization titers shown are the average of 5 animals / group and are calculated as the reciprocal of the serum dilution that inhibits 50% luciferase activity.

[0056] Fig.16A-B Formulas containing sucrose reduce the formation of RSV-F aggregates. Experiments were performed with the DT-crosslinked 428Y mutant (DT-AVR02). After final concentration, analytical size exclusion chromatography (SEC) was performed. As shown in Figure 16A, three peaks were identified - peak C was a trimer, and peaks A and B were aggregates. During the elution process of the purification step, the DT-AVR02 molecular formula containing 10% sucrose appeared to reduce the formation of aggregates. As shown in Figure 16B, in the sample prepared with 10% sucrose, peak C (trimer) still existed, but peaks A and B (aggregates) were not obvious. DETAILED DESCRIPTION

[0057] The present invention provides to some extent a mutant RSV F molecule comprising one or more mutations mutated to tyrosine, which can be cross-linked by or by introducing one or more DTs and stabilized in a pre-fusion conformation. The present invention also provides a nucleic acid molecule encoding the mutant RSV F molecule, a method for preparing the mutant RSV F molecule, a composition comprising the mutant RSV F molecule, and a method for using the mutant RSV F molecule, including but not limited to a vaccination method, a treatment method, and an antibody preparation method.

[0058] definition

[0059] Unless otherwise expressly defined herein, the technical and scientific terms used in the present invention have the meanings commonly understood by those of ordinary skill in the art, and / or their meanings are clear from the context in which the terms are used. Several terms are defined below. Other terms are defined elsewhere in this patent disclosure.

[0060] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. The term "a" (or "an") and the terms "one or more" and "at least one" can be used interchangeably.

[0061] In addition, "and / or" will be regarded as a specific disclosure of each of the two specified features or components, with or without the other feature or component. Thus, the term "and / or" used in phrases 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" used in phrases 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); C (alone).

[0062] Units, prefixes and symbols are expressed in the form accepted by the International System of Units (SI).Numerical ranges provided herein are inclusive of the numbers defining the range.

[0063] As used in this specification, the terms "(about)" and "approximately", when applied to a numerical value, refer to a range of + or - 20% of the indicated value.

[0064] As used herein, the term "exemplary" means serving as an example, instance, or illustration.

[0065] As used herein, the term "RSV F molecule" refers to all forms of RSV F protein, including F1 polypeptide and F2 polypeptide, that is, including F0 precursor polypeptide, trimer of F0 precursor polypeptide, RSV F protomer and mature RSV F trimer-whether membrane-bound or soluble. As used herein, the term "mutant RSV F molecule" refers to an RSV F molecule containing one or more artificially introduced / artificial mutations.

[0066] As used herein, the term "F1 polypeptide" refers to a polypeptide comprising amino acid residues 137 to 513 of the RSV F F0 precursor sequence. Amino acid residues 137 to 513 do not include RSV F transmembrane and cytoplasmic domains. In some embodiments, the F1 polypeptide may also include RSV F transmembrane and cytoplasmic domains (located within residues 514 to 574 of the RSV F F0 precursor sequence).

[0067] As used herein, the term "F2 polypeptide" refers to a polypeptide comprising amino acid residues 26 to 109 of the RSV F F0 precursor sequence.

[0068] Unless otherwise indicated, as used herein, the terms "protein" and "polypeptide" are used interchangeably. As used herein, the term "protein complex" refers to an assembly of two or more proteins or protein subunits (e.g., two or more monomers or protomers). Unless otherwise indicated, all descriptions herein related to proteins or polypeptides also apply to protein complexes, and vice versa.

[0069] As used herein, the terms "nucleic acid molecule," "nucleic acid sequence," and "nucleotide sequence" are used interchangeably.

[0070] As used herein, the terms "stable" and "locked" are used interchangeably, such as with respect to the effect of crosslinking in stabilizing or locking the RSV F protein in its pre-fusion conformation. These terms do not require 100% stability. More specifically, these terms represent the degree of stability that is improved or enhanced. For example, in some embodiments, when the term "stable" is used for RSV F protein crosslinked to its pre-fusion conformation, the term represents that the stability of the pre-fusion conformation is higher than before or without such crosslinking. Stability and relative stability can be measured in various ways described in other parts of the application, such as based on the half-life of the RSV pre-fusion conformation. The improvement or increase of stability can be beneficial to the intended application to any extent, or it is of great significance to the intended application. For example, in some embodiments, stability can be increased by about 10%, 25%, 50%, 100%, 200% (i.e., 2 times), 300% (i.e., 3 times), 400% (i.e., 4 times), 500% (i.e., 5 times), 1000% (i.e., 10 times) or more.

[0071] As used herein, the term "adjuvant" 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).

[0072] 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 S155C, S290C, S190F and V207L ​​mutations.

[0073] As used herein, the term "prefusion conformation" refers to a structural conformation of the RSV F molecule that can be specifically bound by a prefusion-specific antibody.

[0074] As used herein, the term "prefusion-specific antibody" refers to an antibody that specifically binds to the RSV F molecule in its prefusion conformation, but not to the RSV F protein in its postfusion conformation. Prefusion-specific antibodies include but are not limited to D25, AM22, 5C4 and AM14 antibodies.

[0075] As used herein, the term "AM14" refers to the antibody described in WO 2008 / 147196 A2.

[0076] As used herein, the term "AM22" refers to the antibody described in WO 2011 / 043643 A1.

[0077] As used herein, the term "D25" refers to the antibody described in WO 2008 / 147196 A2.

[0078] 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)).

[0079] Mutated RSV-F molecules

[0080] The RSV fusion protein or "F" protein is the envelope glycoprotein of the respiratory syncytial virus. In nature, the RSV F protein is translated into a single precursor polypeptide (referred to as F0). The F0 precursor polypeptide is generally 574 amino acids in length. Amino acids 1 to 25 of the F0 precursor generally contain a signal peptide. The precursor polypeptide F0 forms a precursor trimer that is generally proteolytically hydrolyzed by one or more cellular proteases at a conserved furin consensus cleavage site to produce the Pep 27 polypeptide, the F1 polypeptide, and the F2 polypeptide. The Pep 27 polypeptide (generally amino acids 110 to 136 of the F0 precursor) is removed and does not form part of the mature RSV F trimer. The F2 polypeptide (referred to interchangeably herein as "F2" or "F2 region") generally consists of amino acid residues 26 to 109 of the F0 precursor. The F1 polypeptide (referred to herein alternatively as "F1" or "F1 region") is typically composed of amino acid residues 137 to 574 of the F0 precursor, and includes an extracellular region (typically residues 137 to 524), a transmembrane domain (typically residues 525 to 550), and a cytoplasmic domain (typically residues 551 to 574). The F1 and F2 polypeptides are linked by disulfide bonds to form heterodimers, referred to as RSV-F "protomers." Three such protomers form a mature RSV-F trimer, which is therefore a homotrimer of three protomers. In nature, mature RSV-F trimers are typically membrane-bound. However, a soluble (i.e., non-membrane-bound) version of a mature RSV-F trimer can be prepared by removing the transmembrane and cytoplasmic regions. For example, by truncating the RSV F protein at amino acid position 513 (i.e., by removing amino acids after position 514), it can be converted into a soluble form.

[0081] In nature, the mature RSV-F trimer mediates the fusion of the virus and the cell membrane. The pre-fusion conformation (which may be referred to herein as "pre-F") of the mature RSV-F trimer is highly unstable (metastable). However, once the RSV virus docks with the cell membrane, a series of conformational changes will occur in the RSV-F protein trimer, and it will be converted into a highly stable post-fusion ("post-F") conformation. Known mature RSV F protein can induce effective neutralizing antibodies ("nAb") associated with RSV protection. For example, with RSV-F protein immunization, nAb (such as palivizumab) that has a protective effect on humans can be induced. In the post-fusion form of RSV-F protein, there are multiple neutralizing epitopes (sites I, II and IV). But recently, Magro et al. showed that incubation of human serum with RSV F protein in its post-fusion conformation did not deplete most of the neutralizing activity against F protein, suggesting the presence of a neutralizing antigenic site specific to the pre-fusion conformation (Magro et al. 2012, PNAS 109(8):3089). By x-ray crystallography, the epitopes recognized by palivizumab (Synagis), movizumab (Numax), and the recently discovered 101F monoclonal antibody (McLellan et al., 2010, J. Virol., 84(23):12236-441; and McLellan et al., 2010, Nat. Struct. Mol. Biol., Feb 17(2):248-50) have been mapped. McLellan et al. (Science 340:1113-1117 (2013)) resolved the structure of F protein in its prefusion conformation and revealed a new neutralizing epitope, site -- is only displayed in the prefusion conformation, and a series of antibodies (e.g., 5C4) bind to it with a 50-fold higher neutralizing potency than Synagis and Numax. Accordingly, there is increasing evidence that the site is in this prefusion conformation and displays RSV vaccine immunogens that can elicit effective prophylaxis. However, the highly unstable (metastable) nature of the prefusion conformation of the RSV-F protein has been shown to be a significant obstacle to the development of such a vaccine. Based on a comparison of the prefusion and postfusion RSV F structures by McLellan et al., there appear to be two regions in the F protein that undergo substantial conformational changes. These regions are located at the N-terminus and C-terminus of the F1 subunit (residues 137 to 216 and 461 to 513, respectively). In the crystal structure of the RSV F protein, the C-terminal residues of F1 can be stabilized in the prefusion conformation by adding the foldon trimerization domain, in which the RSV F protein binds to the site Epitope-bound D25 antibody is retained in its pre-fusion conformation. In order to stabilize the N-terminal region of F1, McLellan et al. found that the combination of antibody D25 is sufficient for crystallographic studies. However, in order to produce vaccine immunogens, alternative stabilization schemes are needed, such as those that do not require RSV F protein to be combined with large antibody molecules. An alternative method that has been tried involves introducing paired cysteine ​​mutations (for disulfide bond formation) and cavity filling mutations near the N-terminal of F1 (see McLellan et al. (2013) Science 342: 592-598 described DS-Cav1 RSV F protein variants, which are incorporated herein by reference in their entirety). However, crystallographic analysis of these variants shows that the structure is only partially in the pre-fusion conformation. Therefore, it is necessary to additionally design RSV-F protein to obtain an immunogen for clinical vaccine development.

[0082] The present invention provides some alternative methods to stabilize the RSV F protein in its pre-fusion conformation - based on the introduction of one or more "mutation to tyrosine" mutations and one or more DT crosslinks at specific positions in the RSV F molecule.

[0083] Table 1 and sequence table disclosed in this patent partly provide the amino acid sequences of several exemplary RSV F and mutant RSV F molecules. Most of the sequences provided in Table 1 are shown as "F0" sequences-that is, these sequences include signal peptides and pep27 peptides present in F0 precursors, but not in mature RSV F proteins. Similarly, the multiple sequences provided in Table 1 include natural transmembrane and cytoplasmic domains, and in some embodiments, these domains can be removed to form soluble forms of mature RSV trimers. Therefore, the final / mature forms (that is, forms that may be cross-linked through DT) of these exemplary mutant RSV molecules generally do not include all the amino acids shown in the sequences in Table 1. However, the final / mature forms of these exemplary mutant RSV molecules will include the F2 regions and at least a portion of the F1 regions of these sequences. Typically, the final / mature forms of these exemplary mutant RSV molecules will include the 26th to 109th amino acid residues and the 137th to 513th amino acid residues of these sequences. It should be noted that in all embodiments herein referring to RSV F molecules having specific exemplary amino acid sequences, the entire F0 sequence or amino acid residues 26 to 109 and amino acid residues 137 to 513 thereof are contemplated.

[0084] In this patent disclosure, when a specific amino acid position / residue in a RSV F molecule is referred to by its amino acid residue number (e.g., the 428th amino acid residue), unless otherwise specified, the amino acid number is the numbering of the RSV F amino acid sequence provided in the sequence table and Table 1 of the present application (see, e.g., SEQ ID NOs.1 to 91). This is the same as the numbering system commonly used in the art when describing RSV F sequences. However, it should be noted that it should be understood by those skilled in the art that different numbering systems can be used. For example, if additional amino acid residues are added or removed compared to any one of SEQ ID NOs: 1 to 91. Similarly, it should be understood that when a specific amino acid residue is referred to by its numbering, the description is not limited to the amino acid precisely located at the numbered position when counting from the starting position of a given amino acid sequence, but also refers to the "corresponding" amino acid residue in any and all RSV F sequences-even if the residue is not at the same exact numbered position in a given molecule, for example, 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. For example, by comparing a given RSV F sequence with SEQ ID NO.1 or any other RSV F amino acid sequence provided herein (i.e., SEQ ID NO.1 to 91), those skilled in the art can easily determine the amino acid position "corresponding" to any particular numbered residue described herein. Considering that RSV F sequences are highly conservative between RSV subtypes and RSV strains, this comparison can be easily performed, whether using a computer or using the eyes. The amino acid sequences of a large number of WT / natural RSV F molecules from different RSV subtypes and strains, and the nucleic acid sequences encoding these RSV F molecules, are known in the art. Several exemplary WT / natural RSV F molecules amino acid sequences are provided in SEQ ID NOs: 1 to 20 and 82 to 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. Patent No. 9,738,689 and U.S. Patent No. 9,950,058, and their contents are each incorporated herein by reference.

[0085] WT / natural RSV-F molecules show amazing high sequence conservation between RSV subtypes and RSV strains. For example, see WO2014 / 160463. For example, RSV subtypes A and B have 90% sequence identity for F0 precursor molecules. In a given RSV subtype (such as subtype A or B), the sequence identity between strains is about 98%. In addition, almost all RSV F F0 precursors identified so far are composed of 574 amino acids. There may be some slight differences in length-these differences usually occur in the cytoplasmic domain.

[0086] The mutations described herein are specific mutations of tyrosine and can be introduced into any suitable RSV F "background" sequence.

[0087] In some embodiments, this "background" sequence is the sequence of WT / natural RSV F molecules (i.e., the RSV F molecular sequence present in nature). The amino acid and nucleotide sequences of a large number of WT / natural RSV F molecules from different RSV subtypes and strains are known in the art. The amino acid sequences of several exemplary WT / natural RSV F molecules are provided in SEQ ID NOs: 1 to 20 and 82 to 85 (see Table 1 and sequence table). Other such sequences are described in International Patent Application No. PCT / US2014 / 048086, U.S. Patent No. 9,738,689 and U.S. Patent No. 9,950,058, and their contents are each incorporated herein by reference.

[0088] In some embodiments, this "background" sequence is a sequence of a mutant RSV F molecule-that is, a sequence comprising one or more artificially introduced mutations compared to WT / natural RSV F molecules. For example, in some embodiments, a "background" RSV F sequence may include a "Cav1" mutation, a "DS" mutation, or a combination thereof. Non-limiting examples of suitable mutation background RSV molecules for specific mutations described herein that may be introduced include those sequences having any of the amino acid sequences in SEQ ID Nos 86 to 91, or those sequences comprising any of the 26th to 109th amino acids (i.e., F2) and the 137th to 513th amino acids (i.e., F1) in SEQ ID Nos 86 to 91 (see Table 1 and Sequence Table). Other suitable mutation background RSV molecules for specific mutations described herein may be introduced, including those described in International Patent Application PCT / US2014 / 048086, U.S. Patent No. 9,738,689, and U.S. Patent No. 9,950,058.

[0089] In some embodiments, the "background" RSV F molecule, in which the specific mutations described herein can be introduced, can be a "full-length" RSV F molecule, i.e., including a transmembrane domain and a cytoplasmic domain. Non-limiting examples of suitable "full-length" background RSV molecules in which the specific mutations described herein and cross-links can be introduced include those having an amino acid sequence of any one of SEQ ID Nos 1 to 9, 11 to 29, 31 to 49, 51 to 80, 83, 85, 87, or 89, or those comprising amino acids 26 to 109 (F2) and amino acids 137 to 513 (F1) of any one of SEQ ID Nos 1 to 9, 11 to 29, 31 to 49, 51 to 80, 83, 85, 87, or 89 (see Table 1 and Sequence Listing).

[0090] In some embodiments, the "background" RSV F molecule, into which the specific mutations and crosslinks described herein can be introduced, can be a "soluble" RSV F molecule, i.e., excluding the transmembrane domain and the cytoplasmic domain. Non-limiting examples of suitable "soluble" background RSV molecules into which the specific mutations and crosslinks described herein can be introduced include those having an amino acid sequence of any one of SEQ ID Nos 10, 30, 50, 81, 82, 84, 86, 88, or 90, or those comprising amino acids 26 to 109 (i.e., F2) and amino acids 137 to 513 (i.e., F1) of any one of SEQ ID Nos 10, 30, 50, 81, 82, 84, 86, 88, or 90.

[0091] Similarly, in some embodiments, a soluble "background" RSV F molecule, into which specific mutations and cross-links described herein can be introduced, can be constructed by removing the transmembrane and cytoplasmic domains of a "full-length" RSV F molecule, for example, by removing the sequence after amino acid position 514 of a "full-length" RSV F molecule, such as one of the RSV F molecules described above.

[0092] In some embodiments, the present invention provides mutant RSV F molecules comprising a point mutation at amino acid residue 42 to tyrosine (which may be referred to as a 428Y point mutation), or at an amino acid corresponding to position 428, as determined, for example, by alignment with SEQ ID NO. 1 and / or using the amino acid numbering of SEQ ID NO. 1, to tyrosine.

[0093] In some embodiments, these mutant RSV F molecules comprising the 428Y mutation further comprise a point mutation to tyrosine at amino acid residue 185 (which may be referred to as a 185Y point mutation), or a point mutation to tyrosine at an amino acid residue corresponding to position 185, as determined, for example, by alignment with SEQ ID NO.1 and / or using the amino acid numbering of SEQ ID NO.1. Thus, these mutants comprise a 428Y mutation and a 185Y mutation.

[0094] In some embodiments, these mutant RSV F molecules comprising the 428Y mutation further comprise a point mutation to tyrosine at amino acid residue 226 (which may be referred to as a 226Y point mutation), or a point mutation to tyrosine at an amino acid residue corresponding to position 226, as determined, for example, by alignment with SEQ ID NO.1 and / or using the amino acid numbering of SEQ ID NO.1. Thus, these mutants comprise a 428Y mutation and a 226Y mutation.

[0095] In some embodiments, these mutant RSV F molecules comprising the 428Y mutation further comprise: (a) a point mutation to tyrosine at amino acid residue 226 (which may be referred to as a 226Y point mutation), or a point mutation to tyrosine at an amino acid residue corresponding to position 226, as determined, for example, by alignment with SEQ ID NO.1 and / or using the amino acid numbering of SEQ ID NO.1; and, (b) a point mutation to tyrosine at amino acid residue 185 (which may be referred to as a 185Y point mutation), or a point mutation to tyrosine at an amino acid residue corresponding to position 185, as determined, for example, by alignment with SEQ ID NO.1 and / or using the amino acid numbering of SEQ ID NO.1. Thus, these mutants include 428Y mutations, 185Y mutations, and 226Y mutations.

[0096] 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, that is, it is a soluble (non-membrane-bound) RSV F molecule.

[0097] In some embodiments, the mutant RSV F molecule is a RSV A type or RSV B type molecule.

[0098] In some embodiments, the mutant RSV F molecule is capable of binding to a prefusion specific antibody. In some embodiments, the mutant RSV F molecule is capable of binding to a recognition antigenic site. The invention relates to an antibody that binds to the antigenic site of the leukemia cell, such as one of the antibodies described 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. Non-limiting examples of such antibodies include D25, 5C4, and AM22. Other antibodies that recognize antigenic sites Antibodies are disclosed herein or are known in the art.

[0099] In some embodiments, the mutant RSV F molecule comprises a F2 polypeptide and a F1 polypeptide, wherein the C-terminus of the F2 polypeptide is connected to the N-terminus of the F1 polypeptide by a disulfide bond. In some embodiments, the mutant RSV F molecule comprises a F2 polypeptide and a F1 polypeptide, wherein the C-terminus of the F2 polypeptide is connected to the N-terminus of the F1 polypeptide by an artificially introduced peptide linker.

[0100] In some embodiments, the mutant RSV F molecule includes: (a) an F2 polypeptide comprising or consisting of about 84 amino acid residues; and, (b) an F1 polypeptide comprising or consisting of about 375 amino acid residues.

[0101] In some embodiments, the mutant RSV F molecule comprises: (a) an F2 polypeptide comprising or consisting of about 74-84 amino acid residues; and, (b) an F1 polypeptide comprising or consisting of about 365-375 amino acid residues.

[0102] In some embodiments, the mutant RSV F molecule comprises: (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.

[0103] In some embodiments, the mutant RSV F molecule comprises: (a) an F2 polypeptide comprising or consisting of about 74 to 84 amino acids 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 about 365 to 375 amino acids of amino acid residues 137 to 513 of any one of SEQ ID NOs 21 to 81.

[0104] In some embodiments, the mutant RSV F molecule comprises: (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.

[0105] In some embodiments, the mutant RSV F molecule comprises: (a) an F2 polypeptide comprising or consisting of approximately 74 to 84 amino acids of amino acid residues 26 to 109 of SEQ ID NO 81; and, (b) an F1 polypeptide comprising or consisting of approximately 365 to 375 amino acids of amino acid residues 137 to 513 of SEQ ID NO.81.

[0106] In some embodiments, the mutant RSV F molecule is stabilized in a prefusion conformation by one or more dityrosine cross-links.

[0107] 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 that is stabilized in a prefusion conformation by three or more dityrosine cross-links.

[0108] In some embodiments, the mutant RSV F molecule comprises a dityrosine cross-link between a tyrosine at amino acid position 428 (or an amino acid position corresponding thereto, such as determined by alignment with SEQ ID NO. 1 and / or using the amino acid numbering of SEQ ID NO. 1) and a tyrosine at amino acid position 185 (or an amino acid position corresponding thereto, such as determined by alignment with SEQ ID NO. 1 and / or using the amino acid numbering of SEQ ID NO. 1).

[0109] In some embodiments, the mutant RSV F molecule comprises a dityrosine cross-link between a tyrosine at amino acid position 198 (or an amino acid position corresponding thereto, such as determined by alignment with SEQ ID NO. 1 and / or using the amino acid numbering of SEQ ID NO. 1) and a tyrosine at amino acid position 226 (or an amino acid position corresponding thereto, such as determined by alignment with SEQ ID NO. 1 and / or using the amino acid numbering of SEQ ID NO. 1).

[0110] In some embodiments, the mutant RSV F molecule comprises both of the following: (a) a dityrosine cross-link between a tyrosine at amino acid position 198 (or an amino acid position corresponding thereto, such as determined by alignment with SEQ ID NO.1 and / or using the amino acid numbering of SEQ ID NO.1) and a tyrosine at amino acid position 226 (or an amino acid position corresponding thereto, such as determined by alignment with SEQ ID NO.1 and / or using the amino acid numbering of SEQ ID NO.1); and, (b) a dityrosine cross-link between a tyrosine at amino acid position 428 (or an amino acid position corresponding thereto, such as determined by alignment with SEQ ID NO.1 and / or using the amino acid numbering of SEQ ID NO.1) and a tyrosine at amino acid position 185 (or an amino acid position corresponding thereto, such as determined by alignment with SEQ ID NO.1 and / or using the amino acid numbering of SEQ ID NO.1).

[0111] In some embodiments, the mutant RSV F molecule is a mature RSV F trimer, comprising three dityrosine cross-links, wherein each dityrosine cross-link is between a tyrosine at amino acid position 428 (or an amino acid position corresponding thereto, such as determined by alignment with SEQ ID NO. 1 and / or using the amino acid numbering of SEQ ID NO. 1) and a tyrosine at amino acid position 185 (or an amino acid position corresponding thereto, such as determined by alignment with SEQ ID NO. 1 and / or using the amino acid numbering of SEQ ID NO. 1).

[0112] In some embodiments, the mutant RSV F molecule is a mature RSV F trimer, comprising three dityrosine cross-links, wherein each dityrosine cross-link is between a tyrosine at amino acid position 198 (or an amino acid position corresponding thereto, such as determined by alignment with SEQ ID NO. 1 and / or using the amino acid numbering of SEQ ID NO. 1) and a tyrosine at amino acid position 226 (or an amino acid position corresponding thereto, such as determined by alignment with SEQ ID NO. 1 and / or using the amino acid numbering of SEQ ID NO. 1).

[0113] In some embodiments, the mutant RSV F molecule is a mature RSV F trimer, comprising six dityrosine cross-links, wherein three dityrosine cross-links are between a tyrosine at amino acid position 198 (or an amino acid position corresponding thereto, such as determined by alignment with SEQ ID NO.1 and / or using the amino acid numbering of SEQ ID NO.1) and a tyrosine at amino acid position 226 (or an amino acid position corresponding thereto, such as determined by alignment with SEQ ID NO.1 and / or using the amino acid numbering of SEQ ID NO.1); and, three dityrosine cross-links are between a tyrosine at amino acid position 428 (or an amino acid position corresponding thereto, such as determined by alignment with SEQ ID NO.1 and / or using the amino acid numbering of SEQ ID NO.1) and a tyrosine at amino acid position 185 (or an amino acid position corresponding thereto, such as determined by alignment with SEQ ID NO.1 and / or using the amino acid numbering of SEQ ID NO.1).

[0114] In some embodiments, the RSV F molecule also includes one or more artificially introduced non-DT crosslinks. In some such embodiments, the non-DT crosslinks are disulfide bonds. In some such embodiments, the RSV F molecule includes one or more point mutations that mutate to cysteine. In some such embodiments, one or more disulfide bonds are formed between two cysteines--one or two cysteines are introduced by point mutations. In some such embodiments, the RSV F molecule is included in the point mutation (i.e., 155C mutation) that mutates to cysteine ​​at the 155th amino acid residue. In some such embodiments, the RSV F molecule is included in the point mutation (i.e., 290C mutation) that mutates to cysteine ​​at the 290th amino acid residue. In some such embodiments, the RSV F molecule includes the point mutation (i.e., 155C mutation) that mutates to cysteine ​​at the 155th amino acid residue, and the point mutation (i.e., 290C mutation) that mutates to cysteine ​​at the 290th amino acid residue. In some such embodiments, the RSV F molecule comprises one or more point mutations to cysteine ​​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.

[0115] In some embodiments, the RSV F molecule also includes one or more artificially introduced cavity filling mutations (such as replacements). In some such embodiments, the RSV F molecule is included in the mutation (190F mutation) that mutates to F at the 190th amino acid residue. In some such embodiments, the RSV F molecule is included in the point mutation (207L mutation) that mutates to L at the 207th amino acid residue. In some such embodiments, the RSV F molecule is included in the mutation (190F mutation) that mutates to F at the 190th amino acid residue and the point mutation (207L mutation) that mutates to L at the 207th amino acid residue. In some such embodiments, the RSV F molecule includes one or more cavity filling amino acid replacements, 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 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 herein incorporated by reference in its entirety.

[0116] 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 fibrin domain. Other examples of trimerization domains that can be used include, but are not limited to, those described in the following literature: 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., 2008). etal., 2007. The reovirusσ-1aspartic acid sandwich: Atrimerization motif poised for conformational change. Biol Chem 282(15):11582-11589); Pancera et al. 2005 (Soluble Mimetics of Human Immunodeficiency Virus Type 1Viral Spikes Producedby Replacement of the Native Trimerization Domain with a HeterologousTrimerization Motif:Characterization and Ligand Binding Analysis.J Virol 79(15):9954-9969); Guthe et al. 2004 (Very fast folding and association of atrimerization domain from bacteriophage T4 fibritin.J.Mol.Biol.v337 pp.905-15); and Papaikolopoulou et al. 2008 (Creation of hybrid nanorods from sequences of natural trimeric fibrous proteins using the fibritin trimerizationmotif.Methods Mol Biol 474:15-33). .

[0117] In some embodiments, the mutant RSV F molecule comprises a foldon domain of SEQ ID NO.93.

[0118] In some embodiments, the mutant RSV F molecule also includes one or more labels (e.g., C-terminal labels) for detecting and / or purifying RSV F molecules. Exemplary labels include, but are not limited to, Strep tags, Strep II tags, FLAG tags, glutathione S-transferase (GST) tags, green fluorescent protein (GFP) tags, hemagglutinin A (HA) tags, histidine (His) tags, luciferase tags, maltose binding protein (MBP) tags, c-Myc tags, protein A tags, protein G tags, etc. In some such embodiments, the RSV F molecule includes the His tag of SEQ ID NO.95. In some such embodiments, the RSV F molecule includes the Strep II tags of SEQ ID NO.96. In some such embodiments, the label is a cleavable label, that is, if necessary, they can be cut / removed from the mutant RSV F molecule. In some such embodiments, the label is located near the position of the proteolytic cleavage site (e.g., C-terminal) - so that the label can be removed using protease. In some such embodiments, the RSV F molecule comprises a thrombin cleavage site of SEQ ID NO.94.

[0119] 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 to 4 of SEQ ID NO.92) linker sequences. The linker 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 for detection and / or purification.

[0120] In some embodiments, the mutant RSV F molecule comprises one or more leader sequences, precursor polypeptide sequences, secretion signals, and / or localization signals.

[0121] In the present invention, embodiments of specific exemplary amino acid sequences involving mutant RSV F molecules (e.g., SEQ ID Nos 21-81) or "background" RSV F molecules (e.g., SEQ ID Nos 1-20 or 82-91), or embodiments involving specific regions of these sequences (e.g., F1 and / or F2 regions thereof), variant forms equivalent to these amino acid sequences may also be used. For example, in some embodiments, amino acid sequences having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any RSV F amino acid sequence described herein may be used across its F1 and F2 regions (especially across the 26th to 109th (F2) and the 137th to 513th (part of F1 that does not include transmembrane and cytoplasmic domains) amino acid residues). In some embodiments, amino acid sequences having at least about 80% or at least 85% sequence identity with the amino acid sequence may be used across these F1 and F2 regions. Compared with one or more specific amino acid sequences provided herein, these variant forms may have added, deleted or substituted amino acids.Therefore, their length may be longer or shorter than the specific sequence.

[0122] Similarly, it is possible to remove or change the amino acid residues after the 514th position of each exemplary RSV F amino acid sequence provided herein. For example, in several amino acid sequences provided herein, the amino acid residues after the 514th position include natural RSV F transmembrane and cytoplasmic domains. In some embodiments, the transmembrane and cytoplasmic domains of these natural RSV F can be removed to produce RSV F molecules of soluble (i.e. non-membrane-bound) type, or can be replaced with different transmembrane and / or cytoplasmic domains. Similarly, in several amino acid sequences provided herein, the amino acid residues after the 514th position include the combination of various artificially added C-terminal sequences--such as the artificial C-terminal sequence provided in SEQ ID NO.92, which includes foldon (foldon) domain (SEQ ID NO.93), thrombin cleavage site (SEQ ID NO.94), histidine tag (SEQ ID NO.95), Strep II tag (SEQ ID NO.96) and various joints. In some embodiments, as required, these artificial C-terminal sequences can be removed, modified, rearranged or replaced. 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.

[0123] In some embodiments, one or more amino acid residues in one or specific mutation RSV F molecule described herein can be replaced by another amino acid. In some embodiments, one or more amino acid residues can be replaced by another amino acid with similar polarity and can be used as a functional equivalent, causing silent changes. In some embodiments, the amino acid replacement in the sequence can be selected from other members of the amino acid class, such as to create conservative replacements. 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. This replacement is generally understood as conservative replacement.

[0124] In some embodiments, artificial, synthetic or atypical amino acids or chemical amino acid analogs can be used to make mutant RSV F molecules described herein. Atypical amino acids include, but are not limited to, D-isomers of common amino acids, fluorinated amino acids, and "designer" amino acids, such as β-methyl amino acids, Cγ-methyl amino acids, Nγ-methyl amino acids, and general amino acid analogs. Additional non-limiting examples of atypical amino acids include, but are not limited to: α-aminooctanoic acid, Acpa; (S)-2-aminoethyl-L-cysteine / HCl, Aecys; aminophenylacetic acid, 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-aminophenylalanine, Aphe; b-alanine, Bal; p-bromophenylalanine, Brphe; cyclohexylamine, Cha; citrulline, Cit; β-chloroalanine , Clala; cycloleucine, Cle; p-cholinephenylalanine, Clphe; cysteic acid, Cya; 2,4-diaminobutyric acid, Dab; ​​3-aminopropionic acid and 2,3-diaminopropionic acid, Dap; 3,4-dehydroalanine, Dhp; 3,4-dihydroxyphenylalanine, Dhphe; p-fluorophenylalanine, Fphe; D-glucosamine, 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-methylleucine, Mle; DL-methionine-S-methylsulfonyl chloride, Msmet; 3-(1-naphthyl)alanine, 1Nala; 3-(2-naphthyl)alanine, 2Nala; deleucine, Nle; N-methylalanine, Nmala; norvaline, Nva; O-benzylserine, Obser; O-benzyltyrosine, Obtyr; O-ethyltyrosine, Oetyr; O-methylserine, Omser; O-methyltyrosine, Omtyr; ornithine, Orn; phenylglycine; penicillamine, Pen ; Pyroglutamic acid, Pga; Pipecolic acid, Pip; Sarcosine, Sar; Tert-butylglycine; Tert-butyllalanic acid; 3,3,3-trifluoroalanine, Tfa; 6-hydroxydopa, Thphe; L-vinylglycine, Vig; (-)-(2R)-2-amino-3-(2-aminoethylsulfonyl)propionic acid dihydrochloride, Aaspa; (2S)-2-amino-9-hydroxy-4,7-dioxanonanoic acid, Ahdna; (2S)-2-amino-6-hydroxy-4-oxohexanoic acid, Ahoha; (-)-(2R)-2-amino-3-(2-hydroxyethylsulfonyl)propionic acid, Ahsopa;(-)-(2R)-2-amino-3-(2-hydroxyethylsulfoxide)propionic acid, Ahspa; (2S)-2-amino-12-hydroxy-4,7,10-trioxadodecanoic acid, Ahtda; (2S)-2,9-diamino-4,7-dioxanononanoic acid, Dadna; (2S)-2,12-diamino-4,7,10-trioxadodecanoic acid, Datda; (S)-5,5-difluorodeleucine, Dfnl; (S)-4,4-difluoronorvaline, Dfnv; (3R)-1-1-dioxo-[1,4]thiazine-3-carboxylic acid, Dtca; (S)-4,4,5,5,6,6,6,6-heptafluorodeleucine, Hfnl; (S)-5,5,6,6,6-pentafluorodeleucine, Pfnl; (S)-4,5,5,5-pentafluoronorvaline, Pfnv; and (3R)-1,4-thiazine-3-carboxylic acid. In addition, the amino acid can be D (dextrorotatory) or L (levorotatory). For a review of typical and atypical amino acids, see Sandberg et al. 1998 (Sandberg et al., 1998. New chemical descriptors relevant for the design of biologically active peptides. A multivariate characterization of 87amino acids. J Med Chem 41(14): pp.2481-91). ;

[0125] Characterization of mutant RSV-F molecules

[0126] The mutant RSV F molecules of the present invention, including those containing one of the exemplary amino acid sequences provided herein (e.g., SEQ ID Nos. 21-81), those containing residues 26-109 and / or 137-513 of one of these exemplary amino acid sequences, those containing the F1 and / or F2 polypeptides of one of these exemplary amino acid sequences, and variant forms of these specific amino acid sequences, should: (a) contain F1 polypeptide and F2 polypeptide, (b) be capable of forming or being processed (e.g., from a precursor such as F0) to form a mature RSV F trimer capable of adopting a prefusion conformation (as described / defined herein). In some embodiments, the mutant RSV F molecules of the present invention may also have one or more of the following properties: (1) bind to pre-F specific antibodies, (2) bind to the binding site (3) binds to a neutralizing antibody, (4) binds to a broadly neutralizing antibody, (5) binds to an antibody selected from the group consisting of D25, AM22, 5C4, and 101F, (6) binds to palivizumab (Synagis), (7) binds to and / or activates a B cell receptor, (8) induces an anti-RSV antibody response in an animal, (9) induces a protective anti-RSV antibody response in an animal, (10) induces the production of anti-RSV neutralizing antibodies in an animal, (11) induces the production of anti-RSV broadly neutralizing antibodies in an animal, (12) induces the production of anti-RSV antibodies that recognize a quadruple neutralizing epitope (QNE) in an animal, and / or (13) induces a protective anti-RSV immune response in an animal.

[0127] DT cross-linking

[0128] In some embodiments, the present invention provides mutant RSV F molecules comprising at least one artificially introduced DT cross-link. The DT cross-link helps stabilize the mutant RSV F molecules described herein in their pre-fusion conformation.

[0129] In some embodiments, the DT crosslink can be introduced between two endogenous tyrosine residues, between two artificially introduced tyrosine residues (ie, derived from a "mutation to tyrosine" mutation), or between an artificially introduced tyrosine residue and an endogenous tyrosine residue.

[0130] In some embodiments, at least one tyrosine of at least one DT cross-link is derived from a point mutation that mutates to tyrosine (i.e., introduced by a point mutation that mutates to tyrosine). In some embodiments, the invention provides a mutant trimeric RSV F molecule comprising at least three DT cross-links, wherein at least one tyrosine of each of the at least three DT cross-links is derived from a point mutation that mutates to tyrosine (i.e., introduced by a point mutation that mutates to tyrosine).

[0131] For example, in some embodiments, the invention provides mutant RSV F molecules comprising a DT crosslink between a tyrosine at residue 198 (typically a naturally occurring tyrosine) and an introduced tyrosine at residue 226 (i.e., caused by a 226Y mutation) - i.e., an intramolecular 198Y-226Y DT crosslink. In embodiments where the mutant RSV F molecule is a trimer, the trimer thus comprises three intramolecular 198Y-226Y DT crosslinks.

[0132] In some embodiments, the invention provides mutant RSV F molecules comprising a DT crosslink between a tyrosine introduced at residue 185 (i.e., caused by a 185Y mutation) and a tyrosine introduced at residue 428 (i.e., caused by a 428Y mutation) - i.e., an intermolecular 185Y-428Y DT crosslink. In embodiments where the mutant RSV F molecule is a trimer, the trimer thus comprises three intermolecular 185Y-428Y DT crosslinks.

[0133] In some embodiments, the invention provides mutant RSV F molecules comprising both 198Y-226Y intramolecular DT crosslinks and 185Y-428Y intermolecular DT crosslinks. In embodiments where the mutant RSV F molecule is a trimer, the trimer thus comprises six DT crosslinks—three intramolecular 198Y-226Y DT crosslinks and three intermolecular 185Y-428Y DT crosslinks.

[0134] As described above, each protomer of the mature RSV F trimer contains two different polypeptides - called F1 and F2 - that are non-covalently associated to form the protomer. The bond between the F1 polypeptide and the F2 polypeptide within the same protomer is an example of an intermolecular bond and an intraprotomer bond. The 185Y-428Y DT crosslink is designed to hold the two protomers of the trimer together - that is, it is an intermolecular, interprotomer bond. The tyrosine at position 185 on one protomer forms a dityrosine bond with the tyrosine at position 428 on the other protomer.

[0135] The examples disclosed in this patent provide an exemplary method for performing a DT cross-linking reaction. In addition, methods for performing DT cross-linking are known in the art and are described in, for example, Marshall et al., U.S. Patent Nos. 7,037,894 and 7,445,912, the contents of which are incorporated herein by reference. Dityrosine cross-linking introduces a covalent carbon-carbon bond with minimal variation and zero length. DT cross-linking will not be hydrolyzed under physiological conditions. Dityrosine cross-linking is known to be safe because they can be formed naturally in the body and they are present in large quantities in common foods. For example, DT bonds form the structure of wheat protein powder. Dityrosine bonds do not form spontaneously in vitro. Instead, an enzymatic cross-linking reaction must be performed, in which proteins containing tyrosyl side chains are subjected to reaction conditions that lead to the formation of DT bonds. These conditions are, or become, oxidation reaction conditions, because the DT bond formation reaction is an oxidative cross-linking reaction. In some embodiments, DT cross-linking reaction conditions produce proteins that are not modified or have no detectable modifications. These conditions can be obtained by using an enzyme that catalyzes the formation of H2O2, such as a peroxidase. The formation of DT bonds can be monitored by spectrophotometry using an excitation wavelength of about 320 nm, and fluorescence can be measured at a wavelength of about 400 nm, and the loss of tyrosyl fluorescence can be monitored by standard procedures. When the loss of tyrosyl fluorescence is no longer stoichiometric with the formation of DT bonds, the reaction can be stopped by any method known to those skilled in the art, for example, by adding a reducing agent and then cooling or freezing the sample (on ice) to stop the reaction. U.S. Patents Nos. 7,037,894 and 7,445,912 and the Examples section disclosed in this patent describe other details of suitable DT cross-linking methods.

[0136] Nucleic acid molecules

[0137] In some embodiments, the invention provides nucleic acid molecules encoding mutation RSV F molecules described herein, and vectors comprising the nucleic acid molecules. One of ordinary skill in the art can easily determine the nucleotide sequence of the nucleic acid molecules encoding any mutation RSV F molecules described herein-in view of the generally known and understood properties of genetic code among those of ordinary skill in the art.

[0138] In some embodiments, the nucleic acid molecule encodes a precursor F0 polypeptide that, when expressed in an appropriate cell, will be correctly processed to generate a mature RSV F molecule. For example, in some embodiments, the nucleic acid molecule can encode one of the precursor (F0) polypeptides, whose amino acid sequences are provided in Table 1 and the sequence table. In some embodiments, the nucleic acid molecule can encode only the F2 polypeptide or only the F1 polypeptide.

[0139] Any suitable method known in the art can be used to obtain or prepare nucleic acid molecules encoding mutation RSV F molecules described herein.For example, nucleic acid molecules encoding mutation RSV F molecules can be obtained from cloned DNA, or prepared by chemical synthesis.In some embodiments, nucleic acid molecules can be obtained by reverse transcription RNA, wherein RNA is prepared by any method known to those of ordinary skill in the art.Point mutation, or any other modification as described herein (for example, removing C-terminal sequence, replacing C-terminal sequence, etc.), can be completed by DNA standard recombination methods known and understood by those of ordinary skill in the art.For example, those of ordinary skill in the art are positioned by the nucleotide codons of the specific amino acid residues to be mutated into tyrosine to coding (for example, encoding the nucleotide codons of the 185th, 226th or 428th amino acid residues), if necessary, the nucleotides of the codons can be mutated, to produce the codons encoding tyrosine, can easily carry out "mutation into tyrosine" mutation as described herein.

[0140] No matter how the source is, the nucleic acid molecules encoding the mutant RSV F molecules of the present invention can be cloned into any suitable vector, such as a vector for nucleic acid molecule propagation or a vector for nucleic acid molecule expression. In embodiments where expression is desired, the nucleic acid can be operably linked to a promoter suitable for directional expression in a 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.

[0141] In some embodiments, the nucleic acid molecules encoding the mutant RSV F molecules of the present invention can be codon optimized for expression in cells of specific organisms or species. For example, International Patent Application PCT / US2014 / 048086 provides nucleotide sequences of RSV F molecules for expression in people, hamsters, mice and insect cells, and codon optimized. The codon optimized nucleotide sequences of this coding RSV F protein can be used as "background" sequences for introducing any specific mutations described herein.

[0142] Manufacturing method

[0143] The mutant RSV F molecule of the present invention can be prepared by any suitable method known in the art.Usually, the mutant RSV-F molecule is prepared using the standard method for producing recombinant proteins.For example, the nucleic acid molecules 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 (such as SF9 or Hi5 cells, for example, using baculovirus expression system).The method for expressing protein by nucleic acid molecules is conventional and well known in the art, and any suitable method, carrier, system and cell type known in the art can be used.For example, the nucleic acid molecules encoding the mutant RSV F molecule of the present invention are usually placed in a suitable expression construct comprising a suitable promoter, which is then transferred to the cell for expression.

[0144] In some embodiments, the mutant RSV F molecule of the present invention is a mature RSV F trimer that is stably in a pre-F conformation. In these embodiments, the nucleic acid molecules encoding the mutant RSV F molecule are usually expressed in a soluble form in a cell, then allowed to be assembled into a normal trimer pre-F conformation, and then the molecule carries out an enzymatic DT cross-linking reaction. In some embodiments, before and / or during the enzymatic cross-linking reaction, for example, when cross-linking is performed, it is possible to obtain a mutant RSV F molecule that is in (and / or remains on) a pre-F conformation. In some embodiments, the mutant RSV F molecule can be produced and / or separated in such a way that most or substantially all of the mutant RSV F molecules exist in a pre-F conformation. In some embodiments, the mutant RSV F molecule that is in a pre-F conformation can be separated from a mixed population of RSV F protein molecules, and the mixed population comprises some RSV F molecules that are in a pre-F conformation and some RSV F molecules that are in other conformations. In some embodiments, RSV F protein is expressed in a cell (for example, with its membrane-bound or soluble form), and spontaneously assembles into its normal pre-F conformation. In some embodiments, before DT cross-linking, no additional stabilization is required to maintain the mutant RSV F molecule in its pre-F form. In some embodiments, the mutant RSV F molecule can be maintained under specific conditions or in a specific composition that are conducive to forming and / or maintaining a pre-F conformation. For example, in some embodiments, the mutant RSV F molecule in its pre-F conformation can be maintained in the absence of cells--contact with cells may trigger its conversion to a post-F conformation. The mutant RSV F molecule can be obtained and / or separated and / or maintained in the pre-F conformation using 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 can be expressed in the presence of some molecules, or expressed under co-expression or contact with these molecules, and these molecules bind to RSV F proteins and stabilize them in a pre-F conformation, including but not limited to antibodies, small molecules, peptides and / or peptidomimetics. Non-limiting exemplary antibodies that bind to pre-fusion RSV F proteins include 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, mutant RSV F molecules in pre-F conformation can be obtained, separated, or maintained by controlling the ionic strength of the medium / buffer in which the protein is present (e.g., by using a medium of high or low ionic strength). In some embodiments, mutant RSV F molecules can be obtained, separated, or maintained at one or more temperatures that are conducive to preserving the pre-F conformation.In some embodiments, mutant RSV F molecules can be obtained, isolated, or maintained for a period of time that reduces the extent of loss of the pre-F conformation.

[0145] In some embodiments, analysis can be performed to confirm that a desired conformation, such as a pre-F conformation, has been formed and / or maintained in a mutant RSV F molecule. Such analysis can be performed before cross-linking, during cross-linking, after the cross-linking process, or in any combination of these stages. Such analysis can include any suitable method known in the art for evaluating the three-dimensional structure of a protein or protein complex, including functional analysis, crystallographic analysis, etc. In some embodiments, such analysis can include evaluating the binding of mutant RSV F molecules to certain antibodies, such as antibodies specific for the pre-F conformation as described elsewhere herein and / or antibodies known to bind to the pre-F conformation. Site-binding antibodies include, but are not limited to, 5C4, AM22, and D25 antibodies.

[0146] In some embodiments, the mutant RSV F molecule of the present invention can be purified before, during or after one or more steps in the preparation process. For example, in some embodiments, the mutant RSV F molecule can be purified after completing all preparation steps. In some embodiments, the mutant RSV F molecule can be purified before starting the cross-linking process, or after one or more intermediate method steps in the process, for example: after the mutant RSV F molecule is expressed, after the mutant RSV F molecule is assembled into a mature trimer, after obtaining the mutant RSV F molecule in its pre-F conformation, or during or after the DT cross-linking reaction. The mutant RSV F molecule of the present invention can be separated or purified using any suitable method known in the art. These methods include, but are not limited to, chromatography (such as ion exchange, affinity and / or size column chromatography), ammonium sulfate precipitation, centrifugation, differential dissolution, or any other technology known to those of ordinary skill in the art for purifying proteins. In a specific embodiment, it may be necessary to separate the mature trimer mutant RSV F molecule cross-linked by DT from those not fully cross-linked, or those RSV F molecules that are not fully stable in the pre-F conformation. This can be accomplished using any suitable system known in the art. For example, antibody-based separation methods can be used, using pre-F or post-F specific antibodies, the mutant RSV-F molecule in the pre-F conformation is separated from the mutant RSV-F molecule that is not in the pre-F conformation.Can be from any source for preparing them, purify the mutant RSV F molecule of the present invention.The degree of purification may be different, but in each embodiment, the purified mutant RSV F molecule of the present invention is provided in a form, wherein they comprise about 10%, 20%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 99.9% of total protein in the final composition.In some embodiments, the mutant RSV F molecule of the present invention can be separated and purified from other proteins or any other unwanted product (such as non-crosslinked or non-pre-F RSV F) by standard methods, and the standard methods include but are not limited to chromatography, glycerol gradient method, affinity chromatography, centrifugation, ion exchange chromatography, size exclusion chromatography and affinity chromatography, or by any other standard techniques known in the art for purifying proteins. The sudden change RSV F molecule to be separated can be expressed in high or low ionic medium, or be separated in high or low ionic buffer or solution.The sudden change RSV F molecule of the present invention can also be separated at one or more temperatures that are conducive to keeping the desired conformation.They can also be separated in the time period that reduces the degree of conformation required for formulation loss.Protein formulations keep the degree of one or more required conformations (for example pre-F conformation), can be measured by any suitable method known in the art, including but not limited to biochemistry, biophysics, immunology and virological analysis.These assays include, but are not limited to, for example, 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-electron tomography, calorimetry, surface plasmon resonance (SPR), fluorescence resonance energy transfer (FRET), circular dichroism analysis, small angle x-ray scattering, neutralization assays, antibody-dependent cellular cytotoxicity assays, and / or in vivo challenge studies.

[0147] The yield of the mutant RSV F molecule of the present invention can be determined by any means known in the art, for example, by comparing the amount of the final engineered protein (e.g., cross-linked pre-F RSV) with the amount of the starting material, or with the amount of the material present in any previous step of the production method. Protein concentration can be determined by standard procedures, for example, Bradford (Bradford) or Lowrie (Lowrie) protein assay. Bradford assay has compatibility with reducing agents and denaturants (Bradford, M, 1976. Anal. Biochem. 72: 248). Lowry (Lowry) assay has better compatibility with detergent, and the reaction has a more linear relationship with protein concentration and reading (Lowry, OJ, 1951. Biol. Chem. 193: 265).

[0148] Testing of features

[0149] In some embodiments, the mutant RSV F molecules of the invention or any intermediates in their preparation can be analyzed to confirm that they have desired properties, such as one or more of the properties listed above or identified elsewhere in this patent specification. For example, in some embodiments, in vitro or in vivo testing can be performed to assess the conformational structure, stability (e.g., thermal stability), half-life (e.g., in a subject), polymerization in solution, binding to antibodies (e.g., neutralizing antibodies, pan-neutralizing antibodies; pre-F specific antibodies; recognition sites) of the RSV F protein. The present invention relates to antibodies, 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, the ability to elicit antibody responses, the ability to elicit protective antibodies / immune responses, the ability to elicit neutralizing antibodies, or the ability to elicit pan-neutralizing antibodies. In embodiments where the mutant RSV F molecule of the present invention is tested in vivo in an animal, the animal can be any suitable animal species, including but not limited to mammals (e.g., rodent species (e.g., mice or rats), rabbits, ferrets, pig species, cattle species, horse species, sheep species, or primate species (e.g., humans or non-human primates), or avian species (e.g., chickens).

[0150] Assays for assessing protein conformational structure are well known in the art, and any suitable assay may be used, including, but not limited to, crystallographic analysis (e.g., X-ray crystallography or electron crystallography), sedimentation experiments, analytical ultracentrifugation, electron microscopy (EM), cryo-electron microscopy (cryo-EM), cryo-electron tomography, nuclear magnetic resonance (NMR), small angle x-ray scattering, fluorescence resonance energy transfer (FRET) assays, and the like.

[0151] Assays for evaluating protein stability are well known in the art, and any suitable assay may be used, including, but not limited to, denaturing and native electrophoresis, isothermal titration calorimetry, and time course experiments in which proteins are incubated and analyzed over time at varying protein concentrations, temperatures, pH values, or redox conditions. Proteins may also be analyzed for their susceptibility to proteolytic degradation.

[0152] Assays for evaluating protein binding to antibodies are well known in the art, and any suitable assay may be used, including, but not limited to, immunoprecipitation assays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunosorbent spot assays (ELISPOT), crystallographic analysis (including co-crystallization with antibodies), surface plasmon resonance (SPR) assays, fluorescence resonance energy transfer (FRET) assays, and the like.

[0153] The assay for assessing neutralizing activity is well known in the art, and any suitable assay can be used. For example, testing can be performed to determine the neutralizing activity of the antibody or antiserum produced by vaccination / immunization of an animal with RSV F polypeptide, protein and / or protein complex 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).

[0154] Whether the vaccine immunogen can induce an immune response, and / or the test method proving protective immunity is well known in the art, and any suitable test method can be used.For example, it is possible to test to determine whether immune response and / or protective immunity of anti-RSV infection are provided to animals with RSV F polypeptide, protein and / or protein complex of the present invention.In some embodiments, can be between placebo group and test vaccination group, with regard to its infection rate, seroconversion rate or viral load, compare.

[0155] Assays for evaluating the pharmacokinetics and biodistribution of proteins are also well known in the art, and any suitable assay can be used to evaluate these properties of the RSV F polypeptides, proteins, and / or protein complexes of the invention.

[0156] Composition

[0157] In some embodiments, the present invention provides compositions comprising one or more mutant RSV F molecules described herein. In some embodiments, the composition can be a pharmaceutical composition—that is, comprising components suitable for administration to a living subject.

[0158] In some embodiments, the mutant RSV F molecule of the present invention can be provided in a composition, and the composition 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 can be provided in a composition, such as a pharmaceutical composition, and it includes one or more other components, including but not limited to pharmaceutically acceptable carriers, adjuvants, immunostimulants, wetting agents or emulsifiers, pH buffers, preservatives, and / or any other components suitable for the intended use of the composition. These components can be in the form of solutions, suspensions, emulsions, etc. The term "pharmaceutically acceptable carrier" includes that the mutant RSV F molecule of the present invention can provide various diluents, excipients and / or vehicles provided therein or therewith, and includes but is not limited to, known for safely delivering to humans and / or other animal subjects, and / or the carrier approved by the regulatory agency of the federal or state government, and / or the carrier listed in the U.S. Pharmacopoeia and / or other generally recognized pharmacopeias, and / or obtain special or individual approvals for humans and / or other animals from one or more recognized regulatory agencies. These pharmaceutically acceptable carriers include, but are not limited to, water, aqueous solutions (e.g., physiological saline solutions, buffers, etc.), organic solvents (e.g., certain alcohols and oils, including alcohols and oils of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil), etc. In some embodiments, the composition of the present invention further includes 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), antigen molecule cages (e.g., immunostimulatory complexes ("ISCOMS")), Ag-modified saponin / cholesterol micelles, which form stable cage structures and are transported to draining lymph nodes), bacterial cell wall components, endocytosed nucleic acids (e.g., double-stranded RNA (dsRNA), single-stranded DNA (ssDNA), and unmethylated CpG dinucleotides containing DNA), AUM, aluminum phosphate, aluminum hydroxide, and squalene. In some embodiments, virosomes are used as adjuvants.Other commercially available adjuvants that can be used in accordance with the present invention include, but are not limited to, Ribi Adjuvant System (RAS, an oil-in-water emulsion containing detoxified endotoxin (MPL) and mycobacterial cell wall components in 2% squalene (Sigma M6536)), TiterMax (a stable, metabolizable oil-in-water adjuvant (CytRx Corporation 150 Technology Parkway Technology Park / Atlanta Norcross, Georgia 30092)), Syntex Adjuvant Formulation (SAF, an oil-in-water emulsion stabilized by 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 from 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 many chemical companies); Montanide (an oleic acid ester, ISA Seppic Fairfield, NJ)), AdjuPrime (a carbohydrate polymer), nitrocellulose adsorbed protein, Gerbu adjuvant (CC Biotech, Poway, CA), etc.

[0159] In some embodiments, the mutant RSV F molecules of the present invention may be provided in a composition containing sugar. In some embodiments, the RSV F polypeptides, proteins and / or protein complexes of the present invention may be provided in a composition containing sucrose. In some embodiments, the RSV F polypeptides, proteins and / or protein complexes of the present invention may be provided in a composition containing 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 containing 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 containing 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 containing 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 containing about 25% sucrose. Importantly, it has been found that containing sucrose can reduce the formation of aggregates in the compositions of the RSV F polypeptides, proteins and / or protein complexes of the present invention.

[0160] In some embodiments, the present invention provides compositions of mutant RSV F molecules of the present invention comprising an "effective amount". Similarly, in some embodiments, the present invention provides methods involving administering to a subject a mutant RSV F molecule of an "effective amount" or a composition comprising mutant RSV F. "Effective amount" is the amount required to achieve the desired end result. Examples of expected end results include, but are not limited to: generating a humoral immune response against RSV, generating a neutralizing antibody response against RSV, generating a pan-neutralizing antibody response against RSV, generating protective immunity against RSV, inhibiting or RSV viral replication, and improving one or more RSV infection symptoms. The amount of the mutant RSV F molecule of the present invention or the composition comprising the molecule that effectively achieves the expected end result may depend on a variety of factors, including, but not limited to, the type, subtype and strain of RSV virus, the species of the subject (e.g., humans or some other animal species), the age of the subject, the sex of the subject, the weight of the subject, the planned route of administration, the planned dosing regimen, the severity of any persistent RSV infection (e.g., in the case of therapeutic applications), etc. The effective amount - which may be a range of effective amounts - can be determined without any undue experimentation by standard techniques, for example, using in vitro and / or in vivo tests in the intended test species or any suitable animal model species. Suitable tests include, but are not limited to, those involving extrapolation from dose-response curves, and / or other data obtained from in vitro and / or in vivo model systems. In some embodiments, the effective amount can be determined according to the judgment of a physician or veterinarian based on the specific circumstances.

[0161] Application of mutant RSV-F molecules

[0162] In some embodiments, the mutant RSV F molecule of the present invention can be used as a research tool, a diagnostic tool, a therapeutic agent, a target for producing an antibody reagent or a therapeutic antibody, and / or a component of a vaccine or a vaccine composition. For example, in some embodiments, the mutant RSV F molecule of the present invention can be used as a vaccine immunogen for an animal subject (e.g., a mammalian subject, including the human race). These and other uses of the mutant RSV F molecule of the present invention are described more comprehensively below. It will be appreciated by those skilled in the art that the mutant RSV F molecule of the present invention can also be used for various other applications, and all these applications and uses are intended to fall within the scope of the present invention.

[0163] Tools for studying RSV-F antibodies

[0164] In one embodiment, the mutant RSV F molecule of the present invention can be used as an analyte for testing and / or measuring the binding and / or titer of anti-RSV F antibodies, for example in an ELISA assay, a Biacore / SPR binding assay, and / or any other antibody binding assay known in the art. For example, the mutant RSV F molecule of the present invention can be used for analyzing and / or comparing the effect of anti-RSV F antibodies.

[0165] Tools for producing antibodies

[0166] The mutation RSV F molecule of the present invention can also be used to produce therapeutic antibodies, and / or can be used as the antibody of research tool, or for any other desired purposes.For example, the mutation RSV F molecule of the present invention can be used for immunity, to obtain the RSV F protein antibody used as research tool and / or therapeutic agent.In some embodiments, in order to produce antibody, the mutation RSV F molecule of the present invention can be used for immune non-human animals, such as vertebrates, including but not limited to mice, rats, guinea pigs, rabbits, goats, non-human primates, etc. Then such antibodies can be obtained from animals, which can be monoclonal antibodies or polyclonal antibodies, and / or cells producing such antibodies.For example, in some embodiments, the mutation RSV F molecule of the present invention can be used for immune mice, to produce and obtain monoclonal antibodies, and / or hybridomas producing such monoclonal antibodies.These methods can be implemented using standard methods known in the art for producing mouse monoclonal antibodies, including standard methods for producing hybridomas. In some embodiments, the mutant RSV F molecules of the present invention can be used to produce chimeric (such as partially human), humanized or fully human antibodies, such as using any method currently known in the art for producing chimeric, humanized and fully human antibodies, including but not limited to CDR grafting methods, phage display methods, transgenic mouse methods (such as using mice genetically modified to be able to produce fully human antibodies, such as transgenic mice), and / or any other suitable method known in the art. One or a group of multiple antigens (preferably including the mutant RSV F molecules of the present invention themselves) can be used to characterize the antigenicity of antibodies prepared using this system for the mutant RSV F molecules of the present invention. This antibody can be additionally characterized by any standard method known to those of ordinary 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 determination), and methods known in the art for studying the biodistribution, safety and effectiveness (such as in preclinical and clinical studies) of antibodies.

[0167] Administering drugs to subjects

[0168] In some embodiments, the invention provides methods comprising administering to a subject a mutant RSV F molecule of the invention or a composition comprising the mutant RSV F molecule. These methods may include methods for treating an individual suffering from RSV (i.e., methods of treatment) and / or methods for protecting an individual from future infection by RSV (i.e., methods of prevention).

[0169] The subject of the composition comprising the mutant RSV F molecule of the present invention, or the RSV F polypeptide, protein and / or protein complex, can be applied (e.g., in the process of a therapeutic method or an immunization method), including any and all animal species, especially including those susceptible to RSV infection or providing a model animal system for RSV infection research. 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 subjects include, but are not limited to chickens, such as chickens in poultry farms. In some embodiments, the subject of the composition comprising the mutant RSV F molecule of the present invention or the RSV F molecule either suffers from RSV, or has the risk of RSV infection. In some embodiments, the immune function of the subject declines. In some embodiments, the subject suffers from heart disease or disorder. In some embodiments, the age of the subject is greater than about 50 years, or greater than about 55 years, or greater than about 60 years, or greater than about 65 years, or greater than about 70 years, or greater than about 75 years, or greater than about 80 years, or greater than about 85 years, or greater than about 90 years. In some embodiments, the age of the subject is less than about 1 month, or less than about 2 months, or less than about 3 months, or less than about 4 months, or less than about 5 months, or less than about 6 months, or less than about 7 months, or less than about 8 months, or less than about 9 months, or less than about 10 months, or less than about 11 months, or less than about 12 months, less than about 13 months, or less than about 14 months, or less than about 15 months, or less than about 16 months, or less than about 17 months, or less than about 18 months, or less than about 19 months, or less than about 20 months, or less than about 21 months, or less than about 22 months, or less than about 23 months.

[0170] Various delivery systems are known in the art, and any suitable delivery system can be used to apply 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 applied by any convenient route, such as by infusion or bolus injection, absorbed by epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be applied together with other bioactive agents. Administration can be systemic, or can be local. Pulmonary administration can also be used, for example, by using an inhaler or nebulizer, and using a preparation containing an atomizer.

[0171] In some embodiments, it may be necessary to apply the pharmaceutical composition of the present invention topically to the tissue in which the mutant RSV F molecule can most effectively produce the desired result. This can be by, for example, local infusion, injection, delivery using a catheter, or by an implant, such as a porous, non-porous or gelatinous implant, or an implant comprising one or more membranes (e.g., a silicone membrane) or fibers, from which or by which a protein or protein complex can be locally released. In some embodiments, a controlled release system can be used. In some embodiments, a pump can 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 can be used to promote and / or control the release of mutant RSV F molecules of the invention (see 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, the controlled release system can be placed near the tissue / organ to which the RSV prefusion F protein or polypeptide is to be delivered (see, e.g., Goodson, 1984. Medical Applications of Controlled Release, supra, vol. 2: 115 138). Langer, 1990, Science; vol. 249: pp. 527 1533 describes some suitable controlled release systems that can be used in conjunction with the present invention.

[0172] In some embodiments, the administration of the composition of the present invention can be performed together with the administration of one or more immunostimulants. Non-limiting examples of such immunostimulants include various cytokines, lymphokines and chemokines with immunostimulatory, immunoenhancing 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 factors, Flt3 ligands, B7.1, B7.2. Immunostimulants can be administered in the same formulation as the mutant RSV-F molecule, or they can be administered alone.

[0173] In some embodiments, the mutant RSV F molecules of the present invention or compositions comprising them can be applied to a subject with various RSV vaccination methods or schemes. In some such embodiments, a single dose is used. However, in other embodiments, additional dosages can be used to achieve the desired preventive effect by the same or different approaches. For example, in newborns and infants, multiple administrations may be required to cause sufficient levels of immunity. If necessary, RSV infection protection to a sufficient degree may be maintained, and administration can be continued at intervals throughout the childhood. Similarly, adults who are particularly susceptible to RSV infection, such as the elderly and immunocompromised, may need multiple immunizations to establish and / or maintain a protective immune response. The level of immunity induced can be monitored by, for example, measuring the amount of neutralizing secretion and serum antibodies, and, as required, adjusting the dosage or repeated vaccinations to cause and maintain the required level of protection.

[0174] In some embodiments, the dosage regimen may include a single administration / immunization. In other embodiments, the dosage regimen may include multiple administrations / immunization. For example, the vaccine may be used as the initial immunization, and then one or more reinforcements are performed. In some embodiments of the present invention, this "initial-reinforcement" vaccination regimen may be used. For example, in some such initial-reinforcement regimens, the composition comprising the mutant RSV F molecule described herein may be applied to an individual for many times (e.g., twice, three times or more) at intervals, wherein the first application is "initial" application, and the subsequent application is "reinforcement" application. In other such initial-reinforcement regimens, the composition comprising the virus or DNA vector encoding RSV polypeptide, protein or protein complex may be applied to an individual at first as "initial", and then one or more "reinforcement" applications may be performed using the composition comprising the RSV F polypeptide, protein or protein complex described herein. The composition comprising the mutant RSV F molecule described herein may be applied to an individual. A booster may be delivered by the same and / or different approach as the primary immunization. A booster is generally applied after a period of time after the primary immunization or the previous application of a booster. For example, can be about two weeks or more after the initial immunization to give booster, and / or can be about two weeks or more after the first time to give booster for the second time.Booster can be repeated in a certain time period to give, for example, about two weeks or longer, up to the whole life of the experimenter.Reinforcement can be spaced apart, for example, after the initial immunization or the previous booster about two weeks, about three weeks, about four weeks, about one month, about two months, about three months, about four months, about five months, about six months, about seven months, about eight months, about nine months, about ten months, about eleven months, about one year, about one and a half years, about two years, about two and a half years, about three years, about three and a half years, about four years, about four and a half years, about five years, or longer time.

[0175] In some embodiments, the pharmaceutical composition of the present invention can be conveniently provided in unit dosage form. Unit dosage form refers to a single dose or unit (e.g., effective amount) containing a mutant RSV F molecule of the present invention, or a dosage form of its appropriate ratio. Unit dosage form can be present in a single dose or multiple dose container.

[0176] In some embodiments, the compositions of the invention may be provided in sealed ampoules or vials and may be stored in a freeze-dried (lyophilized) condition, requiring only the addition of a sterile liquid carrier (eg, water) prior to use.

[0177] Reagent test kit

[0178] The present invention also provides a test kit comprising a mutant RSV F molecule of the present invention, or a composition comprising the mutant RSV F molecule. For ease of use of the methods and compositions of the present invention, any component and / or composition described herein, and other components for experiment or treatment or vaccination purposes, can be packaged in the form of a test kit. Typically, in addition to the above-mentioned components, the test kit also includes other materials, which may include, for example, instructions for use of the components, packaging materials, containers and / or delivery devices.

[0179] Table 1 - Exemplary amino acid sequences

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198] In Table 1, SEQ ID NOs 1 to 91 are exemplary WT and mutant RSV F amino acid sequences, and SEQ ID NOs 92 to 96 are exemplary artificial sequences that can be introduced at the C-terminus of RSV F molecules. For SEQ ID NOs 1 to 91, underlined residues are F1 and F2 polypeptides, residues in bold are introduced mutations, residues in italics are residues that can be removed or replaced in some embodiments, and residues with double underlines are artificially introduced C-terminal sequences.

[0199] Table 2 - Summary of exemplary RSV F amino acid sequences

[0200]

[0201] The embodiments of the present invention can also be further described by the following non-limiting examples:

[0202] Example 1

[0203] Selected Materials and Methods

[0204] As described below, mutant RSV F proteins were expressed in human cells and modified by the introduction of a dityrosine bond.

[0205] The cDNA encoding the mutant RSV-F molecule was cloned into the pCDNA3.1 / zeo+ expression vector (Invitrogen) via the 5'BamHI and 3'XhoI restriction endonuclease sites using standard methods (see Figure 1 ).

[0206] HEK 293 cells (ATCC) were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum and 50 μg / ml gentamicin. The cells were seeded in 6-well tissue culture plates (Corning) and cultured to 80% confluence (~24 h). The cells were transfected with 2 μg (micrograms) of RSV-F expression plasmid per well at a DNA to polyethyleneimine (25 kDa, linear) ratio of 1:4 (M / V). After 16 h of transfection, the culture medium was removed and replaced with 2 ml / well of serum-free Freestyle-293 expression medium (Invitrogen). The cells were cultured for another 48 to 72 h at 37 degrees Celsius in 5% CO2 and then collected and analyzed.

[0207] ELISA to detect RSV-F in cell supernatant. After collection, the whole RSV F protein was captured directly from the cell supernatant by keeping it in an EIA / RIA high binding 96-well plate (Corning) at room temperature for 1 hour. The wells containing protein and control were then blocked with PBS-Tween 20 (0.05%) containing 4% skim milk for 1 hour at room temperature. The plate was 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), which recognizes both the pre-fusion and post-fusion forms of RSV F. Using the recognition site Pre-fusion F was detected with a pre-F-specific human monoclonal antibody (2 μg / ml in PBS). The wells were washed again 3 times with PBS-T and then incubated with goat anti-human F(ab)2 conjugated to HRP (Jackson Immunoresearch) diluted 1:5000 in PBS for 1 h at room temperature. The wells were washed 6 times with PBS-T and 100 μl of 3,3',5,5'-tetramethylbenzidine (TMB) was used to generate a colorimetric signal to detect and quantify total RSV-F. An equal volume of 4N sulfuric acid was added to terminate the colorimetric reaction. The final optical density reading was measured at 450 nm using a BioRad Benchmark Plus microplate absorbance spectrophotometer. A 2X serial dilution series of each supernatant was used to determine the linear range of the detectable signal for each sample in order to accurately compare the relative amount of RSV-F between samples.

[0208] Dityrosine cross-linking in cell supernatants . Immediately after collection, 100 μl of transfected and control cell supernatants were transferred to the 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 to be cross-linked. 1 μl of 1.2 mM H2O2 was then added to the control reaction and the DT reaction to give a final reaction concentration of 120 μM H2O2. The reaction was allowed to proceed for 20 minutes at room temperature, after which an equal volume of pH 10 sodium phosphate buffer was added to alkalinize the reaction. Dityrosine-specific fluorescence was read using a fluorescence and chemiluminescence detector (ThermoScientific Fluoroskan Ascent FL) at an excitation wavelength of 320 nm and an emission wavelength of 405 nm.

[0209] 72 hours after transfection, supernatants were cross-linked (DT) or not cross-linked, and total protein was measured by ELISA 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. In some experiments, after 16 days of storage at 4°C, total protein was measured by ELISA using the recognition site PreF-specific human monoclonal antibody (2 μg / ml in PBS) was used to measure the site Display situation.

[0210] Example 2

[0211] Preparation and in vitro characterization of 428Y, 185Y, and 226Y mutant RSV-F molecules

[0212] Figure 2 A schematic diagram of an exemplary mutant RSV F molecule is provided, which comprises three mutations that are tyrosine--428Y mutations, 185Y mutations, and 226Y mutations. 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 the nucleic acid molecule encoding SEQ ID NO.81 is transfected and expressed in cells) comprises the 26th to 109th and 137th to 568th amino acid residues of SEQ ID NO.81--wherein the 26th to 109th amino acid residues are RSV F F2 residues, the 137th to 513th amino acid residues are RSV F F1 residues, and the 514th to 568th amino acid residues are heterologous sequences, and include a foldon domain, a His tag, a StrepII tag, and a thrombin cleavage site. The signal peptide from amino acids 1 to 25 and the pep27 peptide from amino acids 100 to 136 were not present in the final mature trimeric AVR02 molecule.

[0213] Figure 4 A schematic diagram of a method for preparing DT-crosslinked AVR02 (referred to as "DT-AVR02") is provided. Non-crosslinked AVR02 was transiently transfected into 293 Freestyle cells (Invitrogen) using standard methods for PEI and expressed in a secreted / soluble form for 5 days. On the 5th day, cells were harvested and precipitated into blocks by low-speed centrifugal spinning (1000×g×10min) in a swinging bucket rotor, transferred to a second tube, spun at a higher speed to clarify (3000×g×15min), transferred again and passed through a 70μm cell strainer, and then purified. AVR02 was purified using streptavidin lectin resin (IBA Lifesciences) according to the manufacturer's instructions. AVR02 was eluted using a PBS-based elution buffer containing 1mM EDTA and 2.5mM desthiobiotin, concentrated using a tangential flow filtration system (Pall Corporation), and clarified by a final centrifugation step (>20000×g×5min at 4 degrees).

[0214] The cross-linking reaction was performed using 20-40 μg / mL Arthromyces ramosus peroxidase and AVR02 at a concentration of 10-20 μg / mL in phosphate buffer containing 150 mM NaCl. The reaction was started by adding 300-600 μM H2O2 at 39 degrees Celsius, kept for 20 minutes, and quenched by adding ascorbic acid (80 ng / mL-50 μg / mL), at which time imidazole was added to 20 mM. The AVR02 protein was then purified by FPLC on an IMAC column (Ni-NTA) to eliminate peroxidase, followed by size exclusion chromatography (Superdex 200 pg) using PBS as eluent. The fractions containing AVR02 were then pooled, concentrated to 500 μg / mL, and cleaved overnight (20 hours) at 4 degrees Celsius using 2 units / mL biotinylated thrombin (Millipore). The thrombin was then removed according to the manufacturer's instructions and any uncleaved AVR02 protein was eliminated by incubation with streptavidin microbeads for 30 minutes at room temperature in batches. The protein was then passed through a spin filter to remove the microbeads and spun a final time at 4 degrees Celsius before final protein quantification. The final protein assay was a ninhydrin-based protein assay according to the method of Starcher, B (Analytical Biochemistry 292: 125-129 (2001)). The protein was snap frozen in liquid N2 and stored at -80 degrees Celsius before use.

[0215] Figure 3 provides data for the incorporation of two "designs" into AVR02 to form dityrosine bonds. Figure 3A - After the cross-linking reaction was performed, the binding site of D25 (a binding site) was detected by ELISA at 4°C within 2 weeks. Figure 3B - The intermolecular DT bonds formed between 185Y and 428Y (design 2) were determined by Western blotting and compared with WT (motavizumab was the primary antibody). The F1 protein shifted towards the size of the trimer (TM).

[0216] Figure 5A-B Provided, for example, based on site IV / V antibodies (FIG. 5A) and site Data on the conformational integrity of AVR02 and DT cross-linked AVR02 compared to a RSV mutant termed DS-Cav1, which is described in McLellan et al. (2013) Science 342:592-598 and contains S155C, S290C, S190F, and V207L ​​mutations, as determined by binding of antibodies ( FIG. 5B ).

[0217] Figures 6A-B provide data on the temperature stability of DT-AVR02. Figure 6C provides data on the temperature stability of AVR02 after storage at 4°C for 5 weeks. There were no data showing loss in specific mAb (5C4) binding.

[0218] Example 3

[0219] In vivo testing of 428Y, 185Y, and 226Y mutant RSV F molecules in mice

[0220] A study was conducted to test the immunogenicity of DT cross-linked AVR02 (DT-AVR02) compared to DS-Cav1. Details of the AVR02 and DT-AVR02 molecules are described above. DS-Cav1 mutants are described in McLellan et al. (2013) Science 342:592-598 and contain S155C, S290C, S190F, and V207L ​​mutations. Two groups of 10 6-8 week old female pathogen-free balb / c mice (Jackson Labs) were immunized by intramuscular injection in a prime (day 0)-boost (day 21) schedule: Group 1, each animal was injected with 10 μg (micrograms) of DT-AVR02 with alum adjuvant; Group 2, each animal was injected with 10 μg (micrograms) of DS-Cav1 with alum adjuvant. On day 35, two weeks after the second immunization (boost), sera were drawn.

[0221] Heat-inactivated serum was diluted to 1:800 in phenol-free MEM supplemented with glutamax, 5% FBS and penicillin / streptomycin and serially diluted in a 4-fold series (final volume of 50 μl). RSV-Renilla luciferase virus was then diluted to 2×10 4 pfu / mL, and 50 μL of virus-only and motavizumab-containing controls were added to each well (10,000 pfu / well). Viruses and sera were incubated for 2 h at 37°C, 5% CO2. Hep-2 cells (ATCC) were then trypsinized, counted, and diluted to 1 × 10 in the same medium. 6 cells / mL, and 25 μL (2.5×10 4 The cells were then incubated at 37°C, 5% CO2 for 60-72 hours, and luciferase activity was quantified using the Renilla Glo Luciferase Assay System (Promega) according to the manufacturer's instructions. Data were analyzed using nonlinear regression to calculate the IC 50 Concentration (Graphpad Prism).

[0222] Figure 7 Results from this study are presented showing RSV-RenillaLuc neutralization titers in animals vaccinated with alum-loaded DS-Cav1 or DT-AVR02.

[0223] Example 4

[0224] In vivo testing of 428Y, 185Y, and 226Y mutant RSV F molecules in cotton rats

[0225] As used in this example and the accompanying drawings, the term "DT-preF" refers to the molecule referred to by "DT-AVR02" in the preceding examples, and the term DT-preF can be used interchangeably with the term DT-AVR02. Detailed information on the structure of the DT-AVR02 molecule and its preparation method is provided above.

[0226] Summary / Overview

[0227] The aim of this study was to evaluate the protective efficacy and safety of DT-AVR02 against RSV in cotton rats. The effect of adding an adjuvant (2% aluminum hydrogel (ALH), 4 mg / CR) was also evaluated. The vaccine was administered by intramuscular injection using a prime-boost strategy. After RSV / A / Tracy challenge (day +35), lung lavage fluid and nasal washes from the two (2) larger lobes of the right lung were obtained on day +39, and RSV titers were determined by plaque testing. One (1) lobe of the right lung was flash frozen in liquid nitrogen and stored at -80°C. Serum samples were taken throughout the experiment for measurement of neutralizing antibody activity against RSV / A / Tracy and RSV / B / 18537.

[0228] Compared with saline controls, both 2 and 10 μg of DT-AVR02 protein / cotton rat were very effective in reducing RSV / A / Tracy in the lungs by approximately 4 log 10 PFU / g lung, reduced RSV / A / Tracy in the nose by about 1.5 log 10 Total PFU. After the second (boost) vaccination of these naive cotton rats, all vaccines generated RSV / A / Tracy-specific neutralizing antibody responses that were higher than those generated by infection with live RSV / A / Tracy on day 0.

[0229] Using two dosing schedules, the DT-AVR02 (DT-preF) adjuvanted vaccine administered at either 2 μg or 10 μg provided robust immune responses in RSV-naive cotton rats. Both 2 μg and 10 μg doses provided excellent protection against RSV / A / Tracy replication in the lungs, significantly but modestly reduced RSV / A / Tracy replication in the nose, and elicited RSV / A / Tracy-specific and RSV / B / 18537-specific serum neutralizing antibodies. The data suggest that these vaccines should be particularly effective in RSV-preimmune populations, such as the elderly adult population.

[0230] Details of experimental design, results, and conclusions

[0231] We designed and tested a series of RSV F mutants with tyrosine substitutions that were able to introduce targeted dityrosine (DT) crosslinks to conformationally lock the RSV F protein in its prefusion conformation. One of these molecules is referred to herein as DT-AVR02 (also referred to herein as DT-preF). The details of the structure and generation of this molecule are described above in other embodiments. Preliminary studies in mice (see the preceding examples) showed that DT-AVR02 was at least equivalent to DS-Cav1 in terms of immunogenicity, which is currently one of the two "best choices", partially stabilized preF designs (DS-Cav1 and SC-DM) - while also significantly improving thermal stability. Although DS-Cav1 and SC-DM have shown good promise as preF antigens, the proteins remain relatively unstable and lose antigenicity after only a few weeks at 4°C. Our in vitro stability tests showed that DT-AVR02 has higher thermal stability than DS-Cav1 and SC-DM, and its preF antigenicity is maintained for at least 100 hours at 37°C. This suggests that DT-AVR02 may be stable enough for clinical development and that its improved stability should also improve vaccine exposure and efficacy in vivo.

[0232] The purpose of the experiments described in this example was to evaluate the effects of two doses (2 or 10 μg / CR) of the DT-AVR02 / DT-preF RSV prefusion F protein vaccine in cotton rats. The effect of adding the adjuvant 2% aluminum gel (ALH) to the formulation was also evaluated. Vaccination was given by intramuscular injection using a prime-boost strategy. After RSV / A / Tracy challenge (day +35), lung lavage fluid and nasal washes from the two (2) larger lobes of the right lung were obtained on day +39, and RSV titers were determined by plaque assay. Serum samples were taken throughout the experiment for determination of neutralizing antibody activity against RSV / A / Tracy and RSV / B / 18537.

[0233] use Figure 8Vaccination (IM) was performed according to the indicated vaccination schedule with vaccination on day 0, boost on day 21 and challenge 14 days later.

[0234] All animals were challenged with live RSV / A / Tracy on day +35. Group 1, 5 cotton rats (CR) were injected IM with saline (as a positive virus infection control). Group 2, 5 CR were infected with live RSV / A / Tracy on day 0 ("gold standard"). Group 3, 5 CR were injected IM with 2μg DT-preF and 100μg of 2% ALH / CR adjuvant. Groups 4-5, CR were injected IM with 10μg DT-preF and 100μg of 2% ALH / CR adjuvant.

[0235] The cotton rats used had a body weight range of approximately 75-150 g (measured in weeks of age at the start). Body weight was also measured at the end of the experiment. At the start, the animals in all groups had similar weight, age, and sex distribution. The experiments were performed under the guidelines of the NIH and the U.S. Department of Agriculture, Public Health Service Policy on the Humane Care and Use of Laboratory Animals, and the experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC).

[0236] 1.21×10 5 PFU of RSV / A / Tracy (RSV / A / Tracy) (P3 wp3 / 13 / 2015) was administered intranasally (100 μL) to cotton rats lightly anesthetized with isoflurane. After inoculation, the virus inoculum was back-titrated on day 0 (Group 2) and day 35 (all groups) to determine the initial concentration (log 10 TCID 50 / mL).

[0237] Aluminum gel (aluminum hydroxide gel; ALH) was stored at room temperature [21°C]. The concentration of DT-AVR02 stock protein in PBS was 160 μg / mL (stored at -80°C). All steps were performed under a sterile hood. A 2% aluminum gel preparation was used at a dose of 100 μL per animal for a total of 7 doses (700 μL total).

[0238] Protein samples were thawed on wet ice. For the 10 μg dose, the protein was diluted in PBS to a concentration of 105.26 μg / mL (665 μL total, 437 μL DT PreF, 228 μL PBS). For the 2 μg dose, the protein was diluted in PBS to a concentration of 21.05 μg / mL (665, 87.4 μL DT PreF, 577.6 μL PBS). The protein samples were mixed by inversion. Aluminum gel (2%, Brenntag) was thoroughly mixed by inversion. Open the aluminum gel bottle and pipette 35 μL into each dose, mixing by hand to make a uniform suspension. Before inoculation, the prepared solution was mixed several times and repeated before each injection to ensure a uniform solution.

[0239] DT-AVR02 plus adjuvant (100 μL DT-AVR02 plus ALH (Groups 3, 4)) was injected (tuberculin syringe) into the area (IM) of the left tibialis anterior (TA) muscle. The other leg was used for the second vaccination (boost on day +21).

[0240] On day 0, blood was collected from the orbit of one cotton rat in each group. On all other designated days, blood was collected from all cotton rats (5CR / group; 4 groups).

[0241] After euthanasia with CO2, the right lung was ligated. Two lobes were removed from the right lung, rinsed with sterile water to remove external blood stains and weighed. The same two right lobes were lavaged transpleurally using 3 mL Iscove's medium (3 mL syringe with 26G3 / 8 needle) containing 15% glycerol mixed with 2% FBS-MEM (1:1, v:v) and injected at multiple sites to fully expand the lobes. The lavage fluid was recovered by gently pressing on one expanded lobe, and the other lobe was lavaged transpleurally in the same way. The washing fluid was collected and kept on ice until titration. For nasal washes of the upper respiratory tract, the mandible was dislocated. The head was removed and 1 mL Iscove's medium containing 15% glycerol mixed with 2% FBS-MEM (1:1, v:v) was pushed through each nostril (2 mL in total). The effluent was collected from the rear opening of the tray and kept on ice until titration. The samples were not frozen before titration and titration was performed at the end of sample collection. A serum sample was saved for antibody analysis.

[0242] RSV Tracy lung lavage titers (PFU / g lung) and nasal wash titers (total PFU) were determined as follows. Plaque assays were performed 24 h prior to the start of the assay using 24-well tissue culture plates containing subconfluent monolayers of HEp-2 cells (approximately 2 × 10 5 At the beginning of each experiment, the test sample is diluted (usually serially log 10). 0.2 mL of each sample is then added to the wells in duplicate and allowed to adsorb for 90 minutes with occasional gentle shaking. After removing the inoculum, the monolayer is overlaid with 0.75% methylcellulose containing antibiotics, vitamins, and other nutrients. Each experiment includes tissue culture and positive virus controls. The plates are placed in a 36°C, 5% CO2 incubator. On day 6 (+ / - 1 day), the plates are stained with a 0.01% crystal violet / 10% formalin solution (~1.5 mL / well) and placed at room temperature for 24-48 hours. The wells are washed with water. Plaques are easily seen when they appear (clear circles on a dark blue background). All plaques in wells containing approximately 20 to 100 plaques are counted, averaged, and the viral titer is calculated as: for nasal washes, the total log 10 PFU, or log for lung or other organs 10 PFU / g tissue. The detection limit of this method is 0.70log 10 Total PFU or about 1.4log 10 PFU / g lung tissue.

[0243] On days 0, 21, 35 and 39, the anti-RSV neutralizing antibody test was performed as follows. Serum neutralizing antibodies to RSV / A / Tracy and RSV / B / 18537 were detected on 96-well microtiter plates containing HEp-2 cells. Plaque-purified RSV virus was used for microneutralization (Nt) testing. Samples were heat-inactivated at 56°C for 30 min. Starting from 3log2, two-fold serial dilutions were performed in duplicate to determine the neutralizing antibody (Ab) titer of each sample. The neutralizing antibody titer is defined as the serum dilution at which a reduction of ≥50% in viral cytopathic effect (CPE) is observed. CPE is defined as tissue destruction, which can be visually observed after fixing the wells with 10% neutral buffered formalin and staining with crystal violet. Neutralizing antibody (NtAb) titers are provided in absolute logarithms rather than continuous values. The minimum detectable NtAb titer is 2.5log2. Samples with undetectable Nt Ab titers are assigned a value of 2log2. NtAb titers are reported in multiples of 0.5 log2 (e.g., 2.5, 3.0, 3.5, 4.0, etc.). If the NtAb titer of a sample is equal to or exceeds the upper limit of the assay (≥14 log2), the sample is retested to determine the NtAb titer by extending the dilution to 26 log2. As an internal standard, the content of Palivizumab is 40 μg / mL.

[0244] RSV-specific antibody titers were determined by ELISA. Serum samples were stored at -20°C before ELISA testing. First, the viral load or RSV-specific neutralizing antibody level between the control group and the vaccine group was compared by log 10Virus titers or log2-transformed antibody titers were analyzed using two-tailed Student's T test in Excel (Microsoft Office 2013). Additional comparisons were analyzed by ANOVA using InStat3 (GraphPad) with post-hoc Tukey comparisons.

[0245] Further details of the experimental design are given in Table A. The results are summarized below. Further details of the results are given below, including in Table BL.

[0246] Table A

[0247]

[0248] Abbreviations: ALH, 2% Brenntag aluminogel; PFU, plaque forming unit; Nt, neutralization. 1 N = 5 animals / group; 30 animals in total. 2 IM, route of administration. 3 All animals were challenged in (100 μL) with the RSV / A / Tracy pair.

[0249] Table B: Lung and body weight of each group on day +39

[0250]

[0251] 1 There was no statistically significant difference between the two groups (Student's T test, two-tailed). 2 There was no statistically significant difference between the two groups (Student's T test, two-tailed).

[0252] Table C: RSV / A / Tracy titers in nasal washes on day +39

[0253]

[0254] *Minimum detection limit = 0.70log 10 Total PFU. d, death. For statistical analysis (Student's T test, two-tailed), 0 plaques in the undiluted sample were calculated as 0.40 log 10 Total PFU. There are additional significant P values: Group 2 v Groups 3 and 4; P = 0.00004, 0.00024, respectively (Student's T test, two-tailed).

[0255] Table D: RSV / A / Tracy titers in lung lavage fluid on day +39

[0256]

[0257] *Minimum detection limit ~1.4log 10PFU / g lung. d, death. For statistical analysis (Student T test, two-tailed), 0 plaque in undiluted samples was between 0.90 and 1.04 log 10 PFU / g lung. No additional significant P values ​​(Student's T test, two-tailed).

[0258] Table E: RSV / A / Tracy serum neutralization titers on day 0

[0259]

[0260] *Student's t-test, two-tailed. Lower detection limit = 2.5; values ​​< 2.5 were counted as 2 for statistical analysis.

[0261] Table F: RSV / A / Tracy serum neutralization titers on day +21

[0262]

[0263] *Student's T test, two-tailed. Minimum detection limit = 2.5; for statistical analysis, values ​​< 2.5 were counted as 2. Additional significant P value (Student's T test, two-tailed): Group 2 v Groups 3 and 4, P ≤ 0.0036. d, death.

[0264] Table G: RSV / A / Tracy serum neutralization titers on day +35

[0265]

[0266] *Student's T test, two-tailed. Minimum detection limit = 2.5; for statistical analysis, values ​​< 2.5 were counted as 2. Additional significant P values ​​(Student's T test, two-tailed): Group 2 v Groups 3 and 4, P < 0.044; d, death.

[0267] Table H: RSV / A / Tracy serum neutralization titers on day +39

[0268]

[0269] *Student's T test, two-tailed. Minimum detection limit = 2.5; for statistical analysis, values ​​< 2.5 were counted as 2. Additional significant P values ​​(Student's T test, two-tailed): Group 2 v Groups 3 and 4, P ≤ 0.045; d, death.

[0270] Table I: RSV / B / 18537 serum neutralization titers on day 0

[0271]

[0272] *Student's t-test, two-tailed. Lower detection limit = 2.5; values ​​< 2.5 were counted as 2 for statistical analysis.

[0273] Table J: RSV / B / 18537 serum neutralization titers on day +21

[0274]

[0275] *Student's T test, two-tailed. Lower detection limit = 2.5; for statistical analysis, values ​​< 2.5 were counted as 2. No additional significant P value (Student's T test, two-tailed).

[0276] Table K: RSV / B / 18537 serum neutralization titers on day +35

[0277]

[0278] *Student's T test, two-tailed. Minimum detection limit = 2.5; for statistical analysis, values ​​< 2.5 were counted as 2. Additional significant P value (Student's T test, two-tailed): Group 2 v Groups 3 and 4, P ≤ 0.015.

[0279] Table L: RSV / B / 18537 serum neutralization titers on day +39

[0280]

[0281] *Student's T test, two-tailed. Minimum detection limit = 2.5; for statistical analysis, values ​​< 2.5 were counted as 2. Additional significant P value (Student's T test, two-tailed): Group 2 v Groups 3 and 4, P < 0.013.

[0282] Cotton rat 2B was found dead in its cage on day 9. The cause of death was unknown.

[0283] Effect of DT-preF / DT-AVR02 with 2% alum adjuvant on RSV / A / Tracy titers in nasal washes: Infection with live RSV / A / Tracy (Group 2) on day 0 reduced the titer by 4.42 log compared to saline control (Group 1) 10 Total PFU (P < 0.00001; Student's T test, two-tailed) ( Fig. 9 , Table B). All vaccine combinations reduced viral titers by 1.37 to 1.77 log 10 Total PFU (P≤0.00024; Student's T test, two-tailed). There was no statistical difference between the two vaccine combinations.

[0284] Effect of DT-preF / DT-AVR02 with 2% alum adjuvant on RSV / A / Tracy titers in lung lavage fluid: Compared with saline control (group 1), infection with live RSV / A / Tracy on day 0 (group 2) reduced the titer to below the detection limit (4.03 log 10PFU / g lung; P < 0.00001; Student's T test, two-tailed) Fig.10 The RSV titers of the vaccine combination in groups 3 and 4 were reduced to below the detection limit (4.04-4.13 log 10 PFU / g lung; P < 0.00001; Student's T test, two-tailed). There was no statistical difference between the two vaccine combinations.

[0285] Effect of DT-preF / DT-AVR02 with 2% alum adjuvant on the production 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) produced NtAb (about 5.5 log2 / 0.05 mL) on day 21 and remained at this level on day 39 ( Fig.11 ; Tables D-G). At day 21, there was no statistical difference from any DT-AVR02 vaccine (P≤0.0036; Student's T test, two-tailed). However, after the second vaccination on day 21, all vaccines had a robust NtAb response that was higher than that of live RSV / A / Tracy infection.

[0286] RSV / B / 1853 cross-reactive NtAbs were generated by live RSV / A / Tracy infection and two vaccines ( Fig.12 ; Tables H-L). The NtAb response patterns of the two vaccines were similar to those of RSV / A / Tracy, but at a lower 1-2 log2 level.

[0287] Example 5

[0288] Comparison of 428Y and 427Y mutant RSV-F molecules in vitro and in vivo

[0289] Using the methods described in the previous examples, two DT cross-linked mutant RSV F molecules were generated that were stable in the pre-F conformation. The difference between the two mutants was only that one had a point mutation at amino acid 428 that mutated to tyrosine (428Y mutation), and the other had a point mutation at amino acid 427 that mutated to tyrosine (427Y mutation). Both molecules included S190F and V207L ​​mutations (i.e., "Cav1" mutations), mutations at amino acid residues 185 and 226 that mutated to tyrosine (i.e., 185Y mutation and 226Y mutation).

[0290] The 428Y mutant (including V185Y, K428Y, K226Y, S190F and V207L ​​mutations) are interchangeably referred to herein as "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 the nucleic acid molecule encoding SEQ ID NO.81 is transfected and expressed in cells) comprises amino acid residues 26 to 109 and 137 to 568 of SEQ ID NO.81, wherein amino acid residues 26 to 109 are RSV F F2 residues, amino acid residues 137 to 513 are RSV F F1 residues, and amino acid residues 514 to 568 are heterologous sequences, and comprise a foldon domain, a His tag, a StrepII tag, and a thrombin cleavage site. The final mature trimer 428Y mutant molecule does not contain the signal peptide from amino acids 1 to 25 and the pep27 peptide from amino acids 100 to 136.

[0291] The 427Y mutant (including V185Y, K427Y, K226Y, S190F and V207L ​​mutations) are interchangeably referred to herein as "427Y mutations" or "DT-CAV1". The F0 precursor form of the 428Y mutant has the amino acid sequence of SEQ ID NO.81, but 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 of a nucleic acid molecule encoding the modified version of SEQ ID NO.81 in cells) contains amino acid residues 26 to 109 and 137 to 568 of the modified sequence - wherein amino acid residues 26 to 109 are RSV F F2 residues, amino acid residues 137 to 513 are RSV F F1 residues, and amino acid residues 514 to 568 are heterologous sequences, and include a foldon domain, a His tag, a StrepII tag, and a thrombin cleavage site. The final mature trimer 427Y mutant molecule does not contain the signal peptide from amino acids 1 to 25 and the pep27 peptide from amino acids 100 to 136.

[0292] The 427Y and 428Y mutants were expressed and purified as described above and analyzed for conformational integrity by ELISA using motavizumab, normalized to total protein, and stained with conformational 5C4 (site The assay was developed using Ni-NTA coated plates (Qiagen) and one-step TMB reagent (Pierce). As shown in Figure 13, the substitution of N428Y for K427Y significantly increased binding to the site IV-V antibody (Figure 13B and D), and also increased binding to the site IV-V antibody. Binding of antibodies (Figure 13A&C).

[0293] N428Y replaces K427Y and also affects the aggregation state of molecules after cross-linking reaction. Proteins were purified according to the method described elsewhere herein, and two proteins were cross-linked under the same conditions and as described elsewhere herein. In the Superdex 200 26 / 60pg column size exclusion chromatography with PBS as eluent, the chromatogram of cross-linked material was significantly different. As shown in Figure 14A, after cross-linking reaction, 427Y mutant (DT-CAV1) mainly produced high-order aggregate species, as shown in Figure 14 and the corresponding Coomassie staining SDS-PAGE analysis (Figure 14C, two gel bars on the left) of the final product. When 428Y mutant (DT-AVR02) was produced by the same process, most of the proteins were in the trimer fraction, as demonstrated by the corresponding Coomassie staining SDS-PAGE gel (Figure 14C, right gel bars) of the chromatogram (Figure 14B) and the final product.

[0294] Studies were also performed to test the immunogenicity of the 427Y mutant (DT-CAV1), the 427Y mutant (DT-AVR02), and the DS-Cav1 mutant described in McLellan et al. (2013) Science 342:592-598 (which includes S155C, S290C, S190F, and V207L ​​mutations). Two groups of 10 6-8 week old, pathogen-free female CB6 / F1J mice (Jackson Labs) were immunized on a prime (day 0)-boost (day 21) schedule: in both studies, group 1 was vaccinated with 10 μg (micrograms) of DS-Cav1 in alum adjuvant, while group 2 was vaccinated with 10 μg (micrograms) per animal per unit dose of the 427Y mutant (DT-CAV1) in alum adjuvant in the first study and 10 μg (micrograms) per animal per unit dose of the 428Y mutant (DT-AVR02) in alum adjuvant in the second study. Serum was collected on day 35, two weeks after the second vaccination (boost). Results are shown in Table 1. Figure 15A-B As shown, the serum neutralization titer of animals vaccinated with the 428Y mutant (DT-AVR02) ( FIG. 15B ) was significantly higher than that of animals vaccinated with the 427Y mutant (DT-Cav1) ( FIG. 15A ).

[0295] Example 6

[0296] Sucrose-containing formulations reduce RSV-F aggregate formation

[0297] It has been found that the addition of sucrose to the formulation of the mutant RSV F molecules of the present invention significantly reduces the formation of undesirable aggregates. Experiments were performed using the exemplary DT cross-linked 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 of Figure 16A) and aggregates (peak A and peak B of Figure 16A). In the elution process of the purification step, the formulation of the DT-AVR02 molecule with 10% sucrose appears to eliminate the formation of aggregates. As shown in Figure 16B, in the sample formulated with sucrose, peak C (trimer) still exists, but peak A and peak B (aggregates) are not obvious.

[0298] Example 7

[0299] In vivo testing of AVR02 in non-human primates

[0300] Six groups of five non-human primates were immunized by intramuscular injection according to a prime (day 0)-boost (day 28) schedule. Groups 1 and 2 were vaccinated with high doses (50 mg / animal / injection) of DT-AVR02 with (group 1) and without (group 2) adjuvant, respectively; groups 3 and 4 were vaccinated with low doses (10 mg / animal / injection) of DT-AVR02 with (group 3) and without (group 4) adjuvant, respectively; groups 5 and 6 were vaccinated with high doses (50 mg / animal / injection) of our comparator DS-Cav1 with (group 1) and without (group 2) adjuvant, respectively.

[0301] Animals in all groups were injected twice, on day 0 and day 28, respectively. Blood was drawn from all animals before the initial immunization on day 0 and before the secondary immunization on day 28. Blood was drawn / collected again on days 42 and 49. Peripheral blood mononuclear cells were isolated by standard methods to characterize the frequency and phenotype (Th1 / Th2) of RSV-specific T cell responses; serum was analyzed to quantify NHP anti-F responses (IgM, total IgG, IgG1, IgG2, IgG3) by standard methods (site Epitope analysis was performed by competition assay with site II), and neutralization titers were quantified by standard methods.

[0302] Example 8

[0303] Phase I human clinical trial of AVR02

[0304] A randomized, placebo-controlled, observer-blind, dose-ranging study was conducted to evaluate the safety, tolerability, and immunogenicity of the stabilized prefusion RSV F subunit protein vaccine DT-AVR02, alone or with alum adjuvant, in healthy (nonpregnant) adults (18 to 50 years).

[0305] 150 subjects were distributed in 7 cohorts, testing 6 formulations and one placebo. Subjects were randomized 4:1 to vaccine treatment or saline placebo, so each cohort included 20 subjects receiving active vaccine (Groups A, B, C, D, E, and F) and 5 subjects receiving placebo (Group G). 6 groups of 20 (active vaccine) and 5 (placebo) subjects were tested. 6 x 25 = 150 subjects. Low, medium, and high doses (50mcg, 150mcg, and 500mcg per injection), each dose was formulated with and without alum as an adjuvant.

[0306] Subjects received two IM injections: Day 0 and Day 90 (+ / - 5 days). Subjects were followed for all adverse events (AEs), including serious AEs (SAEs) and non-serious AEs, from each vaccination (Day 0 and Day 90) until 30 days after vaccination. In addition, subjects were contacted by phone on Days 60, 150, 180, 210, and 310 to inquire about the occurrence of SAEs and major new medical conditions.

[0307] The primary objective was to evaluate the safety, reactogenicity, and tolerability of three doses of DT-AVR02 alone or with alum when injected IM in healthy adults, monitored over 44 weeks. The secondary objective was to evaluate the antibody response of subjects to three doses of unadjuvanted and adjuvanted DT-AVR02, measured by: total Ab response (including Ab subtyping analysis) by ELISA, epitope-specific response by competition ELISA 2 and 4 weeks after each injection, and neutralization assay 2 and 4 weeks after each injection.

[0308] ***

[0309] Although the above invention has been described in some detail for the purpose of clarity and understanding, it will be apparent to those skilled in the art from reading this disclosure that various changes in form and detail may be made without departing from the true scope of the invention. The invention may also be further defined according to the following claims.

Claims

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

1.

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

1.

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

1.

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

7. A mutant RSV F molecule according to any one of the preceding claims, wherein the molecule is a RSV A type or RSV B type molecule.

8. The mutant RSV F molecule of any one of the preceding claims, wherein the molecule is bound to a prefusion-specific antibody.

9. The mutant RSV F molecule of any one of the preceding claims, wherein the molecule binds to an antibody that recognizes antigenic site φ.

10. The mutant RSV F molecule of any of the preceding claims, 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 the preceding claims, wherein the molecule comprises a F2 polypeptide and a F1 polypeptide, wherein 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 the preceding claims, wherein the molecule comprises a F2 polypeptide and a F1 polypeptide, wherein the C-terminus of the F2 polypeptide is connected to the N-terminus of the F1 polypeptide via an artificially introduced peptide linker.

13. The mutant RSV F molecule of any one of the preceding claims, comprising: an F2 polypeptide of about 84 amino acid residues; and, an F1 polypeptide of about 375 amino acid residues.

14. The mutant RSV F molecule of any one of the preceding claims, comprising: an F2 polypeptide of about 74-84 amino acid residues; and, an F1 polypeptide of about 365-375 amino acid residues.

15. The mutant RSV F molecule of any one of the preceding claims, 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. The mutant RSV F molecule of any one of the preceding claims, comprising: (a) an F2 polypeptide comprising or consisting of about 74 to 84 amino acids from 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 about 365 to 375 amino acids of amino acid residues 137 to 513 of any one of SEQ ID NOs 21 to 81.

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

81.

19. The mutant RSV F molecule of any of the preceding claims, wherein the molecule is stabilized in a prefusion conformation by one or more dityrosine cross-links.

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

21. The mutant RSV F molecule of any one of the preceding claims, wherein the molecule is cross-linked by three or more dityrosines to stabilize the mature RSV F trimer in a prefusion conformation.

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

1.

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

1.

24. The mutant RSV F molecule of any of the preceding claims, wherein the molecule comprises two dityrosine cross-links: (i) a dityrosine cross-link between the tyrosine at amino acid residue 198 and the tyrosine at amino acid residue 226, or between the tyrosines at the amino acid residues corresponding thereto as determined by alignment with SEQ ID NO.1; and, (ii) a dityrosine cross-link between the tyrosine at amino acid residue 428 and the tyrosine at amino acid residue 185, or between the tyrosines at the amino acid residues corresponding thereto as determined by alignment with SEQ ID NO.

1.

25. The mutant RSV F molecule of any one of the preceding claims, wherein the molecule is a mature RSV F trimer comprising three dityrosine cross-links, wherein each dityrosine cross-link is located between the following tyrosines: (a) tyrosine at amino acid residue 428, or at the amino acid residue corresponding thereto as determined by alignment with SEQ ID NO. 1; and, (b) tyrosine at amino acid residue 185, or at the corresponding amino acid residue determined by alignment with SEQ ID NO.

1.

26. The mutant RSV F molecule of any one of the preceding claims, wherein the molecule is a mature RSV F trimer comprising three dityrosine cross-links, wherein each dityrosine cross-link is located between the following tyrosines: (a) tyrosine at amino acid residue 198, or at the amino acid residue corresponding thereto as determined by alignment with SEQ ID NO. 1; and, (b) tyrosine at amino acid residue 226, or at the corresponding amino acid residue determined by alignment with SEQ ID NO.

1.

27. The mutant RSV F molecule of any one of the preceding claims, wherein the molecule is a mature RSV F trimer cross-linked by six dityrosines, wherein: (i) between the tyrosine at amino acid residue 198 and the tyrosine at amino acid residue 226, or between the tyrosines at the amino acid residues corresponding thereto as determined by alignment with SEQ ID NO. 1; and, (ii) three between the tyrosine at amino acid residue 428 and the tyrosine at amino acid residue 185, or between the tyrosines at their respective corresponding amino acid residues determined by alignment with SEQ ID NO.

1.

28. The mutant RSV F molecule of any one of the preceding claims, wherein the molecule further comprises one or more artificially introduced non-DT cross-links.

29. The mutant RSV F molecule of any one of the preceding claims, wherein the molecule further comprises one or more artificially introduced disulfide bonds.

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

31. 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 point mutation.

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

1.

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

1.

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

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

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

1.

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

1.

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

39. 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 the preceding claims, wherein the molecule comprises a heterologous oligomerization domain.

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

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

43. 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 the preceding claims, wherein the molecule comprises one or more tags for detecting the RSV F molecule.

45. The mutant RSV F molecule of any one of the preceding claims, wherein the molecule comprises one or more tags for purifying the RSV F molecule.

46. ​​according to the mutant RSV F molecule described in claim 44 or 45, wherein said tag is selected from the group consisting of Strep tag, Strep II tag, FLAG tag, glutathione S-transferase (GST) tag, green fluorescent protein (GFP) tag, hemagglutinin A (HA) tag, histidine (His) tag, luciferase tag, maltose binding protein (MBP) tag, c-Myc tag, protein A tag and 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 the following amino acid residues: (a) at amino acid residue 428, (b) at amino acid residue 226, and (c) at amino acid residue 185, or the amino acids corresponding thereto as determined by alignment with SEQ ID NO.

1.

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

81.

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

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

81.

54. A soluble mature trimeric RSV F molecule comprising a point mutation to tyrosine at each of the following amino acid residues: (a) at amino acid residue 428, (b) at amino acid residue 226, and (c) at amino acid residue 185, or at the amino acid corresponding thereto as determined by alignment with SEQ ID NO.

1.

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

81.

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

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

81.

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

59. A nucleic acid encoding the mutant RSV F molecule of any of the preceding claims.

60. A vector comprising the nucleic acid according to claim 59.

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

62. A cell comprising the vector of claim 60.

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

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

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

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