Improved RSV vaccine compositions & methods

Abstract

The present invention provides mutant RSV F molecules that can be, or are, stabilized in the pre-fusion conformation (preF) by the introduction of one or more dityrosine (DT) crosslinks. The present invention also provides methods of making such mutant RSV F molecules, vaccine compositions comprising such mutant RSV F molecules, and methods of use of such mutant RSV F molecules, for example in vaccination methods to protect subjects against RSV infection.

Description

[0001] IMPROVED RSV VACCINE COMPOSITIONS & METHODS

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 564,128 filed on March 12, 2024, the content of which is hereby incorporated by reference in its entirety.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under grant number All 12124, AG064107, and AI140941 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] INCORPORATION BY REFERENCE

[0007] For the purposes of only those jurisdictions that permit incorporation by reference, the content of all documents cited herein is hereby incorporated by reference in its entirety.

[0008] BACKGROUND OF THE INVENTION

[0009] Respiratory' Syncytial Virus (RSV) infects humans repeatedly throughout life. Newborn children, the elderly, and immune compromised patients are particularly vulnerable to more severe disease20-22. In newborn children, RSV infection often results in protracted and enhanced respiratory disease, which can result in prolonged respiratory difficulty' throughout childhood and adolescence. In the past, development of an RSV vaccine w as hindered by an early clinical trial in which children vaccinated with a formalin-inactivated vaccine (FI-RSV) experienced an exaggerated immune response to subsequent RSV infection - i.e., vaccine enhanced disease (VED)23. VED was characterized by elevated eosinophilic responses to infection, airway hyper-reactivity, and excessive mucus production23-24. These potentially fatal responses were caused by poorly neutralizing Ab responses with immune complex deposition in the lungs and Th2-type cytokine responses to vaccination in RSV naive children24-25. Eliciting high titers of prefusion-specific, neutralizing antibodies is still considered the best vaccine strategy to protect the elderly and infants via matemal-to-infant vaccination, whereby the importance of T cells to the protection of the elderly should not be discounted26-30. The prefusion conformation of the RSV F (fusion) protein is among the most labile of the fusion proteins. However, an RSV prefusion F (preF) subunit vaccine has been demonstrated to be one of the most promising approaches, since it elicits highly potent neutralizing antibody responses, and because its sequence is highly conserved between strains. In fact, the first two RSV vaccines to obtain licensure in the U.S. are protein subunit vaccines31’33. Significant progress has been made stabilizing RSV preF, starting with the design and characterization of the Vaccine Research Center’s (VRC / NIH) DS-Cavl molecule19’34. DS-Cavl elicits substantially higher neutralizing antibody titers than the RSV F protein in its postfusion conformation19’28’35. Several 2nd-generation RSV subunit vaccines are being developed that further improve upon DS-Cavl 's stability36’42. Current RSV prefusion stabilized first generation vaccines have built upon the success of DS-Cavl and have now achieved full licensure in the U.S. for the elderly and in maternal vaccination31’33 43. Nevertheless, ample room for improvement exists. For example, Pfizer’s maternal vaccine does not substantially outperform the protection afforded by monoclonal antibody administration in infants, and GSK’s leading vaccine for the elderly does not adequately protect in the 80+ elderly or the frail where they achieve only 34 and 14% efficacy, respectively, and lack statistical significance. These immunosenescent populations are precisely the most likely to be hospitalized and have poor clinical outcomes and vaccines therefore need to elicit potent responses in these groups. Accordingly, there are still several important and unmet medical needs in the field of vaccination against RSV. The present invention addresses these needs.

[0010] BRIEF SUMMARY OF THE INVENTION

[0011] The present invention is based, in part, on certain new and surprising discoveries that are described in the Detailed Description and Examples section of this patent disclosure. These discoveries provide various developments of, and improvements to, work described previously by Marshall et al. in patent applications PCT / US2014 / 048086 and PCT / US 18 / 045463, the contents of each of which are incorporated by reference herein.

[0012] PCT / US2014 / 048086 and PCT / US 18 / 045463 (and the various granted national patents related thereto, including, but not limited to, U.S. PatentNos. 10,125,172. 11,267,848 and 11,926,649) describe engineered RSV F molecules in which mutations to tyrosine are introduced at certain specifically designed locations, allowing the formation of dityrosine crosslinks at these specific locations, which stabilizes the RSV fusion (F) protein in its pre-fusion (preF) conformation. As described further in Examples 1 and 2 herein, it has now been discovered that the efficacy of such engineered RSV F molecules as vaccine immunogens for protection against RSV can be significantly improved when some, but not all, of the introduced tyrosine residues are crosslinked. This can be achieved when the dityrosine crosslinking reaction is performed under conditions that lead to incomplete crosslinking, allowing the production of improved vaccine compositions containing a mixed population of RSV F molecules having varying levels of dT crosslinking (i.e., compositions containing a distribution of species of RSV F molecules having varying levels of DT crosslinking).

[0013] Furthermore, and as described in Example 1 herein, it has now also been discovered that, using engineered RSV F molecules as described in PCT / US2014 / 048086 and PCT / US 18 / 45463, and / or the improved mixed vaccine compositions as described in Example 1 and 2 and elsewhere herein, it is possible to achieve unprecedented levels of protection against RSV infection across age groups (providing 11 times greater potency than DS-Cavl stabilized prefusion RSV F protein) and, importantly, to overcome immunosenescence leading to high levels of protection against RSV infection in elderly individuals.

[0014] Building on these surprising discoveries, the present invention provides various new and useful compositions and methods, some aspects of which are summarized below, and other aspects of which are described in further detail in other sections of this patent disclosure.

[0015] Accordingly, in some embodiments the present invention provides vaccine compositions that comprise soluble, trimeric, RSV F molecules di-tyrosine crosslinked in the pre-fusion (pre-F) conformation, wherein about 25-35% of the RSV F molecules in the composition have no intermolecular di-tyrosine crosslinks, and about 65-75% of the RSV F molecules in the composition have at least 2 intermolecular di-tyrosine crosslinks. Similarly, in some embodiments, the present invention provides vaccine compositions that comprise soluble, trimeric, RSV F molecules di-tyrosine crosslinked in the pre-fusion (pre-F) conformation, wherein the average number of di-tyrosine crosslinks between paired tyrosine residues is 4-5 per trimer.

[0016] In some such embodiments the trimeric RSV F molecules comprises a “to-tyrosine” mutation at two or more of amino acid residues: 77, 88, 97, 147, 150, 155, 159, 183, 185, 187, 220, 222, 223, 226, 255, 427, 428, and 469, or at or at amino acid residues that correspond to these amino acid residues as determined by alignment to, and using the amino acid numbering of, SEQ ID NO. 1. In preferred embodiments the trimeric RSV F molecules comprise a tyrosine residue at each of amino acid positions 185, 198, 226 and 428.

[0017] Similarly, in some such embodiments the trimeric RSV F molecules comprise di-tyrosine crosslinks between at least two of the following paired tyrosine residues: 147 and 286; 198 and 220; 198 and 222; 198 and 223; 198 and 226; 33 and 469; 77 and 222; 88 and 255; 97 and 159; 183 and 427; 185 and 427; 185 and 428; and 187 and 427, or at or at amino acid residues that correspond to these amino acid residues as determined by alignment to, and using the amino acid numbering of, SEQ ID NO. l. In preferred embodiments, the trimeric RSV F molecules comprise an intermolecular di-tyrosine crosslink connecting a tyrosine at amino acid residue 185 of one monomer within the trimer with a tyrosine at amino acid residue 428 of a different monomer within the trimer, and an intramolecular di-tyrosine crosslink connecting a tyrosine at amino acid residue 226 with a tyrosine at amino acid residue 198. In some such embodiments about 25-35% of the RSV molecules in the composition have no intermolecular di-tyrosine crosslinks connecting a tyrosine at amino acid residue 185 of one monomer within the trimer with a tyrosine at amino acid residue 428 of a different monomer withing the trimer, and about 65-75% of the RSV molecules in the composition have at least 2 intermolecular di-tyrosine crosslinks, wherein the at least 2 intermolecular di-tyrosine crosslinks connect a tyrosine at amino acid residue 185 of one monomer within the trimer with a tyrosine at amino acid residue 428 of a different monomer withing the trimer.

[0018] In other embodiments, the present invention provides methods of vaccinating subjects against RSV infection. In some of such embodiments, such methods comprise administering to the subjects an effective amount of a vaccine composition as described above or elsewhere herein. In some embodiments the subjects are human subjects. In some embodiments the subjects are elderly human subjects, for example of at least 75 years in age, or at least 80 years in age.

[0019] These and other aspects of the present invention are described further in the Detailed Description, Examples, Claims, Figures, and Brief Description of the Figures sections of this patent application - all of which sections are intended to be read in conjunction with one another. Furthermore, one of skill in the art will recognize that the various embodiments of the present invention described above and elsewhere throughout this patent disclosure can be combined in various different ways, and that such combinations are within the scope of the present invention.

[0020] BRIEF DESCRIPTION OF THE FIGURES

[0021] Fig. 1A-D: Screening of dityrosine pairs within the RSV F protein for expression, conformation, and dityrosine bond formation. Panels: (Fig. 1A) RSV F DT-mutants were transfected into 293T cells in a 96-well plate format, harvested on day 3, and supernatants were used in ELISA based total protein (Motavizumab), and conformational (AM14, D25, MPE8) analysis. Crosslinking analysis by fluorescence intensity measurement after crosslinking of proteins in supernatants was also included in the screen. (Fig. IB) Graphs of ELISA data showing a representative subset of the initial screening of expression level and conformation using Motavizumab (Site II), D25 (Site 0), AM14 (Site IV / V) and MPE8 (Site III) respectively. This experiment was performed twice. (Fig. 1C) Graphs of ELISA data of second-tier screening for expression using Motavizumab and pre-F antigenicity using AM 14 done in conjunction with panel D. (Fig. ID) Fluorescence intensity (ex320nm / em405nm) measurements were taken before and after crosslinking of potential hits using supernatants from transfected 293T cells, where protein signal was normalized to the DS-Cavl-V185N428 variant (demonstrating low expression). This graph indicates the mutant-specific signal post background subtraction of mock-transfected samples.

[0022] Fig. 2A-D: Design and characterization of dityrosine crosslinked DT-preF molecule. Panels: (Fig. 2A) TOP: RSV F parent molecule (Cavl) with Cavl mutations indicated: Bottom Left: DS-Cavl successor of Cavl parent with additional DS mutations indicated. Bottom Right: preFC with tyrosine mutations indicated for dityrosine pairing. Note: K226Y pairs with endogenous tyrosine Y198. Amino acid numbering is indicated below the stick diagram. The “H ’, “S”, and scissors represent 6X His tag, Strep-II tag, and Thrombin cleavage site, respectively. (Fig. 2B) Crystal structure of DS-Cavl protein with preFC mutations modeled on the structure. DT bond locations are circled indicating the intramolecular crosslink preserving site 0 (5C4, D25) and the intermolecular crosslink preserving the site IVA interface (AM14). (Fig. 2C) Coomassie Blue Protein staining of SDS-PAGE separated preFC and DTpre-F under reducing conditions before and after crosslinking. Monomer, trimer, and F2 are indicated. Similar gel shifts have been observed consistently (>100 independent experiments). (Fig. 2D) ELISA binding curves of preFC and DT-preF using primary antibodies Motavizumab and AM 14 before and after crosslinking. Source data are provided as a Source Data file, the average data from 2 different crosslinking experiments is graphed.

[0023] Fig. 3A-C: Tyrosine modification analysis of DT-preF using NanoLC-ESI-MS / MS (Fig. 3A) MS / MS spectrum of conjugated peptide indicating a Y-Y linkage (Y185-Y428) between the ‘X’ peptide (AVVSLSDGYSV 177-187) and Y’ peptide (YRG 428-430) following digestion of the glycoprotein with trypsin, chymotrypsin, and pepsin. The ion series is indicated at the top right of the figure, while the mass spectrum is beneath with the ion series labeled. (Fig. 3B) Zoomed in view of the Left (L) insert (Fig. 3C) Zoomed in view of the Right (R) insert. Mass spectrometry analysis to find crosslinked species was performed a single time due to 98.5% coverage of the protein.

[0024] Fig. 4A-B: Biochemical / Biophysical characterization of the DT-preF molecule. (Fig. 4A). Amino acid analysis of DT-preF. Samples were acid-hydrolyzed and analyzed for amino acid content using a CONCISE AMINOSEP BECKMAN STYLE Na+ column and a Hitachi analytical HPLC. Table (top) with calculated Dityrosine (Di-Tyr) bonds in the DT-preF sample utilizing the experimentally determined percentage recovery' and quantification information. Chromatogram (bottom left) indicating the detection of the dityrosine peak in the DT-preF sample. Table (bottom right) indicating list of amino acids detected in the analysis with the respective peak attributes / quantification. Amino acid analysis was performed a single time. (Fig. 4B) Chromatograms of uncrosslinked F protein containing the DT mutations, preFC (top left) and DT-preF (bottom left) showing fluorescence and UV traces at increasing retention times (min) following separation using analytical size exclusion chromatography under reducing and denaturing conditions. Chromatogram showing fluorescence and UV traces of DT-preF (top right) and its corresponding SDS-PAGE analysis gel (bottom right) indicating separation of the dimer / trimer peak from the monomer peak, as indicated. This separation between multimers and monomers observed on SDS-PAGE analysis gels has been observed and reproduced in > 3 independent experiments.

[0025] Fig. 5A-C: In vitro stability of dityrosine crosslinked DT-preF molecule and its effects on potency in vivo. Panels: (Fig. 5A) In vitro stability' of DS-Cavl (top) and DT-preF (bottom) incubated at 4°C vs unincubated (fresh) at the indicated timepoints. Binding curves for AMI 4 (performed in triplicate) are presented on Motavizumab-normalized proteins for each timepoint. Data are presented as mean values + / - SD. ELIS As were run in triplicate using 2 different crosslinked protein preparations. (Fig. 5B) Dot plots of the neutralization titer data as reciprocal of IC50's with mean titers indicated as a bar. Groups of animals were compared on the distribution of the outcome using the Wilcoxon Rank Sum test. Sample size ‘n’ to derive statistics=10, 6-8-week-old, CB6F1 / J female mice / group. * Indicates p=0.0420 significance. Neutralization assays were run in quadruplicate. (Fig. 5C) Graphical representation of the data in B with DT-preF and DS-Cavl groups' potency loss as a percentage of their freshly thawed values (set at 100%) was compared in terms of mean neutralization titers after incubation at 4 C for 4 weeks. The percentage loss after 4 weeks of each sample is shown in light grey (top portion of each bar) and the remaining potency is in dark grey (bottom portion of each bar).

[0026] Fig. 6A-E: DT-preF is a highly potent immunogen in mice. Panels: (Fig. 6A) Mouse serum neutralization titers were measured by Renilla Luciferase assay after vaccination with 2.3 pg of the indicated immunogens formulated on Alhydrogel to compare potency of DT-preF and DS-Cavl. The reciprocal of the IC50 neutralizing antibody titers are graphed as dot plots. Geometric mean titers are graphed, and the numerical value is specified on the graph as a bar. Error bars represent the geometric SD. Groups of animals were compared on the distribution of the outcome using the Mann-Whitney test with a p value of 0.0002. Sample size ‘n’ to derive statistics= 10, 15-week-old. female CB6F1 / J mice / group; assays were run in duplicate. (Fig. 6B) Anti prefusion F binding titers were measured by ELISA using the serum obtained in (Fig. 6A) and prefusion F as the capture antigen. The EC50’s were plotted as dot plots with the geometric mean titers graphed, represented as a bar and the numerical value is specified on the graph. Sample size ‘n’ to derive statistics= 10, 15 -week-old. female CB6F1 / J / group; assays were run in duplicate. (Fig. 6C) Young (4 months) and old (17 months) female, BALB / c mice were immunized with our DT-preF immunogen on alum at a low dose (LD; lOug) and a high dose (HD; 45ug), respectively. Anti- preF antibody responses were quantified from serum samples collected on day 0 (pre-bleed), day 21 (prime only) and day 35 (prime-boost) by ELISA-based assay and plotted as bar graphs with the individual titers indicated by dots. Mean comparisons were performed by a 2-tailed paired (within group) or unpaired (between groups) Student’s t test using GraphPad Prism software. Error bars represent the SEM. p values of 1- and 2-star significances are p=0.0186 and p=0.00I9, respectively. (Fig. 6D) B-cell responses were measured for young, low dose and old, high dose vaccination groups by measuring vaccine-specific IgGs in antibody secreting cells (ASC) using a CTL ELISpot scanner. The mean data is shown as a bar graph with error bars to indicate the SEM. Mean comparisons were performed by a 2-tailed paired Student's t test using GraphPad Prism software but were not significant. The F Binding and ELISPOT analysis was performed at an earlier timepoint after 3 young and 6 aged biologically independent animal data had been accumulated. (Fig. 6E) Serum from day35 (prime-boost) was used to determine neutralizing antibody titers using a luminescence-based microneutralization assay. Dot plots of neutralizing antibody titers, expressed as the reciprocal of the IC50 serum dilution with geometric means graphed and indicated as bars, were used to compare young, low dose vaccination vs old, low dose and old, high dose vaccinations. Statistical analysis was performed using a two-sample t-test (p=0.041). Neutralization assays were run on duplicate plates and averaged prior to IC50 calculation with 4-month-old (young) and 17-month-old (old) male and female Balb / c mice totaling 12 or 13 biologically independent animals per group.

[0027] Fig. 7A-D: DT-preF is a highly potent immunogen in cotton rats. (Fig. 7A) Schematic describing the experimental design of the study. (Fig. 7B) Cotton rat lung titers were analyzed by plaque assay after vaccination with DT-preF (2 and 10 pg / animal) and the indicated controls at 4 days post challenge with 105 pfu / animal RSV / A / Long. The limit of detection is indicated, individual animal values are plotted as dot plots, and the error bars represent the SEM. (Fig. 7C) and (D) RSV / A and RSV / B cotton rat neutralizing antibody titers were measured in individual groups listed at the indicated timepoints. Serum antibody levels were assessed up to 39 days, which includes day 4 post challenge. For all panels, N = 5 animals / group in a single cotton rat study. A two-tailed Student’s t-test was used for statistical analysis. Minimal detection = 2.5.

[0028] Fig. 8A-B: Characterization of DT-preF by antigenic analysis and fluorimetry. (Fig. 8A) ELISA binding curves of DT-preF vs DS-Cavl proteins using primary antibodies Motavizumab, D25, and MPE8 which are specific to epitopes in Site II, Site 0, and Site III, respectively. (Fig. 8B) Protein concentration-based, fluorescence intensity analysis of crosslinked and purified DT-preF under native conditions. Data are presented as mean values + / - SD.

[0029] Fig. 9A-C: Control protein analysis of DS-Cavl and Cavl exposed to crosslinking conditions. (Fig. 9A) Coomassie-stained SDS-PAGE gel analysis of crosslinking reactions pre and post crosslinking run under denaturing conditions for DS-Cavl, DT-preF and Cavl. Monomer, Dimer, trimer, and ARP protein bands are indicated. (Fig. 9B. left) Western Blot and Coomassie stained protein gel run under reducing conditions with purified Cavl protein exposed to the crosslinking conditions. Monomer and F2 species are indicated by the arrow. (Fig. 9B, right) UV absorbance chromatogram (top) at 205 nm and fluorescence chromatogram (bottom) at Ex 320nm / Em 405nm generated by SEC-HPLC under denaturing conditions. (Fig. 9C, right) Western Blot and Coomassie stained protein gel under reducing conditions for a representative DT-preF protein. Trimer, Dimer, Monomer and F2 species are indicated. (Fig. 9C, left) UV absorbance chromatogram (top) at 205 nm and fluorescence chromatogram (bottom) at Ex 320nm / Em 405nm generated by SEC-HPLC under denaturing conditions.

[0030] Fig. 10 A-B: Additional stability characterization of DT-preF. (Fig. 9A) Second derivative analysis of a Differential Scanning Fluorimetry (DSF) experiment of uncrosslinked (preFc) and crosslinked (DT-preF). An increase of +25°C in the melting temperature is observed when DT-preF is analyzed and it ultimately reaches a melting temperature of 79.63°C as compared to uncrosslinked protein’s melting temperature of 54.19°C. (Fig. 9B) ELISA-based stability' analysis of dityrosine crosslinked DT-preF and DS-Cavl proteins incubated at 4°C vs unincubated (fresh) proteins at the indicated timepoints. A highly prefusion-specific sandwich ELISA (5C4 / AM14) and D25 direct binding ELISA’s were performed with binding curves presented on Motavizumab-normalized proteins.

[0031] Fig. 11: Coverage map of the uncrosslinked, mature preFcprotein (Signal peptide and p27 region eliminated). Tyrosine residues involved in the dityrosine bond formation are indicated with a box (mutation) or a circle (endogenous tyrosine). The position of these residues is shifted from the GenBank sequence and correspond to Y 160- Y403 and Y 173 (endogenous)- Y201 in this amino acid sequence. The portion highlighted in dark grey indicates the coverage area by Trypsin cleavage, and the region highlighted in light grey (ELPR) indicates coverage by Chymotrypsin only cleavage.

[0032] Fig. 12: Extent of crosslinking impacts potency in mice. Mice were immunized IM in a prime:boost regimen with 2 ug of AdvaxSMformulated DT-preF. Protein was prepared in a stirred tank bioreactor to achieve higher and lower amounts of crosslinking as indicated. Serum from immunized mice was harvested 2-weeks post boost, heat inactivated and used to perform neutralization assays using the RSV Renilla Luciferase virus. Neutralization titers (NTso) are reported as the reciprocal of the serum dilution resulting in 50% inhibition of the Renilla luciferase reporter virus growth as determined using the Renilla-glo luciferase assay kit. Mean neutralization titers are indicated as a bar with their actual values adjacent to the bar. These data are the average of neutralization titers run in triplicate.

[0033] DETAILED DESCRIPTION OF THE INVENTION

[0034] The present invention provides, in part, mutant RSV F molecules that comprise one-or-more to-tyrosine mutations and that can be, or are, stabilized in a pre-fusion conformation by the introduction of one-or-more DT crosslinks. The present invention also provides vaccine compositions comprising such RSV F molecules, methods of making such mutant RSV F molecules and vaccine compositions, and methods of use of such mutant RSV F molecules and vaccine compositions - including, but not limited to, vaccination methods including methods for vaccination of the elderly and the frail against RSV.

[0035] Definitions

[0036] The technical and scientific terms used in the present disclosure have the meanings commonly understood by those of ordinary skill in the art and / or their meaning is clear from the context in which the terms are used - unless specifically defined otherwise herein.

[0037] Several terms are defined below. Other terms are defined elsewhere in the text of this patent disclosure.

[0038] 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 terms “a” (or “an”) as well as the terms “one or more” and “at least one” can be used interchangeably.

[0039] Furthermore, “and / or” is to be taken as specific disclosure of each of the tw o specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” is intended to include A and B, A or B, A (alone), and B (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C A (alone); B (alone); and C (alone). Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges provided herein are inclusive of the numbers defining the range.

[0040] As used in the present specification the terms “about’' and “approximately,” when used in relation to numerical values, mean within + or - 10% of the stated value.

[0041] As used herein the term “effective amount” refers to an amount of a specified agent (e.g., RSV F molecule) or a specified composition (e.g., vaccine composition) that is sufficient to achieve the stated outcome (e.g.. the generation of a humoral immune response against RSV, the generation of a neutralizing antibody response against RSV, protection against RSV infection, and the like) to a detectable degree, or, in some cases to a specifically stated degree (e.g., a stated degree of protection against RSV infection). In some embodiments the effective amount is specified (e.g., in terms of a number of micrograms (mcgs), or a number of micrograms per kg bodyweight). In other embodiments an appropriate “effective amount” may be determined empirically, for example using standard techniques known in the art, such as dose escalation studies, and may be determined taking into account such factors as the planned route of administration, desired frequency of administration, the age of the subj ect, etc. Furthermore, an “effective amount” may be determined using studies such as those described in the Examples section of this patent disclosure.

[0042] As used herein the term “exemplary” means serving as an example, instance, or illustration.

[0043] As used herein the term “RSV F molecules” refers to all forms of the RSV F protein that comprise an Fl polypeptide and an F2 polypeptide - i.e. including F0 precursor polypeptides, trimers of F0 precursor polypeptides, RSV F protomers, and mature RSV F trimers - whether membrane bound or soluble. As used herein the term “mutant RSV F molecules” refers to RSV F molecules that contain one or more artificially introduced / man-made mutations. An

[0044] As used herein the term “Fl polypeptide” refers to a polypeptide comprising amino acid residues 137-513 of an RSV F F0 precursor sequence. Amino acid residues 137-513 do not include the RSV F transmembrane and cytoplasmic domains. In some embodiments Fl polypeptides may also include the RSV F transmembrane and cytoplasmic domains (located within residues 514-574 of the RSV F F0 precursor sequence). As used herein the term "F2 polypeptide” refers to a polypeptide comprising amino acid residues 26-109 of an RSV F F0 precursor sequence.

[0045] As used herein the terms “mutant” and “engineered” are used interchangeably to refer to molecules (such as RSV F molecules) that comprise at least one mutation introduced into a reference molecule. The reference molecule may be a naturally occurring molecule (such as a RSV F molecule that occurs in nature) or a non-naturally occurring molecule (such as the man-made DS-Cavl RSV F molecule) or any of the reference molecules for which sequences are provided in PCT / US2014 / 048086 and / or PCT / US 18 / 45463. The mutation may be a substitution, deletion, or insertion. For example, many of the embodiments of the present invention involve “mutant” or “engineered” RSV F molecules in which one or more nontyrosine residues are substituted with a tyrosine residue. Similarly, many of the embodiments of the present invention involve “mutant” or “engineered” RSV F molecules that comprise introduced trimerization domains, linker domains, protease cleavage sites, and / or tags useful purification and / or detection. As used herein the terms “protein” and “polypeptide” are used interchangeably, unless otherwise stated. As used herein the term “protein complex” refers to an assembly of two or more proteins or protein subunits, such as two or more monomers or protomers. Unless otherwise stated, all description herein that relates to proteins or polypeptides applies equally to protein complexes, and vice versa.

[0046] As used herein, the terms “nucleic acid molecules,” nucleic acid sequences,” and “nucleotide sequences” are used interchangeably .

[0047] As used herein the terms “stabilized” and “locked” are used interchangeably, for example in relation to the effect of crosslinking in stabilizing or locking the RSV F protein in its prefusion conformation. These terms do not require 100% stabili ty. Rather these terms denote a degree of improved or increased stability. For example, in some embodiments, when the term “stabilized” is used in relation to a RSV F protein crosslinked in its pre-fusion conformation, the term denotes that the pre-fusion conformation has greater stability than it would have had prior to or without such crosslinking. Stability, and relative stability', may be measured in various ways as described in other sections of this application, for example based on the half-life of the RSV pre-fusion conformation. The improvement or increase in stability may be to any degree that is useful or significant for the intended application. For example, in some embodiments, stability may be increased by about 10%, 25%, 50%, 100%, 200% (i.e. 2-fold), 300% (i.e. 3-fold), 400% (i.e. 4-fold), 500% (i.e. 5-fold), 1000% (i.e. 10- fold). or more.

[0048] As used herein the term "adjuvant" refers to a substance capable of enhancing, accelerating, or prolonging the body's immune response to an immunogen or immunogenic composition, such as a vaccine

[0049] As used herein the term "DS-Cavl" refers to the mutant RSV F protein described in McLellan, et al., Science, 342(6158), 592-598, 2013 - which comprises S155C, S290C, S190F. and V207L mutations.

[0050] As used herein the term "pre-fusion conformation" refers to a structural conformation of an RSV F molecule that can be specifically bound by a pre-fusion-specific antibody.

[0051] As used herein the terms “inter-moleculaf ’ (or intermolecular) and ’‘inter-protomeric” (or interprotomeric) are used interchangeably.

[0052] As used herein the term "pre-fusion-specific antibody" refers to an antibody that specifically binds to an RSV F molecule in its pre-fusion conformation but does not bind to the RSV F protein in a post-fusion conformation. Prefusion-specific antibodies include, but are not limited to the D25, AM22, 5C4, and AM14 antibodies. As used herein the term "AM14" refers to an antibody described in WO 2008 / 147196 A2. As used herein the term "AM22" refers to an antibody described in WO 2011 / 043643 Al. As used herein the term "D25" refers to an antibody described in WO 2008 / 147196 A2. As used herein the term "5C4" refers to an antibody as described in McLellan et al., 2010, Nat. Struct. Mol. Biol., Feb 17(2): 248-50) were mapped. McLellan et al. (Science 340: 1113-1117 (2013)).

[0053] Numbers in superscript following text herein refer to the numbered references found in the Reference List section of this patent disclosure.

[0054] Mutant RSV F Molecules

[0055] The RSV Fusion or “F” protein is an envelope glycoprotein of respiratory syncytial viruses. In nature the RSV F protein is translated as a single precursor polypeptide (designated F0). The F0 precursor polypeptide is generally 574 amino acids in length. Amino acids 1-25 of the F0 precursor generally comprise a signal peptide. The precursor polypeptide F0 forms a precursor trimer, which is typically proteolytically cleaved by one or more cellular proteases at conserved furin consensus cleavage sites to yield a Pep 27 polypeptide, an Fl polypeptide and an F2 polypeptide. The Pep 27 polypeptide (generally amino acids 1 10-136 of the F0 precursor) is excised and does not form part of the mature RSV F trimer. The F2 polypeptide (which may alternatively be referred to herein as “F2” or the “F2 region’") generally consists of amino acid residues 26-109 of the F0 precursor. The Fl polypeptide (which may alternatively be referred to herein as "‘Fl” or the “Fl region”) generally consists of amino acid residues 137-574 of the F0 precursor and comprises an extracellular region (generally residues 137-524), a transmembrane domain (generally residues 525-550), and a cytoplasmic domain (generally residues 551-574). The Fl and F2 polypeptides are linked by disulfide- bonds to form a heterodimer which is referred to as an RSV F “protomer.” Three such protomers form the mature RSV F trimer - which is thus a homotrimer of the three protomers. In nature the mature RSV F trimer is generally membrane-bound. However, soluble (i.e. non-membrane bound) versions of the mature RSV F trimer can be made by removing the transmembrane and cytoplasmic regions. For example, conversion to a soluble form can be accomplished by truncating the RSV F protein at amino acid 513 (i.e. by removing amino acids 514 onwards).

[0056] In nature the mature RSV F trimer mediates fusion of viral and cellular membranes. The prefusion conformation of the mature RSV F trimer (which may be referred to herein as “pre-F”) is highly unstable (metastable). However, once the RSV virus docks with the cell membrane, the RSV F protein trimer undergoes a series of conformational changes and transitions to a highly stable post-fusion (“post-F”) conformation. The mature RSV F protein is known to induce potent neutralizing antibodies (“nAbs”) that correlate with RSV protection. For example, immunization with the RSV F protein induces nAbs that are protective in humans (e.g. Synagis). Several neutralizing epitopes (sites I, II and IV) are present on the post-fusion form of RSV F protein. Recently, however, Magro et al. showed that incubation of human sera with the RSV F protein in its post-fusion conformation failed to deplete the majority of neutralizing activity against the F protein, indicating the presence of neutralizing antigenic sites unique to the pre-fusion conformation (Magro et al. 2012, PNAS 109(8): 3089). By x- ray crystallography, the epitopes recognized by palivizumab (Synagis), motavizumab (Numax), and that of the more 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) were mapped. McLellan et al. (Science 340: 1113-1117 (2013)) solved the structure of the F protein in its pre-fusion conformation, which revealed a novel neutralizing epitope - site 0 - that is only displayed in the pre-fusion conformation, and to which a series of antibodies bind, e.g. 5C4, that are up to 50-fold more potently neutralizing than Synagis and Numax. Accordingly, there is mounting evidence that an RSV vaccine immunogen in this pre-fusion conformation and displaying site 0 could elicit effective protection. However, the highly unstable (metastable) nature of the pre-fusion conformation of the RSV F protein has proved to be a significant barrier to the development of such a vaccine. Based on a comparison of the pre- and post-fusion RSV F structures of McLellan et al. there appear to be two regions of the F protein that undergo large conformational changes (>5 A). These regions are located at the N- and C-termini of the F l subunit (residues 137-216 and 461-513, respectively). In the crystal structure of the RSV F protein held in its pre-fusion conformation by the D25-antibody bound to the site o epitope, the C-terminal Fl residues can be stabilized in the pre-fusion conformation by appending a foldon trimerization domain. To stabilize the N-terminal region of Fl, McLellan et al. found that binding of the antibody D25 was sufficient for crystallographic studies. However, for production of a vaccine immunogen alternative stabilization strategies are needed, such as those that do not require the RSV F protein to be bound to a large antibody molecule. One alternative approach that has been attempted involved the introduction of paired cysteine mutations (for disulfide bond formation) and cavity-filling mutations near the Fl N -terminus (see the DS-Cavl RSV F protein variant described in McLellan et al. (2013) Science 342:592-598, which is hereby incorporated by reference in its entirety)- However, crystallographic analysis of such variants revealed that the structure was only partially in the pre-fusion conformation. Accordingly, additional engineering of the RSV F protein is needed in order to achieve an immunogen for clinical vaccine development.

[0057] The present invention provides certain alternative approaches for stabilizing the RSV F protein in its pre-fusion conformation - based on the introduction of one or more ‘iotyrosine” mutations and one or one or more DT crosslinks at specified locations in RSV F molecules.

[0058] The amino acid sequences of several exemplary RSV F and mutant RSV F molecules are provided in Table 1 and in PCT / US2014 / 048086 and PCT / US 18 / 45463. Most of the sequences provided in Table 1, PCT / US2014 / 048086 and PCT / US 18 / 45463 are presented as “FO” sequences - i.e. these sequences comprise a signal peptide and a pep27 peptide that is present in FO precursors but that is not present in mature RSV F proteins. Similarly, several of the sequences provided in Table 1 and PCT / US2014 / 048086, and / or PCT / US18 / 45463 include native transmembrane and cytoplasmic domains that, in some embodiments, can be removed to form soluble versions of mature RSV trimers. Thus, the final / mature versions of these exemplary mutant RSV molecules (i.e. the versions that may be DT crosslinked) typically will not comprise all of the amino acids shown in the sequences in Table 1 and PCT / US2014 / 048086 and / or PCT / US 18 / 45463. However, the final / mature versions of these exemplary mutant RSV molecules will comprise the F2 region and at least a portion of the Fl region of these sequences. Typically, the final / mature versions of these exemplary mutant RSV molecules will comprise amino acid residues 26-109 and amino acid residues 137-513 of these sequences. It should be noted that, in all embodiments herein that refer to an RSV F molecule having a specific exemplary amino acid sequence, either the full FO sequence or amino acid residues 26-109 and amino acid residues 137-513 therefore are contemplated.

[0059] Throughout the present patent disclosure, when specific amino acid positions / residues in an RSV F molecule are referred to by their amino residue number (such as amino acid residue 428 for example), and unless otherwise stated, the amino acid numbering is that used for the RSV F amino acid sequences provided in Table 1 of the present application (see, e.g., SEQ ID NOs. 1-91) and in the Sequence Listings of PCT / US2014 / 048086 and / or

[0060] PCT / US 18 / 45463. This is the same numbering system that is used routinely in the art when describing RSV F sequences. However, it should be noted, and one of skill in the art will understand, that different numbering systems can be used. For example, if there are additional amino acid residues added or removed as compared to any of SEQ ID NO: 1-91. As such, it is to be understood that when a specific amino acid residue is referred to by its number, the description is not limited to only amino acids located at precisely that numbered position when counting from the beginning of a given amino acid sequence, but rather that the '‘corresponding” amino acid residue in any and all RSV F sequences is intended - even if that residue is not at the same precise numbered position in a given molecule, for example if the RSV sequence is shorter or longer than SEQ ID NO. 1, or has insertions or deletions as compared to SEQ ID NO. 1. One of skill in the art can readily determine what is the “corresponding” amino acid position to any of the specific numbered residues recited herein, for example by aligning a given RSV F sequence to SEQ ID NO. 1 or to any of the other RSV F amino acid sequences provided herein (i.e. SEQ ID NOs. 1-91). Such alignments can be readily performed - whether by computer or by eye - given the highly conserved nature of RSV F sequences across RSV subtypes and RSV strains. The amino acid sequences of a large number of WT / native RSV F molecules from different RSV subtypes and strains, as well as nucleic acid sequences encoding such RSV F molecules, are known in the art. Amino acid sequences of several exemplary WT / native RSV F molecules are provided in SEQ ID NOs: 1- 20 and 82-85. Other such sequences can be found in public sequence databases. Other such sequences are described in International Patent Application No. PCT / US2014 / 048086 U.S. Patent No. 9,738, 689, and U.S. Patent No. 9,950,058 - the contents of each of which are hereby incorporated by reference.

[0061] WT / native RSV F molecules exhibit a strikingly high level of sequence conservation - both across RSV subtypes and across RSV strains. See, for example, WO2014 / 160463. For example. RSV subtypes A and B share 90% sequence identity across the F0 precursor molecule. Within a given RSV subtype (e.g. subtype A or B) the sequence identity’ across strains is about 98%. Furthermore, nearly all RSV F F0 precursors identified to date consist of 574 amino acids. There can be some minor differences in length - such differences generally occurring in the cytoplasmic domain.

[0062] The specific to-tyrosine mutations described herein can be introduced into any suitable RSV F “background” sequence.

[0063] In some embodiments such “background” sequences are those of WT I native RSV F molecules (i.e. those that exist in nature). The amino acid and nucleotide sequences of a large number of WT / native RSV F molecules from different RSV subty pes and strains are known in the art. Amino acid sequences of several exemplary’ WT / native RSV F molecules are provided in SEQ ID NOs: 1-20 and 82-85 (see Table 1). 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 - the contents of each of which are hereby incorporated by reference.

[0064] In some embodiments such “background” sequences are those of mutant RSV F molecules - i.e. those that comprise one or more artificially introduced mutations as compared to WT / native RSV F molecules. For example, in some embodiments the "background" RSV F sequence may comprise a "Cav I " mutation, a “DS” mutation, or a combination thereof. Non-limiting examples of suitable mutant background RSV molecules into which the specific mutations described herein may be introduced include those having the amino acid sequences of any of SEQ ID Nos 86-91, or those comprising amino acids 26-109 (i.e. F2) and amino acids 137-513 (i.e. Fl) of any of SEQ ID Nos 86-91 (see Table 1). Other suitable mutant background RSV molecules into which the specific mutations described herein may be introduced include those described in International Patent Application No.

[0065] PCT / US2014 / 048086 U.S. Patent No. 9,738, 689, and U.S. Patent No. 9,950,058.

[0066] In some embodiments the “background” RSV F molecule - into which the specific mutations described herein may be introduced - may be a “full-length” RSV F molecule, i.e. comprising a transmembrane domain and a cytoplasmic domain. Non-limiting examples of suitable “full length” background RSV molecules into which the specific mutations and crosslinks described herein may be introduced include those having the amino acid sequences of any of SEQ ID Nos 1-9, 11-29, 31-49, 51-80, 83, 85, 87, or 89, or those comprising amino acids 26-109 (F2) and amino acids 137-513 (Fl) of any of SEQ ID Nos 1-9, 11-29, 31-49, 51-80, 83, 85, 87, or 89 (see Table 1).

[0067] In some embodiments the “background” RSV F molecule - into which the specific mutations and crosslinks described herein may be introduced - may be a “soluble” RSV F molecule, i.e not. comprising a transmembrane domain and a cytoplasmic domain. Non-limiting examples of suitable “soluble” background RSV molecules into which the specific mutations and crosslinks described herein may be introduced include those having the amino acid sequences of any of SEQ ID Nos 10, 30, 50, 81, 82, 84, 86, 88, or 90 or those comprising amino acids 26-109 (i.e. F2) and amino acids 137-513 (i.e. Fl) of any of SEQ ID Nos 10, 30, 50, 81, 82, 84, 86, 88, or 90.

[0068] Similarly, in some embodiments the soluble “background” RSV F molecule - into which the specific mutations and crosslinks described herein may be introduced - may be created by removing the transmembrane domain and cytoplasmic domain of a “full-length” RSV F molecule, for example by removing amino acids 514 onwards of a “full-length” RSV F molecule - such as one of those described above. In some embodiments the present invention provides mutant / engineered RSV F molecules comprising a point mutation to tyrosine at one or more of amino acid positions: 77, 88, 97, 147, 150, 155, 159, 183, 185, 187, 220, 222, 223, 226, 255, 427, 428, or 469, or at amino acid residues that corresponds to these amino acid positions - for example as determined by alignment to, and / or using the amino acid numbering of, SEQ ID NO. 1.

[0069] In some embodiments the present invention provides mutant / engineered RSV F molecules comprising a point mutation to tyrosine at two or more of amino acid positions: 77, 88, 97, 147, 150, 155, 159, 183, 185, 187, 220, 222, 223, 226, 255, 427, 428, or 469, or at ammo acid residues that corresponds to these amino acid positions - for example as determined by alignment to, and / or using the amino acid numbering of, SEQ ID NO. 1 .

[0070] In some embodiments the present invention provides mutant / engineered RSV F molecules comprising a point mutation to tyrosine at three or more of amino acid positions: 77, 88. 97. 147, 150. 155, 159, 183. 185, 187. 220, 222, 223, 226, 255. 427, 428. or 469. or at ammo acid residues that corresponds to these amino acid positions - for example as determined by alignment to, and / or using the amino acid numbering of, SEQ ID NO. 1.

[0071] In some embodiments the present invention provides mutant / engineered RSV F molecules comprising a point mutation to tyrosine at four or more of amino acid positions: 77, 88, 97, 147, 150, 155, 159, 183, 185, 187, 220, 222, 223, 226, 255, 427, 428, or 469, or at ammo acid residues that corresponds to these amino acid positions - for example as determined by alignment to. and / or using the amino acid numbering of, SEQ ID NO. 1.

[0072] In some preferred embodiments the present invention provides mutant RSV F molecules comprising a point mutation to ty rosine at amino acid residue 428 (which may be referred to as a 428Y point mutation), or at an amino acid that corresponds to position 428 - for example as determined by alignment to, and / or using the amino acid numbering of, SEQ ID NO. 1.

[0073] In some preferred embodiments such mutant RSV F molecules comprising a 428Y mutation further comprise a point mutation to ty rosine at amino acid residue 185 (which may be referred to as a 185 Y point mutation), or at an amino acid that corresponds to position 185 - for example as determined by alignment to, and / or using the amino acid numbering of, SEQ ID NO.l. Such mutants thus comprise both a 428Y mutation and a 185Y mutation. In some preferred embodiments such mutant RSV F molecules comprising a 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 at an amino acid that corresponds to position 226 - for example as determined by alignment to, and / or using the amino acid numbering of, SEQ ID NO.l. Such mutants thus comprise both a 428Y mutation and a 226Y mutation.

[0074] In some embodiments such mutant RSV F molecules comprising a 428Y mutation further comprise both (a) a point mutation to tyrosine at amino acid residue 226 (which may be referred to as a 226Y point mutation), or at an amino acid that corresponds to position 226 - for example as determined by alignment to, 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 at an amino acid that corresponds to position 185 - for example as determined by alignment to, and / or using the amino acid numbering of, SEQ ID NO.l. Such mutants thus comprise a 428Y mutation, a!85Y mutation, and a 226Y mutation. By way of example. SEQ ID NO. 81, and the RSV molecule referred to as "preFC" in the Examples, comprise a 428Y mutation, a 185Y mutation, and a 226Y mutation.

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

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

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

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

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

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

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

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

[0083] In some embodiments the mutant RSV F molecule comprises (a) an F2 polypeptide comprising or consisting of amino acid residues 26-109 of SEQ ID NO 81, and (b) an Fl polypeptide comprising or consisting of amino acid residues 137-513 of SEQ ID NO. 81. By way of example, the RSV molecule referred to as “preFC" in the Examples comprises an F2 polypeptide comprising or consisting of amino acid residues 26-109 of SEQ ID NO 81, and an Fl polypeptide comprising or consisting of amino acid residues 137-513 of SEQ ID NO. 81.

[0084] In some embodiments the mutant RSV F molecule comprises (a) an F2 polypeptide comprising or consisting of approximately 74-84 amino acids of amino acid residues 26-109 of SEQ ID NO 81, and (b) an Fl polypeptide comprising or consisting of approximately 365- 375 amino acids of amino acid residues 137-513 of SEQ ID NO. 81. In some embodiments the mutant RSV F molecules of the present invention are stabilized in a prefusion conformation by one or more di-tyrosine crosslinks.

[0085] In some embodiments the mutant RSV F molecule is a RSV F trimer. In some such embodiments the RSV F molecule is a RSV F trimer stabilized in a prefusion conformation by di-tyrosine crosslinks.

[0086] In some embodiments the mutant RSV F molecule comprises at least one di-tyrosine crosslink, wherein one or both tyrosines of the crosslink originate(s) from a point mutation to tyrosine, and wherein the crosslinks are located between one or more paired tyrosine amino acid residues selected from the following pairs: 147 and 286; 198 and 220; 198 and 222; 198 and 223; 198 and 226; 33 and 469; 77 and 222; 88 and 255; 97 and 159; 183 and 427; 185 and 427; 185 and 428; and 187 and 427. In some embodiments, the mutant RSV F molecule comprises at least two di-tyrosine crosslinks, wherein one or both tyrosines of each of the two crosslinks originate(s) from a point mutation to tyrosine, and wherein the at least two crosslinks are located between one or more of the paired tyrosine amino acid residues listed in the previous sentence.

[0087] In some embodiments the mutant RSV F molecule comprises an inter-molecular (also referred to as an inter-protomeric) di-tyrosine crosslink between a tyrosine at amino acid position 428 (or an amino acid position corresponding thereto - for example as determined by alignment to, 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 - for example as determined by alignment to, and / or using the amino acid numbering of, SEQ ID NO. 1).

[0088] In some embodiments the mutant RSV F molecule comprises an intra-molecular (also referred to as an intra-protomeric) di-tyrosine crosslink between a tyrosine at amino acid position 198 (or an amino acid position corresponding thereto - for example as determined by alignment to, 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 - for example as determined by alignment to, and / or using the amino acid numbering of. SEQ ID NO. 1).

[0089] In some embodiments the mutant RSV F molecule comprises both: (a) a di-tyrosine crosslink between a tyrosine at amino acid position 198 (or an amino acid position corresponding thereto - for example as determined by alignment to, 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 - for example as determined by alignment to, and / or using the amino acid numbering of, SEQ ID NO. 1), and (b) a di-tyrosine crosslink between a tyrosine at amino acid position 428 (or an amino acid position corresponding thereto - for example as determined by alignment to, and / or using the amino acid numbering of, SEQ ID NO.l) and a tyrosine at amino acid position 185 (or an amino acid position corresponding thereto - for example as determined by alignment to, and / or using the amino acid numbering of, SEQ ID NO. l).

[0090] In some embodiments the mutant RSV F molecule is a RSV F trimer comprising three dityrosine crosslinks - each of which is between a tyrosine at an amino acid position 428 (or an amino acid position corresponding thereto - for example as determined by alignment to, and / or using the amino acid numbering of, SEQ ID NO. 1) and a tyrosine at an amino acid position 185 (or an amino acid position corresponding thereto - for example as determined by alignment to. and / or using the amino acid numbering of, SEQ ID NO. l).In some embodiments the mutant RSV F molecule is a RSV F trimer comprising up to two di-tyrosine crosslinks between a tyrosine at an amino acid position 428 (or an amino acid position corresponding thereto - for example as determined by alignment to, and / or using the amino acid numbering of. SEQ ID NO.l) and a tyrosine at an amino acid position 185 (or an amino acid position corresponding thereto - for example as determined by alignment to, and / or using the amino acid numbering of, SEQ ID NO. 1).

[0091] In some embodiments the mutant RSV F molecule is a RSV F trimer comprising no more than one di-tyrosine crosslinks between a tyrosine at an amino acid position 428 (or an amino acid position corresponding thereto - for example as determined by alignment to, and / or using the amino acid numbering of, SEQ ID NO. 1) and a tyrosine at an amino acid position 185 (or an amino acid position corresponding thereto - for example as determined byalignment to. and / or using the amino acid numbering of, SEQ ID NO. 1).

[0092] In some embodiments the mutant RSV F molecule is a RSV F trimer comprising no dityrosine crosslinks between a tyrosine at an amino acid position 428 (or an amino acid position corresponding thereto - for example as determined by alignment to, and / or using the amino acid numbering of, SEQ ID NO. l) and a tyrosine at an amino acid position 185 (or an amino acid position corresponding thereto - for example as determined by alignment to, and / or using the amino acid numbering of, SEQ ID NO. 1).

[0093] In some embodiments the mutant RSV F molecule is a RSV F trimer comprising three dityrosine crosslinks - each of which is between a tyrosine at an amino acid position 198(or an amino acid position corresponding thereto - for example as determined by alignment to, and / or using the amino acid numbering of, SEQ ID NO. 1) and a tyrosine at an amino acid position 226 (or an amino acid position corresponding thereto - for example as determined by alignment to, and / or using the amino acid numbering of, SEQ ID NO. 1).

[0094] In some embodiments the mutant RSV F molecule is a RSV F trimer comprising up to two di -tyrosine crosslinks between a tyrosine at an amino acid position 198(or an amino acid position corresponding thereto - for example as determined by alignment to, and / or using the amino acid numbering of, SEQ ID NO. 1) and a tyrosine at an amino acid position 226 (or an amino acid position corresponding thereto - for example as determined by alignment to. and / or using the amino acid numbering of, SEQ ID NO.l).

[0095] In some embodiments the mutant RSV F molecule is a RSV F trimer comprising no more than one di -tyrosine crosslink between a tyrosine at an amino acid position 198 (or an ammo acid position corresponding thereto - for example as determined by alignment to, and / or using the amino acid numbering of, SEQ ID NO. 1) and a tyrosine at an amino acid position 226 (or an amino acid position corresponding thereto - for example as determined by alignment to. and / or using the amino acid numbering of, SEQ ID NO. 1).

[0096] In some embodiments the mutant RSV F molecule is a RSV F trimer comprising no dityrosine crosslinks between a tyrosine at an amino acid position 198 (or an amino acid position corresponding thereto - for example as determined by alignment to, and / or using the amino acid numbering of, SEQ ID NO. 1) and a tyrosine at an amino acid position 226 (or an amino acid position corresponding thereto - for example as determined by alignment to, and / or using the amino acid numbering of, SEQ ID NO.l).

[0097] In some embodiments the mutant RSV F molecule is a RSV F trimer comprising six dityrosine crosslinks, of which three are between a tyrosine at an amino acid position 198 (or an amino acid position corresponding thereto - for example as determined by alignment to, and / or using the amino acid numbering of, SEQ ID NO. 1) and a tyrosine at an amino acid position 226 (or an amino acid position corresponding thereto - for example as determined by alignment to. and / or using the amino acid numbering of, SEQ ID NO. 1), and three are between a tyrosine at an amino acid position 428 (or an amino acid position corresponding thereto - for example as determined by alignment to, and / or using the amino acid numbering of, SEQ ID NO. 1) and a tyrosine at an amino acid position 185 (or an amino acid position corresponding thereto - for example as determined by alignment to, and / or using the amino acid numbering of, SEQ ID NO. 1).

[0098] In some embodiments the mutant RSV F molecule is a RSV F trimer comprising 1, 2, 3, 4, or 5 di-tyrosine crosslinks, selected from the three possible crosslinks between a tyrosine at an amino acid position 198 (or an amino acid position corresponding thereto - for example as determined by alignment to, and / or using the amino acid numbering of, SEQ ID NO.l) and a ty rosine at an amino acid position 226 (or an amino acid position corresponding thereto - for example as determined by alignment to, and / or using the amino acid numbering of, SEQ ID NO. 1), and the three possible crosslinks between a tyrosine at an amino acid position 428 (or an amino acid position corresponding thereto - for example as determined by alignment to, and / or using the amino acid numbering of, SEQ ID NO. 1) and a tyrosine at an amino acid position 185 (or an amino acid position corresponding thereto - for example as determined by alignment to. and / or using the amino acid numbering of, SEQ ID NO. 1).

[0099] In some embodiments, the mutant RSV F molecules further comprise one or more artificially introduced non-DT crosslinks. In some such embodiments such non-DT crosslinks are disulfide bonds. In some such embodiments such RSV F molecules comprise one-or-more point mutations to cysteine. In some such embodiments one or more of the disulfide bonds is formed between two cysteines - one or both of which has been introduced by point mutation. In some such embodiments the RSV F molecule comprises a point mutation to cysteine at amino acid residue 155 (i.e. a 155C mutation). In some such embodiments the RSV F molecule comprises a point mutation to cysteine at amino acid residue 290 (i.e. a 290C mutation). In some such embodiments the RSV F molecule comprises both a point mutation to cysteine at amino acid residue 155 (i.e. a 155C mutation) and a point mutation to cysteine at amino acid residue 290 (i.e. a 290C mutation). In some such embodiments the RSV F molecule comprises one or more of the point mutations to cysteine disclosed in either U.S. Patent No. 9,738, 689, U.S. Patent No. 9,950,058, or McLellan et al. (2013) Science 342:592- 598, each of which is hereby incorporated by reference in its entirety for this purpose.

[0100] In some embodiments, the mutant RSV F molecules further comprise one or more artificially introduced cavity-filling mutations (e.g. substitutions). In some such embodiments the RSV F molecule comprises a mutation to F at amino acid residue 190 (a 190F mutation). In some such embodiments the RSV F molecule comprises a point mutation to L at amino acid residue 207 (a 207L mutation). In some such embodiments the RSV F molecule comprises both a mutation to F at amino acid residue 190 (a 190F mutation) and a point mutation to L at amino acid residue 207 (a 207L mutation). In some such embodiments the RSV F molecule comprises one or more cavity-filling amino acid substitutions selected from the group consisting of: 58W, 83W, 87F, 90L, 153W, 190F, 203W, 207L, 220L, 260W, 296F, and 298L. In some such embodiments the RSV F molecule comprises one or more of the cavityfilling amino acid substitutions disclosed in either U.S. Patent No. 9,738, 689, U.S. Patent No. 9,950,058. or McLellan et al. (2013) Science 342:592-598. each of which is hereby incorporated by reference in its entirety for this purpose. In some embodiments the mutant RSV F molecules comprise an oligomerization domain. In some embodiments the mutant RSV F molecules comprise a trimerization domain. In one embodiment the trimerization domain is, or comprises, a foldon domain, a GCN4 domain, a T4 fibrinitin domain, a trimerization domain comprising the 6-hehcal bundle formed in the post-fusion form of viral fusion proteins (wherein such viral fusion proteins include, but are not limited to, retroviral fusion proteins, Syncytin-1 & 2, HERV-K ENV, ERV3), and the human C-propeptide of al (I) collagen (Trimer-Tag). In some embodiments the mutant RSV F molecules comprise a trimerization domain that comprises a self-assembling protein with 3-fold symmetry axes and outward projecting N termini that can be sequence- and structure-adjusted and fused to protomers and hold the protomers in a trimeric complex.

[0101] Examples of other trimerization domains that can be used include, but are not limited to. those described in Habazettl et al., 2009 (Habazettl et al., 2009. NMR Structure of a Monomeric Intermediate on the Evolutionarily Optimized Assembly Pathway of a Small Trimerization Domain. J.Mol.Biol. pp. null); Kammerer et al., 2005 (Kammerer et al., 2005. A conserved trimerization motif controls the topology of short coiled coils. Proc Natl Acad Sci USA 102 (39): 13891-13896); Innamorati et al., 2006. (Innamorati et al., 2006. An intracellular role for the Clq-globular domain. Cell signal 18(6): 761-770); Schelling et al., 2007 (Schelling et al., 2007. The reovirus o-l aspartic acid sandwich: A trimerization motif poised for conformational change. Biol Chem 282(15): 1 1582-11589); Pancera et al., 2005. (Soluble Mimetics of Human Immunodeficiency Virus Type 1 Viral Spikes Produced by Replacement of the Native Trimerization Domain with a Heterologous Trimerization Motif: Characterization and Ligand Binding Analysis. J Virol 79 (15): 9954-9969); Guthe et al., 2004. (Very fast folding and association of a trimerization domain from bacteriophage T4 fibritin. J.Mol.Biol. v337 pp. 905-15); and Papanikolopoulou et al., 2008 (Creation of hybrid nanorods from sequences of natural trimeric fibrous proteins using the fibritin trimerization motif. Methods Mol Biol 474: 15-33).

[0102] In some embodiments, the mutant RSV F molecules comprise the foldon domain of SEQ ID NO. 93.

[0103] In some embodiments, the mutant RSV F molecules further comprise one or more tags useful for detection and / or purification of the RSV F molecules (e.g. C-terminal tags). Exemplary tags 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, and the like. In some such embodiments such RSV F molecules comprise the His tag of SEQ ID NO. 95. In some such embodiments such RSV F molecules comprise the Strep II tag of SEQ ID NO. 96. In some such embodiments such tags are cleavable tag - i.e. they can be cleaved / removed from the mutant RSV F molecule if desired. In some such embodiments such tags are located adjacent to (e.g. C-terminal to) a proteolytic cleavage site - such that a protease can be used to remove the tag. In some such embodiments such RSV F molecules comprise the thrombin cleavage site of SEQ ID NO. 94.

[0104] In some embodiments, the mutant RSV F molecules further comprise one or more peptide “linker” sequences. Suitable linker sequences include, but are not limited to, G, GG, GGG, GS, and SAIG (amino acids 1-4 of SEQ ID NO. 92) linker sequences. Such linkers may be provided; between the C-terminal end of F2 and the N-terminal end of Fl, between the C- terminal end of Fl and the N-terminal end of any artificial trimerization domains, and / or between any other artificially introduced sequences - such as trimerization domains, proteolytic cleavage domains, and tags useful for detection and / or purification. In some embodiments the mutant RSV F molecules comprise one or more leader sequences, precursor polypeptide sequences, secretion signals, and / or localization signals.

[0105] In those embodiments of the present invention that relate to specific exemplary' amino acid sequences of mutant RSV F molecules (e.g. those of SEQ ID Nos 21-81) or of “background'’ RSV F molecules (e.g. those of SEQ ID Nos 1-20 or 82-91), or that related to specific regions of such sequences (e.g. Fl and / or F2 regions thereof) variant forms of such amino acid sequences that are equivalent thereto can also be used. For example, in some embodiments amino acid sequences that have at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity any of the RSV F amino acid sequences described herein across their Fl and F2 regions (specifically across amino acid residues 26-109 (F2) and 137-513 (part of Fl excluding the transmembrane and cytoplasmic domains) can be used. In some embodiments amino acid sequences that have at least about 80% or at least 85% sequence identity to such amino acid sequences across these Fl and F2 regions can be used. Such variant forms may have amino acids added, removed, or substituted as compared to one or more of the specific amino acid sequences provided herein. Thus, they can be longer or shorter in length than the specified sequences.

[0106] Similarly, amino acid residues 514 onwards of each of the exemplary RSV F amino acid sequences provided herein may be removed or varied. For example, in several of the amino acid sequences provided herein amino acid residues 514 onwards comprise native RSV F transmembrane and cytoplasmic domains. In some embodiments these native RSV F transmembrane and cytoplasmic domains can be removed to generate a soluble (i.e. nonmembrane bound) version of the RSV F molecule or can be replaced with different transmembrane and / or cytoplasmic domains. Similarly, in several of the amino acid sequences provided herein amino acid residues 514 onwards comprise a combination of various artificially added C-terminal sequences - such as the artificial C-terminal sequence provided in SEQ ID NO. 92. which comprises a foldon domain (SEQ ID NO. 93), a thrombin cleavage site (SEQ ID NO. 94), a Histidine tag (SEQ ID NO. 95), a Strep II tag (SEQ ID NO. 96), and various linkers. In some embodiments such artificial C-terminal sequences can be removed, modified, rearranged or replaced - as needed. 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. In some embodiments one or more amino acid residues within one or the specific mutant RSV F molecules described herein can be substituted with another amino acid. In some embodiments, one or more amino acid residues can be substituted by another amino acid having a similar polarity and that may act as a functional equivalent, resulting in a silent alteration. In some embodiments substitutions for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs e.g. to create a conservative substitution. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such substitutions are generally understood to be conservative substitutions.

[0107] In some embodiments artificial, synthetic, or non-classical amino acids or chemical amino acid analogs can be used to make the mutant RSV F molecules described herein. Non- classical amino acids include, but are not limited to, the D-isomers of the common amino acids, fluoro-amino acids, and “designer” amino acids such as -methyl amino acids, Cy - methyl amino acids, Ny -methyl amino acids, and amino acid analogs in general. Additional non-limiting examples of non-classical amino acids include, but are not limited to: a- aminocaprylic acid, Acpa; (S)-2-aminoethyl-L-cysteine / HCl, Aecys; aminophenylacetate, Afa; 6-amino hexanoic acid, Ahx; y-amino isobutyric acid and a-aminoisobytyric acid, Aiba; alloisoleucine, Aile; L-allylglycine, Alg; 2-amino butyric acid, 4-aminobutyric acid, and a - aminobutyric acid, Aba; p-aminophenylalanine, Aphe; b-alanine, Bal; p-bromophenylalaine, Brphe; cyclohexylalanine, Cha; citrulline, Cit: p-chloroalanine, Gala; cycloleucine, Cle; p- cholorphenylalanine, Clphe; cysteic acid, Cya; 2,4-diaminobutyric acid. Dab; 3-amino propionic acid and 2,3-diaminopropionic acid, Dap; 3,4-dehydroproline, Dhp; 3,4- dihydroxylphenylalanine, Dhphe; p-flurophenylalanine, Fphe; D-glucoseaminic acid, Gaa; homoarginine, Hag; 8-hydroxylysine / HCl, Hlys; DL- -hydroxynorvaline, Hnvl; homoglutamine, Hog; homophenylalanine, Hoph; homoserine, Hos; hydroxyproline, Hpr; p- iodophenylalanine, Iphe; isoserine, Ise; a-methylleucine, Mie; DL-methionine-S- methylsulfoniumchloide, Msmet; 3-(l -naphthyl) alanine, INala; 3-(2-naphthyl) alanine, 2Nala; norleucine, Nle; N-methylalanine, Nmala; Norvaline, Nva; O-benzylserine. Obser; O- benTyltyrosine, Obtyr; O-ethyltyrosine, Oetyr; O-methyl serine, Omser; O-methylthreonine, Omthr; O-methyltyrosine. Omtyr; Ornithine, Om; phenylglycine; penicillamine, Pen; pyroglutamic acid. Pga; pipecolic acid, Pip; sarcosine, Sar; t-butylglycine; t-butylalanine; 3,3,3-trifluroalanine, Tfa; 6-hydroxydopa, Thphe; L-vinylglycine, Vig; (-)-(2R)-2-amino-3- (2-aminoethylsulfonyl) propanoic acid dihydroxochloride, Aaspa; (2S)-2-amino-9-hydroxy- 4,7-dioxanonanoic acid, Ahdna; (2S)-2-amino-6-hydroxy-4-oxahexanoic acid, Ahoha; (-)- (2R)-2-amino-3-(2-hydroxy ethylsulfonyl) propanoic acid, Ahsopa; (-)-(2R)-2-amino-3-(2- hydroxy ethylsulfanyl) propanoic acid, Ahspa; (2S)-2-amino-12-hydroxy-4,7,10- trioxadodecanoic acid, Ahtda; (2S)-2,9-diamino-4,7-dioxanonanoic acid, Dadna; (2S)-2,12- diamino-4,7,10-trioxadodecanoic acid, Datda; (S)-5,5-difluoronorleucine, Dfnl; (S)-4,4- difluoronorvaline, Dfnv; (3R)-l-l-dioxo-[l,4]thiaziane-3-carboxylic acid, Dtca; (S)- 4,4,5,5.6.6.6-heptafluoronorleucine. Hfhl; (S)-5,5,6,6.6-pentafluoronorleucine. Pfhl; (S)- 4,4,5,5,5-pentafluoronorvaline, Pfnv; and (3R)-l ,4-thiazinane-3-carboxylic acid, Tea. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary). For a review of classical and non-classical 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 87 amino acids. J Med Chem 41(14): pp. 2481-91).

[0108] In some embodiments the present invention provides mutant RSV F molecules that comprise at least one artificially introduced DT crosslink. Such DT crosslinks serve to stabilize the mutant RSV F molecules described herein in their pre-fusion conformation.

[0109] In some embodiments such DT crosslinks may be introduced between two endogenous tyrosine residues, between two artificially introduced tyrosine residues (i.e. originating from ■‘to-tyrosine” mutations), or between an artificially-introduced tyrosine residue and an endogenous tyrosine residue.

[0110] In some preferred embodiments at least one tyrosine of the at least one DT crosslink derives from (i.e. was introduced by) a point-mutation to tyrosine.

[0111] In some embodiments the present invention provides a mutant trimeric RSV F molecule comprising at least one, or at least two, or at least three, or at least four, or at least five, or at least six DT crosslinks, wherein at least one tyrosine of each DT crosslinks derives from (i.e. was introduced by) a point-mutation to tyrosine. In some embodiments the present invention provides compositions (such as vaccine compositions) comprising mutant trimeric RSV F molecules wherein the mutant trimeric RSV F molecules comprise populations of RSV molecules having differing numbers of DT crosslinks per trimer (i.e., the compositions comprise a distribution of species of RSV F molecules having varying levels of DT crosslinking).

[0112] For example, in some embodiments, the present invention provides compositions (such as vaccine compositions) comprising a population of RSV molecules having at least one DT crosslink, and / or a population of RSV molecules having at least two DT crosslinks, and / or a population of RSV molecules having at least three DT crosslinks, and / or a population of RSV molecules having at least four DT crosslinks, and / or a population of RSV molecules having at least five DT crosslinks, and / or a population of RSV molecules having at least six DT crosslinks, wherein at least one tyrosine of each of each DT crosslink derives from (i.e. was introduced by) a point-mutation to tyrosine.

[0113] In some embodiments, the present invention provides compositions (such as vaccine compositions) comprising populations of RSV molecules having differing numbers of DT crosslinks per trimer, wherein the average number of DT crosslinks per trimer in the RSV molecules in the composition is 1.

[0114] In some embodiments, the present invention provides compositions (such as vaccine compositions) comprising populations of RSV molecules having differing numbers of DT crosslinks per trimer, wherein the average number of DT crosslinks per trimer in the RSV molecules in the composition is 2.

[0115] In some embodiments, the present invention provides compositions (such as vaccine compositions) comprising populations of RSV molecules having differing numbers of DT crosslinks per trimer, wherein the average number of DT crosslinks per trimer in the RSV molecules in the composition is 3.

[0116] In some embodiments, the present invention provides compositions (such as vaccine compositions) comprising populations of RSV molecules having differing numbers of DT crosslinks per trimer, wherein the average number of DT crosslinks per trimer in the RSV molecules in the composition is 4. In some embodiments, the present invention provides compositions (such as vaccine compositions) comprising populations of RSV molecules having differing numbers of DT crosslinks per trimer, wherein the average number of DT crosslinks per trimer in the RSV molecules in the composition is 5.

[0117] In some embodiments, the present invention provides compositions (such as vaccine compositions) comprising populations of RSV molecules having differing numbers of DT crosslinks per trimer, wherein the average number of DT crosslinks per trimer in the RSV molecules in the composition is 6.

[0118] In preferred embodiments, the present invention provides compositions (such as vaccine compositions) comprising populations of RSV molecules having differing numbers of DT crosslinks per trimer, wherein the average number of DT crosslinks per trimer in the RSV molecules in the composition is from about 4 to about 5.

[0119] For example, in some embodiments the present 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. resulting from a 226Y mutation) -i.e. an intramolecular 198Y-226Y DT crosslink. In embodiments where the mutant RSV F molecule is a trimer, the trimer may comprise one, two, or three of such intramolecular 198Y- 226Y DT crosslinks.

[0120] In some embodiments the present invention provides mutant RSV F molecules comprising a DT crosslink between an introduced tyrosine at residuel85 (i.e. resulting from a!85Y mutation) and an introduced tyrosine at residue 428 (i.e. resulting from a 428Y mutation) - i.e. a 185Y-428Y intermolecular DT crosslink. In embodiments where the mutant RSV F molecule is a trimer, the trimer may comprise one, two or three of such intermolecular 185Y- 428Y DT crosslinks.

[0121] And in some embodiments the present invention provides mutant RSV F molecules comprising both at least one 198Y-226Y intramolecular DT crosslink and at least one 185Y- 428Y intermolecular DT crosslink. In such embodiments where the mutant RSV F molecule is a trimer, the trimer thus comprises from two to six DT crosslinks - i.e. from one to three intramolecular 198Y-226Y DT crosslinks and from one to three intermolecular 185Y-428Y DT crosslinks. As described above, each protomer of the mature RSV F trimer comprises two distinct polypeptides - termed Fl and F2 - which associate non-covalently to form a protomer. A bond between a Fl polypeptide and a F2 polypeptide within the same protomer is an example of an inter-molecular bond and an intra-protomer bond. The 185Y-428Y DT crosslink is designed to hold two protomers of the trimer together - i.e. it is an inter-molecular or interprotomer bond. The tyrosine at position 185 on one protomer forms a di-tyrosine bond with a tyrosine at position 428 on a different protomer.

[0122] Exemplary methods of performing a DT crosslinking reaction are provided in the Examples section of this patent disclosure. Furthermore, methods of performing DT crosslinking are known in the art and are described in, for example, Marshall et al. US Patent Nos. 7,037,894, 7,445,912, 10,125,172, 11,267,848, and 11,926,649 - the contents of which are hereby incorporated by reference. Di-tyrosine crosslinking introduces covalent carbon-carbon bonds that are minimally altering and zero-length. DT crosslinks are not hydrolyzed under physiological conditions. Di-tyrosine crosslinks are known to be safe, as they form naturally in vivo, and as they are present in large quantities in common foods. For example, DT bonds form the structure of wheat gluten. Di-tyrosine bonds do not form spontaneously in vitro. Rather, an enzymatic crosslinking reaction must be performed in which proteins with tyrosyl side chains are subjected to reaction conditions that lead to the formation of DT bonds. Such conditions are, or become, oxidative reaction conditions, as the DT bond formation reaction is an oxidative crosslinking reaction. In some embodiments the DT crosslinking reaction conditions yield proteins that are otherwise not, or not detectably, modified. Such conditions may be obtained by use of enzymes that catalyze the formation of H2O2, such as peroxidases. DT bond formation may be monitored by spectrophotometry with an excitation wavelength of around 320 nm, and fluorescence measured at a wavelength of around 400 nm, and loss of ty rosyl fluorescence may be monitored by standard procedures. When loss of tyrosyl florescence is no longer stoichiometric with DT bond formation, the reaction may be stopped by any methods known to one skilled in the art, such as, for example, by the addition of a reducing agent and subsequent cooling (on ice) or freezing of the sample.

[0123] In some aspects, the present invention provides RSV F molecules, and compositions (such as vaccine compositions) comprising populations of RSV F molecules, where not all of the ty rosines residues that could theoretically be linked with a DT crosslink (e.g., based on their structural proximity to one another) are connected. The degree of DT crosslinking can be controlled by adjusting one or more of the DT crosslinking reaction conditions. For example, in some embodiments, the degree of DT crosslinking can be controlled by adjusting the total amount or concentration of the catalyst (e.g. peroxidase enzyme (e.g. Arthromyces ramosus peroxidase) or Nickel-peptide complex) used, the total amount or concentration of the oxidant (e.g.. hydrogen peroxide or or magnesium monoperoxyphthalate) used, the reaction temperature, and / or the reaction time. For example, in some embodiments the DT crosslinking reaction is conducted as described in Example 1 and is allowed to proceed for 5- 50 minutes. In some embodiments the DT crosslinking reaction is conducted as described in Example 1 and is allowed to proceed for about 5 minutes, or about 10 minutes, or about 15 minutes, or about 20 minutes, or about 25 minutes, or about 30 minutes, or about 35 minutes, or about 40 minutes, or about 45 minutes, or about 50 minutes. Similarly, in some embodiments the DT crosslinking reaction is conducted as described in Example 1 and is allowed to proceed for 5-10 minutes, or 10-15 minutes, or 15-20 minutes, or 20-25 minutes, or 25-30 minutes, or 30-35 minutes, or 35-40 minutes, or 40-45 minutes, or 45-50 minutes. Similarly, in some embodiments the DT crosslinking reaction is conducted as described in Example 1 and is allowed to proceed for 5-15 minutes, or 15-25 minutes, or 25-35 minutes, or 35-45 minutes, or 45-55 minutes. The degree of DT cross liking achieved can be assessed using one or more of the assays described in Example 1 (such as the SDS PAGE gel shift, DT fluorescence, mass spectrometry and / or HPLC assays described therein) or known in the art.

[0124] The mutant RSV F molecules of the present invention - including those comprising an amino acid sequence of one of the exemplary amino acid sequences provided herein (e.g. SEQ ID Nos. 21-81), and those comprising residues 26-109 and / or 137-513 of one of such exemplary amino acid sequences, and those comprising an Fl and / or F2 polypeptide of one of such exemplary amino acid sequences, and variant forms of such specific amino sequences - should: (a) comprise an Fl polypeptide and an F2 polypeptide, and (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 pre-fusion conformation (as described / defined herein). The mutant RSV F molecules of the present invention may, in some embodiments, also have one or more of the following properties: (1) binding to a pre-F specific antibody, (2) binding to an antibody that binds to site o, (3) binding to a neutralizing antibody, (4) binding to a broadly neutralizing antibody, (5) binding to an antibody selected from the group consisting of D25, AM22, 5C4, 10 IF, (6) binding to palivizumab (Synagis), (7) binding to and / or activating a B cell receptor, (8) eliciting an anti-RSV antibody response in an animal, (9) eliciting a protective anti-RSV antibody response in an animal, (10) eliciting production of anti-RSV neutralizing antibodies in an animal, (11) eliciting production of anti-RSV broadly neutralizing antibodies in an animal, (12) eliciting production of anti-RSV antibodies that recognize quaternary neutralizing epitopes (QNEs) in an animal, and / or (13) eliciting an anti-RSV protective immune response in an animal.

[0125] Nucleic Acid Molecules

[0126] In some embodiments the present invention provides nucleic acid molecules that encode the mutant RSV F molecules described herein, as well vectors comprising such nucleic acid molecules. One of ordinary7skill in the art can readily determine the nucleic acid sequence of a nucleic acid molecule that encodes any one of the mutant RSV F molecules described herein - given the universally known and understood nature of the genetic code amongst those of ordinary skill in the art.

[0127] 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 may encode one of the precursor (F0) polypeptides for which the amino acid sequences are provided in Table 1 and in the Sequence Listing. In some embodiments, the nucleic acid molecules may encode only a F2 polypeptide or only a Fl polypeptide.

[0128] Nucleic acid molecules that encode the mutant RSV F molecules described herein can be obtained or made using any suitable method known in the art. For example, nucleic acid molecules encoding the mutant RSV F molecules may be obtained from cloned DNA or made by chemical synthesis. In some embodiments the nucleic acid molecules may be obtained by reverse transcribing RNA prepared by any of the methods known to one of ordinary7skill in the art. Point mutations, or any of the other modifications described herein (e.g. removing C-terminal sequences, substituting C-terminal sequences, etc.), can be made by standard recombinant DNA methodologies that are well known and understood to those of ordinary skill in the art. For example, one of ordinary skill in the art can readily make a “to- tyrosine” mutation as described herein by locating the nucleotide codon that encodes the specific amino acid residue to be mutated to tyrosine (e.g. that encodes amino acid residue 185, 226. or 428), and mutating the nucleotides of that codon as necessary to generate a tyrosine-encoding codon.

[0129] Whatever the source, a nucleic acid molecule encoding a mutant RSV F molecule of the present invention can be cloned into any suitable vector, such as those to be used for propagation of the nucleic acid molecule or those to be used for expression of the nucleic acid molecule. In embodiments requiring expression, the nucleic acid can be operatively linked to a promoter suitable for directing expression in the desired cell ty pe, such as a mammalian cell or an insect cell, and may be incorporated into any suitable expression vector, such as a mammalian or insect expression vector.

[0130] In some embodiments nucleic acid molecules encoding the mutant RSV F molecules of the present invention can be codon optimized for expression in cells of a particular organism or species. For example. International Patent Application No. PCT / US2014 / 048086 provides nucleotide sequences of RSV F molecules that have been codon-optimized for expression in human, hamster, mouse, and insect cells. Such codon-optimized nucleotide sequences encoding the RSV F protein can be used as ‘‘background’' sequences for introduction of any of the specific mutations described herein.

[0131] Methods of Manufacture

[0132] The mutant RSV F molecules of the invention can be made by any suitable means known in the art. Generally, the mutant RSV F molecules are made using standard methods used for the production of recombinant proteins. For example, nucleic acid molecules encoding a mutant RSV F molecule of the 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, e.g. using a baculovirus expression system). Methods for expressing proteins from nucleic acid molecules are routine and well known in the art, and any suitable methods, vectors, systems, and cell ty pes known in the art can be used. For example, ty pically nucleic acid molecules encoding the mutant RSV F molecules of the invention will be placed into a suitable expression construct containing a suitable promoter, which will then be delivered to cells for expression. In some embodiments the mutant RSV F molecules of the invention are mature RSV F trimers stabilized in the pre-F conformation. In such embodiments typically nucleic acid molecules encoding the mutant RSV F molecules are expressed in cells in soluble form, and then allowed to assemble into the normal trimeric pre-F conformation before subjecting the molecules to the enzymatic DT crosslinking reaction. In some embodiments, prior to and / or during the enzy matic crosslinking reaction, the mutant RSV F molecules may be obtained in (and / or maintained in) the pre-F conformation, for example while crosslinking is performed. In some embodiments the mutant RSV F molecules may be produced and / or isolated in such a way that most, or substantially all, of the mutant RSV F molecules are present in the pre-F conformation. In some embodiments mutant RSV F molecules in the pre-F conformation may be separated from a mixed population of RSV F protein molecules comprising some that are in the pre-F conformation and some that are in other conformations. In some embodiments, the RSV F protein is expressed in cells (for example as its membrane bound or soluble form) and spontaneously assembles into its normal pre-F conformation. In some embodiments no additional stabilization is necessary to retain the mutant RSV F molecule in its pre-F form prior to DT crosslinking. In some embodiments the mutant RSV F molecule may be kept under particular conditions, or in particular compositions, that favor formation and / or maintenance of the pre-F conformation. For example, in some embodiments the mutant RSVF molecule in its pre-F conformation may be maintained in the absence of cells - contact with which might otherwise trigger a switch to the post-F conformation. The mutant RSV F molecules may be obtained and / or isolated 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 methods. In some embodiments the mutant RSV F molecules may be expressed in the presence of, co-expressed with, or contacted with, molecules that bind to the RSV F protein and stabilize it in its pre-F conformation, including, but not limited to, antibodies, small molecules, peptides, and / or peptidomimetics. Nonlimiting examples of antibodies that bind to the pre-fusion RSV F protein include the 5C4, AM22, and D25 antibodies (see McLellan et al. (2013) Science 342:592-598, which is hereby incorporated by reference in its entirety). In some embodiments, the mutant RSV F molecule may be obtained, isolated, or maintained in its pre-F conformation by controlling the ionic strength of the media / buffer in which the protein is present (such as by using high or low ionic strength media). In some embodiments the mutant RSV F molecules may be obtained. isolated, or maintained at one or more temperatures that favor preservation of the pre-F conformation. In some embodiments the mutant RSV F molecules may be obtained, isolated, or maintained over a period of time that diminishes the degree to which the pre-F conformation is lost.

[0133] In some embodiments analysis may be performed to confirm that the desired conformation, such as the pre-F conformation, has been formed and / or maintained in the mutant RSV F molecules. Such analysis may be performed prior to crosslinking, during the crosslinking process, after the crosslinking process, or at any combination of such stages. Such analysis may comprise any suitable methods known in the art for assessing the 3-dimensional structure of a protein or protein complex, including functional analysis, crystallographic analysis, and the like. In some embodiments such analysis may include assessing binding of the mutant RSV F molecules to certain antibodies, such as those that are specific to the pre-F conformation and / or those that are known to bind to the o site, as described elsewhere herein, including, but not limited to the 5C4, AM22, and D25 antibodies.

[0134] In some embodiments the mutant RSV F molecules of the invention may be purified before, during, or after, one or more steps in the manufacturing process. In some embodiments the mutant RSV F molecules may be purified before commencing the crosslinking process, or after one or more of the intermediate method steps in the process, for example: after expression of the mutant RSV F molecule, after assembly of the mutant RSV F molecule into a mature trimer, after obtaining the mutant RSV F molecule in its pre-F conformation, or during or after performing a DT crosslinking reaction. The mutant RSV F molecules of the invention may be isolated or purified using any suitable method known in the art. Such methods include, but are not limited to, chromatography (e.g. ion exchange, affinity, and / or sizing column chromatography), ammonium sulfate precipitation, centrifugation, differential solubility, or by any other technique for the purification of proteins known to one of ordinary skill in the art. In specific embodiments it may be necessary to separate DT crosslinked mature tri meric mutant RSV F molecules from those that were not sufficiently crosslinked, or those in which the pre-F conformation was not sufficiently stabilized. This can be done using any suitable system known in the art. For example, mutant RSV F molecules in the pre-F conformation can be separated from those that are not in the pre-F conformation using antibody-based separation methods using pre-F or post-F specific antibodies. The mutant RSV F molecules of the invention may be purified from any source used to produce them. The degree of purity may vary, but in various embodiments, the purified mutant RSV F molecules of the invention are provided in a form in which is they comprise more than about 10%, 20%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.9% of the total protein in the final composition. In some embodiments the mutant RSV F molecules of the invention may be isolated and purified from other proteins, or any other undesirable products (such as non-crosslinked or non-pre-F RSV F), by standard methods including, but not limited to, chromatography, glycerol gradients, affinity chromatography, centrifugation, ion exchange chromatography, size exclusion chromatography, and affinity' chromatography, or by any other standard technique for the purification of proteins known in the art. The mutant RSV F molecules to be isolated may be expressed in high or low ionic media, or isolated in high or low ionic buffers or solutions. The mutant RSV F molecules of the invention may also be isolated at one or more temperatures that favor preservation of the desired conformation. They may also be isolated over a period of time that diminishes the degree to which a preparation would have lost the desired conformation. The degree to which a preparation of proteins retains one or more desired conformations (such as the pre-F conformation) may be assayed by any suitable method known in the art, including, for example, but not limited to, biochemical, biophysical, immunologic, and virologic analyses. Such assays include, for example, but are not limited to, immunoprecipitation, enzyme-linked immunosorbent assays (ELIS As), or enzyme-linked immunosorbent spot (ELISPOT) assays, crystallographic analysis (including co-crystallization with antibodies), sedimentation, analytical ultracentrifugation, dynamic light scattering (DLS), electron microscopy (EM), cryo-EM tomography, calorimetry, surface plasmon resonance (SPR), fluorescence resonance energy transfer (FRET), circular dichroism analysis, and small angle x-ray scattering, neutralization assays, antibody-dependent cellular cytotoxicity assays, and / or virologic challenge studies in vivo.

[0135] The yield of the mutant RSV F molecules of the invention can be determined by any means know n in the art, for example, by comparing the amount of the final engineered proteins (such as crosslinked pre-F RSV) as compared to the amount of the starting material, or as compared to the amount of the materials present in any preceding step of the production methods. Protein concentrations can be determined by standard procedures, such as. for example, Bradford or Lowrie protein assays. The Bradford assay is compatible with reducing agents and denaturing agents (Bradford, M, 1976. Anal. Biochem. 72: 248). The Lowry assay has better compatibility with detergents and the reaction is more linear with respect to protein concentrations and read-out (Lowry, O J, 1951. Biol. Chem. 193: 265).

[0136] Assays for Properties

[0137] In some embodiments the mutant RSV F molecules of the invention, or any intermediates in their manufacture, may 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 assays can be performed to assess the RSV F protein’s conformational structure, stability (e.g. thermostability), half-life (e.g. inside the body of a subject), aggregation in solution, binding to an antibody (such as a neutralizing antibody, broadly neutralizing antibody; pre-F-specific antibody; antibody that recognizes site o, conformationally-specific antibody, antibody that recognizes a metastable epitope, D25. AM22. 5C4, 101F or palivizumab), binding to a B cell receptor, activation of a B Cell receptor, antigenicity, immunogenicity, abi 1 i ty to elicit an antibody response, ability to elicit a protective antibody / immune response, ability to elicit production of neutralizing antibodies, or ability to elicit aproduction of broadly neutralizing antibodies. In embodiments where the mutant RSV F molecules of the invention are tested in an animal in vivo, the animal may be any suitable animal species, including, but not limited to a mammal (such as a rodent species (e.g. a mouse or rat), a rabbit, a ferret, a porcine species, a bovine species, an equine species, an ovine species, or a primate species (e.g. a human or a non-human primate), or an avian species (such as a chicken).

[0138] Assays for assessing a protein’s conformational structure are well known in the art and any suitable assay can be used, including, but not limited to. crystallographic analysis (e.g. X-ray crystallography or electron crystallography), sedimentation analysis, analytical ultracentrifugation, electron microscopy (EM), cryo-electron microscopy (cryo-EM). cryo- EM tomography, nuclear magnetic resonance (NMR), small angle x-ray scattering, fluorescence resonance energy transfer (FRET) assays, and the like.

[0139] Assays for assessing a protein’s stability are well known in the art and any suitable assay can be used, including, but not limited to, denaturing and non-denaturing electrophoresis, isothermal titration calorimetry, and time-course experiments in which proteins are incubated and analyzed over time at varying protein concentrations, temperatures, pHs or redox conditions. Proteins may also be analyzed for susceptibility to proteolytic degradation.

[0140] Assays for assessing binding of proteins to antibodies are well know n in the art, and any suitable assay can be used, including, but not limited to, immunoprecipation assays, enzyme- linked immunosorbent assays (ELISAs), enzyme-linked immunosorbent spot assays (ELISPOTs), crystallographic assays (including co-crystallization with antibodies), surface plasmon resonance (SPR) assays, fluorescence resonance energy transfer (FRET) assays, and the like.

[0141] Assays for assessing neutralization activity are well known in the art, and any suitable assay can be used. For example, assays can be performed to determine the neutralizing activity of antibodies or antisera generated by vaccination / immunization of animals with the RSV F polypeptides, proteins, and / or protein complexes of the 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 gpl20 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 gpl40, trimeric cleavagedefective gpl40 and momomeric gpl20. Virol 360: 329-340).

[0142] Assays for assessing whether a vaccine immunogen is capable of eliciting an immune response and / or proving protective immunity are well known in the art, and any suitable assay can be used. For example, assays can be performed to determine whether vaccination / immunization of animals with the RSV F polypeptides, proteins, and / or protein complexes of the invention provide an immune response and / or protective immunity against infection with RSV. In some embodiments comparisons may be made between placebo and test vaccinated groups with regard to their rates of infection or sero-conversion or viral loads.

[0143] Assays for assessing a protein's pharmacokinetics and bio-distribution are also well known in the art. and any suitable assay can be used to assess these properties of the the RSV F polypeptides, proteins, and / or protein complexes of the invention.

[0144] Compositions In some embodiments the present invention provides compositions comprising one or more of the mutant RSV F molecules described herein, or a population of RSV molecules as described herein. In some embodiments such compositions may be vaccine compositions - i.e., compositions comprising the RSV F molecules or populations of RSV molecule described herein, and optionally one or more components that are suitable for administration to living subjects, in a form suitable for administration to a subject, or in a form suitable for reconstitution and subsequent administration to a subject. In some embodiments the vaccine composition is in liquid form. In some embodiments the vaccine composition is in lyophilized form.

[0145] In some embodiments the mutant RSV F molecules of the invention may be provided in a vaccine composition that comprises one or more additional active components, such as one or more additional vaccine immunogens or therapeutic agents. In some embodiments the mutant RSV F molecules of the invention may be provided in a composition, such as a vaccine composition, that comprises one or more other components, including, but not limited to, pharmaceutically acceptable carriers, adjuvants, immunostimulatory agents, wetting or emulsifying agents, pH buffering agents, preservatives, and / or any other components suitable for the intended use of the compositions. Such compositions can take the form of solutions, suspensions, emulsions and the like. The term "pharmaceutically acceptable carrier" includes various diluents, excipients and / or vehicles in which, or with which, the mutant RSV F molecules of the invention can be provided and includes, but is not limited to, carriers known to be safe for delivery to human and / or other animal subjects, and / or approved by a regulatory agency of the Federal or a state government, and / or listed in the U.S. Pharmacopeia, and / or other generally recognized pharmacopeia, and / or receiving specific or individual approval from one or more generally recognized regulator}' agencies for use in humans and / or other animals. Such pharmaceutically acceptable carriers, include, but are not limited to, water, aqueous solutions (such as saline solutions, buffers, and the like), organic solvents (such as certain alcohols and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil), and the like. In some embodiments the compositions of the invention also comprise one or more adjuvants. Exemplary adjuvants include, but are not limited to, alum adjuvants, inorganic or organic adjuvants, oil-based adjuvants, virosomes, liposomes, lipopolysaccharide (LPS). molecular cages for antigens (such as immune-stimulating complexes ("ISCOMS”)), Ag- modified saponin / cholesterol micelles that form stable cage-like structures that are transported to the draining lymph nodes), components of bacterial cell walls, endocytosed nucleic acids (such as double-stranded RNA (dsRNA), single-stranded DNA (ssDNA), and unmethylated CpG dinucleotide-containing DNA), AUM, aluminum phosphate, aluminum hydroxide, and Squalene. In some embodiments virosomes are used as the adjuvant. Additional commercially available adjuvants that can be used in accordance with the present invention include, but are not limited to, the 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 water-in-oil adjuvant (CytRx Corporation 150 Technology Parkway Technology Park / Atlanta Norcross, Georgia 30092)), Syntex Adjuvant Formulation (SAF. an oil-in-water emulsion stabilized by Tween 80 and pluronic polyoxyethlene / polyoxypropylene block copolymer LI 21 (Chiron Corporation, Emeryville, CA)), Freund’s Complete Adjuvant, Freund's Incomplete Adjuvant, ALUM - aluminum hydroxide, A1(OH)3 (available as Alhydrogel, Accurate Chemical & Scientific Co, Westbury. NY). SuperCarrier (Syntex Research 3401 Hillview Ave. P.O. Box 10850 Palo Alto, CA 94303), Elvax 40Wl,2(an ethylene-vinyl acetate copolymer (DuPont Chemical Co. Wilmington, DE)), L-tyrosine co-precipitated with the antigen (available from numerous chemical companies); Montanide (a manide-oleate, ISA Seppic Fairfield, NJ)), AdjuPrime (a carbohydrate polymer), Nitrocellulose-absorbed protein, Gerbu adjuvant (C-C Biotech, Poway, CA), and the like.

[0146] In some embodiments the mutant RSV F molecules of the invention may be provided in a composition that comprises a sugar. In some embodiments the RSV F polypeptides, proteins, and / or protein complexes of the invention may be provided in a composition that comprises sucrose. In some embodiments the RSV F polypeptides, proteins, and / or protein complexes of the invention may be provided in a composition that comprises about 5% sucrose. In some embodiments the RSV F polypeptides, proteins, and / or protein complexes of the invention may be provided in a composition that comprises about 10% sucrose. In some embodiments the RSV F polypeptides, proteins, and / or protein complexes of the invention may be provided in a composition that comprises about 15% sucrose. In some embodiments the RSV F polypeptides, proteins, and / or protein complexes of the invention may be provided in a composition that comprises about 20% sucrose. In some embodiments the RSV F polypeptides, proteins, and / or protein complexes of the invention may be provided in a composition that comprises about 25% sucrose. Importantly, it has been found that inclusion of sucrose reduces the formation of aggregates in compositions of the RSV F polypeptides, proteins, and / or protein complexes of the invention.

[0147] In some embodiments the present invention provides compositions that comprise an “effective amount” of a mutant RSV F molecule of the invention. Similarly, in some embodiments the present invention provides methods that involve administering an “effective amount” of a mutant RSV F molecule, or a composition comprising a mutant RSV F, to a subject.

[0148] Uses of Mutant RSV F Molecules

[0149] In some embodiments, the mutant RSV F molecules of the invention may be useful as research tools, as diagnostic tools, as therapeutic agents, as targets for the production of antibody reagents or therapeutic antibodies, and / or as vaccines or components of vaccine compositions. For example, in some embodiments the mutant RSV F molecules of the invention are useful as vaccine immunogens in animal subjects, such as mammalian subjects, including humans. These and other uses of the mutant RSV F molecules of the invention are described more fully below. Those of skill in the art will appreciate that the mutant RSV F molecules of the invention may be useful for a variety of other applications also, and all such applications and uses are intended to fall within the scope of this invention.

[0150] Tools for Studying RSV F Antibodies

[0151] In one embodiment, the mutant RSV F molecules of the invention may be useful as analytes for assaying and / or measuring binding of, and / or titers of. anti-RSV F antibodies, for example in ELISA assays, Biacore / SPR binding assays, and / or any other assays for antibody binding known in the art. For example, the mutant RSV F molecules of the invention could be used to analyze, and / or compare the efficacy of anti-RSV F antibodies.

[0152] Tools for Generation of Antibodies

[0153] The mutant RSV F molecules of the invention may also be useful for the generation of therapeutic antibodies and / or antibodies that can be used as research tools or for any other desired use. For example, the mutant RSV F molecules of the invention can be used for immunizations to obtain antibodies to the RSV F protein for use as research tools and / or as therapeutics. In some embodiments the mutant RSV F molecules of the invention can be used to immunize a non-human animal, such as a vertebrate, including, but not limited to, a mouse, rat, guinea pig, rabbit, goat, non-human primate, etc. to generate antibodies. Such antibodies, which may be monoclonal or polyclonal, and / or cells that produce such antibodies, can then be obtained from the animal. For example, in some embodiments mutant RSV F molecules of the invention may be used to immunize a mouse and to produce and obtain monoclonal antibodies, and / or hybridomas that produce such monoclonal antibodies. Such methods can be carried out using standard methods known in the art for the production of mouse monoclonal antibodies, including standard methods for hybridoma production. In some embodiments mutant RSV F molecules of the invention may be used for the production of a chimeric (e.g. part-human), humanized, or fully-human antibody, for example using any of the methods currently known in the art for production of chimeric, humanized and fully human antibodies, including, but not limited to, CDR grafting methods, phage-display methods, transgenic mouse methods (e.g. using a mouse that has been genetically altered to allow for the production of fully human antibodies, such as the Xenomouse) and / or any other suitable method known in the art. Antibodies to the mutant RSV F molecules of the invention made using such systems can be characterized antigenically using one or a set of several antigens, preferably including the mutant RSV F molecules of the invention themselves. Additional characterization of such antibodies may be carried out by any standard methods known to one 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, iso-electric point determination), and methods know n in the art for studying biodistribution, safety, and efficacy of antibodies - for example in preclinical and clinical studies.

[0154] Administration to Subjects & Vaccination Methods

[0155] In some embodiments, the present invention provides methods that comprise administering the mutant RSV F molecules of the invention, or vaccine compositions comprising such mutant RSV F molecules, to subjects. Such methods may comprise methods for treating individuals having RSV (i.e. therapeutic methods) and / or methods for protecting individuals against future RSV infection (i.e. prophylactic / vaccination methods). For example, in some embodiments the present invention provides a method of vaccinating a subject against RSV, the method comprising administering to the subject an RSV F molecule as described herein, or a vaccine composition as described herein.

[0156] Subjects to which the mutant RSV F molecules of the invention, or compositions comprising such RSV F molecules, can be administered (for example in the course of a method of treatment or a method of vaccination) include any and all animal species, including, in particular, those that are susceptible to RSV infection or that can provide model animal systems for the study of RSV infection. In some embodiments, the subjects are mammalian species. Mammalian subjects include, but are not limited to, humans, non-human primates; members of the artiodactyla order, members of the 46 ovidae family, members of the bovinae subfamily, medium to large-sized ungulates, cattle, bison, African buffalo, and water buffalos; rodents; cotton rats; rabbits; and ferrets.

[0157] In some embodiments the subjects to which the mutant RSV F molecules of the invention, or compositions comprising such mutant RSV F molecules are administered, either have RSV, or are at risk of RSV infection.

[0158] In preferred embodiments the subjects are human. In some such embodiments, the human subjects are immuno-compromised. In some such embodiments, the human subjects are immunosenescent. In some such embodiments, the human subjects are frail. In some such embodiments, the human subjects have a heart disease, or a heart disorder. In some such embodiments, the human subjects have asthma. In some such embodiments, the human subjects have diabetes. In some embodiments, the subjects have COPD. In some embodiments, the subjects have chronic heart failure. In some embodiments, the subjects have advanced liver or kidney disease. In some embodiments, the subjects have chronic respiratory disease or a chronic pulmonary disease.

[0159] In some embodiments, the subject is a human of greater than about 50 years in age, or greater than about 55 years in age, or greater than about 60 years in age, or greater than about 65 years in age, or greater than about 70 years in age, or greater than about 75 years in age, or greater than about 80 years in age, or greater than about 85 years in age, or greater than about 90 years in age.

[0160] In some embodiments, the subject is a human of less than about 1 day in age, or less than about 2 days in age, or less than about 3 days in age, or less than about 5 days in age, or less than about 10 days in age, or less than about 15 days in age, or less than about 20 days in age, or less than about 25 days in age, or less than about 1 month in age. or less than about 2 months in age, or less than about 3 months in age, or less than about 4 months in age, or less than about 5 months in age, or less than about 6 months in age, or less than about 7 months in age, or less than about 8 months in age, or less than about 9 months in age, or less than about 10 months in age, or less than about 11 months in age, or less than about 12 months in age, or less than about 13 months in age, or less than about 14 months in age, or less than about 15 months in age, or less than about 16 months in age, or less than about 17 months in age, or less than about 18 months in age, or less than about 19 months in age, or less than about 20 months in age, or less than about 21 months in age, or less than about 22 months in age, or less than about 23 months in age, or less than about 24 months in age, or less than about 3 years in age, or less than about 4 years in age, or less than about 5 years in age, or less than about 6 years in age, or less than about 7 years in age, or less than about 8 years in age, or less than about 9 years in age, or less than about 10 years in age, or less than about 11 years in age, or less than about 12 years in age.

[0161] Typically, the amount of the RSV F molecule or vaccine composition administered according to the methods of the present invention is an “effective amount.”

[0162] In some embodiments the effective amount is an amount effective to prevent lung and lower airway infection from RSV in the subject. In some embodiments the effective amount is an amount effective to prevent lower respiratory tract disease (LRDT) caused by RSV infection in the subject.

[0163] In some embodiments, the amount of the RSV F molecule administered to a human subject is about 1 microgram (mcg), or about 2 micrograms (mcgs), or about 3 mcgs, or about 5 mcgs, or about 10 mcgs, or about 20 mcgs, or about 30 mcgs, or about 40 mcgs, or about 50 mcgs, or about 60 mcgs, or about 70 mcgs, or about 80 mcgs, or about 90 mcgs, or about 100 mcgs, or about 110 mcgs, or about 120 mcgs, or about 130 mcgs, or about 140 mcgs, or about 150 mcgs, or about 160 mcgs, or about 170 mcgs, or about 180 mcgs, or about 190 mcgs, or about 200 mcgs, or about 225 mcgs, or about 250 mcgs, or about 275 mcgs, or about 300 mcgs, or about 350 mcgs, or about 400 mcgs, or about 500 mcgs, or about 600 mcgs, or about 750 mcgs, or about 1 milligram, or about 2 milligrams. One important finding of the present invention, as described in Example 1 , is that, unlike the RSV vaccines licensed to date, very good levels of protection can be achieved in elderly subjects (as used herein the term “elderly” when used with reference to human subjects refers to subjects of greater than about 70 years in age, particularly when the amount of the RSV F molecule administered is increased to about 4.5 times the dose that is effective to provide protection in younger adult subjects. Thus, in some embodiments, the amount of the RSV F molecule administered to an elderly human subject is about 20 micrograms (mcgs), or about 40 mcgs, or about 80 mcgs, or about 120 mcgs, or about 160 mcgs, or about 200 mcgs, or about 240 mcgs, or about 280 mcgs, or about 320 mcgs, or about 360 mcgs, or about 400 mcgs, or about 440 mcgs, or about 480 mcgs, or about 520 mcgs, or about 560 mcgs, or about 600 mcgs, or about 640 mcgs, or about 680 mcgs, or about 720 mcgs, or about 760 mcgs, or about 800 mcgs, or about 840 mcgs, or about 880 mcgs, or about 920 mcgs, or about 960 mcgs, or about 1 milligram, or about 1.1 milligrams (mgs), or about 1.2 mgs, or about 1.3mgs, or about 1.5mgs, or about 2mgs, or about 3mgs, or about 5mgs, or about lOmgs.

[0164] Various delivery routes and delivery systems are known in the art and any suitable delivery route or delivery systems can be used to administer the compositions (e.g. vaccine compositions) of the present invention to subjects. Such delivery' routes and systems include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral delivery routes and systems. The compositions of the present invention may be administered by any convenient route or system, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. Pulmonary administration can also be employed, e.g, by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. In preferred embodiments the compositions (e.g. vaccine compositions) of the present invention are administered to human subjects by intramuscular injection.

[0165] In some embodiments it may be desirable to administer the compositions (e.g.. vaccine compositions) of the invention locally to a tissue in which the mutant RSV F molecules may be most effective in generating a desirable outcome. This may be achieved by, for example, local infusion, injection, delivery' using a catheter, or by means of an implant, such as a porous, non-porous, or gelatinous implant or an implant comprising one or more membranes (such as sialastic membranes) or fibers from or through which the protein or protein complexes may be released locally. In some embodiments a controlled release system may be used. In some embodiments a pump may be used (see Langer, supra, Sefton, 1987. CRC Crit. Ref. Biomed. Eng. 14: 201 ; Buchwald et al. , 1980. Surgery 88: 507; Saudek etal.,

[0166] 1989. N. Engl. J. Med. 321: 574). In some embodiments polymeric materials may be used to facilitate and / or control release of the 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 a controlled release system can be placed in proximity to 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). Some suitable controlled release systems that may be used in conjunction with the present invention are described Langer,

[0167] 1990, Science; vol. 249: pp. 527-1533.

[0168] In some embodiments, administration of the compositions (e.g., vaccine compositions) of the invention can be performed in conjunction with administration of one or more immunostimulatory agents. Non-limiting examples of such immunostimulatory agents include various cytokines, lymphokines and chemokines with immunostimulatory, immunopotentiating, and pro-inflammatory 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 immunostimulatory agents, such as macrophage inflammatory factor, Flt3 ligand. B7. 1; B7.2. The immunostimulatory agents can be administered in the same formulation as the mutant RSV F molecules, or can be administered separately.

[0169] In some embodiments, the mutant RSV F molecules of the invention, or compositions comprising them, can be administered to subjects in a variety of different RSV vaccination methods or regimens. In some such embodiments, administration of a single dose is performed. However, in other embodiments, additional dosages can be administered, by the same or different route, to achieve the desired prophylactic effect. In neonates and infants, for example, multiple administrations may be required to elicit sufficient levels of immunity. Administration can continue at intervals throughout childhood, as necessary to maintain sufficient levels of protection against RSV infection. Similarly, adults who are particularly susceptible to RSV infection, such as, for example, the elderly and immunocompromised individuals, may require multiple immunizations to establish and / or maintain protective immune responses. Levels of induced immunity can be monitored, for example, by measuring amounts of neutralizing secretory and serum antibodies, and dosages adjusted or vaccinations repeated as necessary to elicit and maintain desired levels of protection.

[0170] In some embodiments, dosing regimens may comprise a single administration / immunization. In other embodiments, dosing regimens may comprise multiple administrations / immunizations. For example, vaccines may be given as a primary immunization followed by one or more boosters. In some embodiments of the present invention such a '‘prime-boosf ’ vaccination regimen may be used. For example, in some such prime-boost regimens a composition comprising a mutant RSV F molecule as described herein may be administered to an individual on multiple occasions (such as two, three, or even more occasions) separated in time, with the first administration being the “priming’7administration and subsequent administrations being “booster” administrations. In other such prime-boost regimens a composition comprising mutant RSV F molecules as described herein may be administered to an individual after first administering to the individual a composition comprising a viral or DNA vector encoding an RSV polypeptide, protein or protein complex as a “priming” administration, with one or more subsequent “booster” administrations of a composition comprising a RSV F polypeptide, protein or protein complex as described herein. Boosters may be delivered via the same and / or different route as the primary immunization. Boosters are generally administered after a time period after the primary immunization or the previously administered booster. For example, a booster can be given about two weeks or more after a primary immunization, and / or a second booster can be given about two weeks or more after the first boosters. Boosters may be given repeatedly at time periods, for example, about two weeks or greater throughout up through the entirety of a subject's life. Boosters may be spaced, for example, about two weeks, about three w eeks, 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 more after a primary immunization or after a previous booster.

[0171] In some embodiments the vaccinecompositions of the invention may be conveniently provided in unit dosage forms. Unit dosage forms are those containing a single dose or unit (e.g. an effective amount), or an appropriate fraction thereof, of the mutant RSV F molecules of the invention. The unit dosage forms may be presented in single-dose or multi-dose containers.

[0172] 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 (e.g. water) immediately prior to use.

[0173] Kits

[0174] The present invention further provides kits comprising mutant RSV F molecules of the invention, or compositions containing such mutant RSV F molecules. To facilitate use of the methods and compositions of the invention, any of the components and / or compositions described herein, and additional components useful for experimental or therapeutic or vaccine purposes, can be packaged in the form of a kit. Typically, the kit contains, in addition to the above components, additional materials which can include, e.g., instructions for using the components, packaging material, a container, and / or a delivery device.

[0175] Table 1 - Exemplary Amino Acid Sequences

[0176] In Table 1 SEQ ID NOs 1-91 are exemplary WT and mutant RSV F amino acid sequences and SEQ ID Nos 92-96 are exemplary artificial sequences that can be introduced at the C- terminal of an RSV F molecule. For SEQ ID NOs 1-91 underlined residues are Fl and F2 polypeptides, residues in bold font are introduced mutations, residues in italics are residues that may be removed or replaced in some embodiments, and double-underlined residues are artificially introduced C-terminal sequences.

[0177] Table 2 - Summary of Exemplary RSV F Amino Acid Sequences

[0178] Additional Description

[0179] The invention may also be further described as set forth in the following numbered paragraphs.

[0180] Numbered Paragraphs Relating to Compositions

[0181] 1. A vaccine composition comprising soluble, trimeric, RSV F molecules di-tyrosine crosslinked in the pre-fusion (pre-F) conformation, wherein each monomer in the trimeric RSV F molecules comprises: an Fl domain, an F2 domain, and a trimerization domain, and wherein each monomer in the trimeric RSV F molecules comprises at least two introduced to-tyrosine mutations, and wherein the di-tyrosine crosslinks connect paired tyrosine residues in the RSV F molecules, wherein at least one tyrosine residue in each pair is an introduced to-tyrosine mutation, and wherein (a) about 25-35% of the RSV F molecules in the composition have no intermolecular di-tyrosine crosslinks, and (b) about 65-75% of the RSV F molecules in the composition have at least 2 intermolecular di-tyrosine crosslinks.

[0182] 2. A vaccine composition according to paragraph 1, wherein the average number of dityrosine crosslinks between paired tyrosine residues is 4-5 per trimer.

[0183] 3. A vaccine composition according to paragraph 1, wherein the average number of dityrosine crosslinks between paired tyrosine residues is from about 3.5 to about 4.4 per trimer.

[0184] 4. A vaccine composition according to paragraph 1, wherein the average number of dityrosine crosslinks between paired tyrosine residues is from about 4.5 to about 5.4 per trimer.

[0185] 5. A vaccine composition according to any of the preceding paragraphs, wherein each monomer in the trimeric RSVF molecule comprises a to-tyrosine mutation at two or more of amino acid residues: 77, 88, 97, 147, 150. 155, 159, 183, 185, 187, 220, 222, 223, 226. 255, 427, 428, and 469, or at or at amino acid residues that correspond to these amino acid residues as determined by alignment to, and using the amino acid numbering of, SEQ ID NO. 1. A vaccine composition according to any of the preceding paragraphs, wherein the trimeric RSVF molecule comprises di-tyrosine crosslinks between at least two of the following paired tyrosine residues: 147 and 286; 198 and 220; 198 and 222; 198 and 223; 198 and 226; 33 and 469; 77 and 222; 88 and 255; 97 and 159; 183 and 427; 185 and 427; 185 and 428; and 187 and 427, or at or at amino acid residues that correspond to these amino acid residues as determined by alignment to. and using the amino acid numbering of, SEQ ID NO. 1. A vaccine composition according to any of the preceding paragraphs, wherein the trimeric RSVF molecule comprises a tyrosine residue at each of amino acid positions 185, 198, 226 and 428. A vaccine composition according to any of the preceding paragraphs, comprising: (a) trimeric RSV F molecules that have an intermolecular di-tyrosine crosslink connecting a tyrosine at amino acid residuel85 of one monomer within the trimer with a tyrosine at amino acid residue 428 of a different monomer within the trimer, and (b) trimeric RSV F molecules that have an intramolecular di-tyrosine crosslink connecting a tyrosine at amino acid residue 226 with a tyrosine at amino acid residue 198. The vaccine composition according to paragraph 7 or paragraph 8, wherein: (a) about 25-35% of the RSV molecules in the composition have no intermolecular di-tyrosine crosslinks connecting a tyrosine at amino acid residue 185 of one monomer within the trimer with a tyrosine at amino acid residue 428 of a different monomer withing the trimer, and about 65-75% of the RSV molecules in the composition have at least 2 intermolecular di-tyrosine crosslinks, wherein the at least 2 intermolecular di-tyrosine crosslinks connect a tyrosine at amino acid residuel85 of one monomer within the trimer with a tyrosine at amino acid residue 428 of a different monomer withing the trimer. The vaccine composition of any of the preceding paragraphs, wherein each monomer in the soluble, mature, trimeric RSVF molecules comprises: (a) an F2 polypeptide consisting of amino acid residues 26-109 of SEQ ID NO 81, and (b) an Fl polypeptide consisting of amino acid residues 137-513 of SEQ ID NO. 81. 11. The vaccine composition of any of the preceding paragraphs, further comprising an adjuvant selected from the group consisting of alum, CpG, alum+CpG, and Advax.

[0186] 12. The vaccine composition of any of the preceding paragraphs, wherein the trimerization domain comprises: a foldon domain, a GCN4 domain, a T4 fibrinitin domain, a human C-propeptide of al (I) collagen, or a 6-helical bundle domain from the post-fusion form of a retroviral fusion protein, Syncytin-1, Syncytin-2, HERV-K ENV, or ERV3.

[0187] 13. The vaccine composition of any of the preceding paragraphs, wherein the trimerization domain comprises the foldon domain of SEQ ID NO. 93.

[0188] 14. A method of vaccinating a subject against RSV infection, wherein the method comprises administering to the subject an effective amount of a vaccine composition according to any of the preceding paragraphs.

[0189] 15. The method of paragraph 13, wherein the subject is a human subject.

[0190] 16. The method of paragraph 14, wherein the subject is a non-elderly human adult subject.

[0191] 17. The method of paragraph 15, wherein the amount of the RSVF molecules administered is from about 1 mcg to about 2 mgs.

[0192] 18. The method of paragraph 14, wherein the subject is an elderly human subject.

[0193] 19. The method of paragraph 17, wherein the amount of the RSVF molecules administered is from about 20 mcgs to about 10 mgs.

[0194] 20. The method of paragraph 17, wherein the amount of the RSVF molecules administered is about 4.5 times the amount administered to a non-elderly adult human subject.

[0195] 21. The method of any of paragraphs 17-19, wherein the subject is at least 75 years old.

[0196] 22. The method of any of paragraphs 17-19, wherein the subject is at least 80 years old.

[0197] 23. The method of any of paragraphs 17-19, wherein the subject is at least 85 years old.

[0198] 24. The method of any of paragraphs 17-19, wherein the subject is at least 90 years old.

[0199] 25. The method of any of paragraphs 13-33, wherein the vaccination method results in protection of greater than 40% against disease caused by RSV infection.

[0200] 26. The method of any of paragraphs 13-33, wherein the vaccination method results in protection of greater than 50% against disease caused by RSV infection. 27. The method of any of paragraphs 13-33, wherein the vaccination method results in protection of greater than 60% against disease caused by RSV infection.

[0201] 28. The method of any of paragraphs 13-33, wherein the vaccination method results in protection of greater than 70% against disease caused by RSV infection.

[0202] 29. The method of any of paragraphs 13-33. wherein the vaccination method results in protection of greater than 80% against disease caused by RSV infection.

[0203] 30. The method of any of paragraphs 13-33. wherein the vaccination method results in protection of greater than 90% against disease caused by RSV infection.

[0204] 31. The method of any of paragraphs 13-33, wherein the vaccination method results in protection of greater than 95% against disease caused by RSV infection.

[0205] Numbered Paragraphs Relating to treatment of Elderly Subjects

[0206] 1. A method of vaccinating an elderly subject against RSV infection, wherein the method comprises administering to the subject an effective amount of a vaccine composition comprising soluble, trimeric, RSV F molecules di-tyrosine crosslinked in the pre-fusion (pre-F) conformation, wherein each monomer in the trimeric RSV F molecules comprises: an Fl domain, an F2 domain, and a trimerization domain, and wherein each monomer in the trimeric RSV F molecules comprises at least two introduced to-tyrosine mutations, and wherein the di-tyrosine crosslinks connect paired tyrosine residues in the RSV F molecules, wherein at least one tyrosine residue in each pair is an introduced to- tyrosine mutation.

[0207] 2. The method of paragraph 1, wherein the subject is a human subject.

[0208] 3. The method of paragraph 2, wherein the amount of the RSVF molecules administered is from about 20 mcgs to about 10 mgs.

[0209] 4. The method of paragraph 2, wherein the amount of the RSVF molecules administered is about 4.5 times the amount administered to a non-elderly adult human subject.

[0210] 5. The method of any of paragraphs 1-4, wherein the subject is at least 75 years old.

[0211] 6. The method of any of paragraphs 1-4, wherein the subject is at least 80 years old. 7. The method of any of paragraphs 1-4, wherein the subject is at least 85 years old.

[0212] 8. The method of any of paragraphs 1-4, wherein the subject is at least 90 years old.

[0213] 9. The method of any of paragraphs 1-8, wherein the vaccination method results in protection of greater than 40% against disease caused by RSV infection.

[0214] 10. The method of any of paragraphs 1-8, wherein the vaccination method results in protection of greater than 50% against disease caused by RSV infection.

[0215] 11. The method of any of paragraphs 1-8, wherein the vaccination method results in protection of greater than 60% against disease caused by RSV infection.

[0216] 12. The method of any of paragraphs 1-8, wherein the vaccination method results in protection of greater than 70% against disease caused by RSV infection.

[0217] 13. The method of any of paragraphs 1-8, wherein the vaccination method results in protection of greater than 80% against disease caused by RSV infection.

[0218] 14. The method of any of paragraphs 1-8, wherein the vaccination method results in protection of greater than 90% against disease caused by RSV infection.

[0219] 15. The method of any of paragraphs 1-8, wherein the vaccination method results in protection of greater than 95% against disease caused by RSV infection.

[0220] 16. The method of any of the preceding paragraphs, wherein the

[0221] 17. The method according to any of the preceding paragraphs, wherein each monomer in the trimeric RSVF molecules in the vaccine comprises a to-tyrosine mutation at two or more of amino acid residues: 77, 88, 97, 147, 150, 155, 159, 183, 185, 187, 220, 222, 223, 226, 255, 427, 428, and 469, or at or at amino acid residues that correspond to these amino acid residues as determined by alignment to, and using the amino acid numbering of, SEQ ID NO. 1.

[0222] 18. The method according to any of the preceding paragraphs, wherein the trimeric RSVF molecules in the vaccine composition comprise di-tyrosine crosslinks between at least two of the following paired tyrosine residues: 147 and 286; 198 and 220; 198 and 222; 198 and 223; 198 and 226; 33 and 469; 77 and 222; 88 and 255; 97 and 159; 183 and 427; 185 and 427; 185 and 428; and 187 and 427, or at or at amino acid residues that correspond to these amino acid residues as determined by alignment to, and using the amino acid numbering of, SEQ ID NO. 1.

[0223] 19. The method according to any of the preceding paragraphs, wherein the trimeric RSVF molecules in the vaccine composition comprise a tyrosine residue at each of ammo acid positions 185, 198, 226 and 428.

[0224] 20. The method according to any of the preceding paragraphs, wherein the trimeric RSVF molecules in the vaccine composition comprise (a) an intermolecular di-tyrosine crosslink connecting a tyrosine at amino acid residuel85 of one monomer within the trimer with a tyrosine at amino acid residue 428 of a different monomer within the trimer, and ( b) an intramolecular di-tyrosine crosslink connecting a tyrosine at amino acid residue 226 with a tyrosine at amino acid residue 198.

[0225] 21. The method according to any of the preceding paragraphs, wherein each monomer in the trimeric RSVF molecules in the vaccine composition comprises: (a) an F2 polypeptide consisting of amino acid residues 26-109 of SEQ ID NO 81, and (b) an Fl polypeptide consisting of amino acid residues 137-513 of SEQ ID NO. 81.

[0226] 22. The method according to any of the preceding paragraphs, wherein the vaccine composition further comprises an adjuvant selected from the group consisting of alum. CpG, alum+CpG, and Advax.

[0227] 23. The method according to any of the preceding paragraphs, wherein the trimerization domain comprises: a foldon domain, a GCN4 domain, a T4 fibrinitin domain, a human C-propeptide of al (I) collagen, or a 6-helical bundle domain from the post-fusion form of a retroviral fusion protein. Syncytin-1, Syncytin-2, HERV-K ENV, or ERV3.

[0228] 24. The method according to any of the preceding paragraphs, wherein the trimerization domain comprises the fol don domain of SEQ ID NO. 93.

[0229] Various embodiments of the present invention may also be further described by the following non-limiting examples:

[0230] EXAMPLES

[0231] Example 1 Engineered dityrosine-bonding of the RSV prefusion F protein imparts stability and potency advantages

[0232] Overview

[0233] Viral fusion proteins facilitate cellular infection by fusing viral and cellular membranes, which involves dramatic transitions from their pre- to post-fusion conformations. These proteins are among the most protective viral immunogens, but they are metastable which often makes them intractable as subunit vaccine targets. Adapting a natural enzymatic reaction, we have harnessed the structural rigidity that targeted dityrosine crosslinks impart to covalently stabilize fusion proteins in their native conformations. In this example, we show that the prefusion conformation of respiratory syncytial virus fusion protein can be stabilized with two engineered dityrosine crosslinks (DT-preF), markedly improving its stability7and shelf-life and resulting in 11 times greater potency as compared with the DS-Cavl stabilized prefusion F protein in immunogenicity7studies and the ability to overcome immunosenescence in aged mice with a high-dose formulation.

[0234] Introduction

[0235] Dityrosine bonds naturally7form to provide structural rigidity in proteins such as elastin, collagen, and resilin. They are also found in bamboo, the joints of grasshoppers and dragonflies, and the human aortal-5. Additionally, they have been engineered into proteins such as silk fibroin to improve visco-elastic properties and resiliences. Dityrosine bonds are stable even under extreme conditions, including acid hydrolysis and boiling in reducing sample buffer?, 8. Their formation can be catalyzed by several different mechanisms including enzymatic (peroxidase) processes7-13. The enzymatic dityrosine crosslinking reaction propagates through a resonance-stabilized free radical mechanism. The reaction only forms crosslinks between tyrosine sidechains, and because dityrosine bonds are zerolength they7can only form between tyrosine residues in structural proximity7. Therefore, this reaction can be used to identify protein: protein interactions (e.g. in capsid studies of the adeno-associated virus)14. We have leveraged the specificities of this reaction and developed a technology to engineer targeted dityrosine crosslinks at specific positions in a protein structure in order to stabilize proteins and lock desirable protein conformations 15-17. Unlike di-sulfide bonds, dityrosine bonds are introduced through an enzy matic step in manufacturing by which the protein is stabilized after it is fully folded, minimizing misfolding / aggregation that often occurs with engineered disulfide bondsl8.

[0236] Many human pathogenic viruses (e.g. influenza, coronaviruses, Ebola, HIV, and the respiratory syncytial virus - RSV) are enveloped, meaning they have an outer lipid bilayer derived from the host cell. Therefore, fusion of viral and cellular membranes is a key step in the entry of all enveloped viruses and is accomplished by virally encoded, fusion proteins that fuse lipid bilayers by transitioning from their prefusion to their postfusion conformations. In recent years it has become clear that fusion proteins elicit antibody (Ab) responses that neutralize viral infection, a powerful and predictive measure of vaccine efficacy. Fusion proteins in their prefusion conformation expose potently neutralizing epitopes to the immune system and these prefusion-specific epitopes give rise to the most potently neutralizing and protective Ab responses, likely because only fusion proteins in their prefusion conformation on the surface of infecting virus particles mediate viral entry. Antibodies that bind the prefusion conformation with high avidity and affinity block the fusion protein’s function, and thus prevent infection. However, one of the major challenges to developing recombinant, soluble prefusion vaccine immunogens is that the prefusion conformation is spring-loaded and metastable, and often readily transitions irreversibly into the postfusion conformation typostF”).

[0237] Structure-based design has yielded proteins that provide proof of principle that prefusion vaccine immunogens are, indeed, the most potent (e.g., RSV DS-Cavl)19. However, these designs are often only partially stabilized, and in addition to prolonging shelf-life and reducing cold-chain requirements, we believe that further improving thermostability also prolongs in vivo exposure of prefusion-specific epitopes, strengthens the antigenic signal of these epitopes, and thus enhances B cell responses and affinity maturation. Thus, fusion proteins better stabilized in the prefusion conformation should yield higher-affinity and higher-titer Ab responses to prefusion-specific epitopes and provide better protection. Dityrosine (“DT”) crosslinking provides a means to stabilize fusion proteins in their prefusion conformation that goes beyond the limits of traditional, mutation-based designs (for example, disulfide engineering, and cavity filling mutations) 18, 19. By focusing Ab responses on prefusion-specific epitopes, dityrosine-stabilized immunogens can elicit more potently neutralizing Ab responses, resulting in better vaccine immunogens. Respiratory Syncytial Virus (RSV) infects humans repeatedly throughout life. Newborn children, the elderly, and immune compromised patients are particularly vulnerable to more severe disease20-22. In newborn children, RSV infection often results in protracted and enhanced respiratory' disease, which can result in prolonged respirator}7difficult}7throughout childhood and adolescence. However, development of an RSV vaccine was hindered by an early clinical trial in which children vaccinated with a formalin-inactivated vaccine (FI-RSV) experienced an exaggerated immune response to subsequent RSV infection - i.e. vaccine enhanced disease (VED)23. VED was characterized by elevated eosinophilic responses to infection, airway hyper-reactivity, and excessive mucus production23,24. These potentially fatal responses were caused by poorly neutralizing Ab responses with immune complex deposition in the lungs and Th2-type cytokine responses to vaccination in RSV naive children24,25. Eliciting high titers of prefusion-specific, neutralizing antibodies is still considered the best vaccine strategy to protect the elderly and infants via maternal -to-infant vaccination, whereby the importance of T cells to the protection of the elderly should not be discounted26-30.

[0238] The prefusion conformation of the RSV F (fusion) protein is among the most labile of the fusion proteins. However, an RSV prefusion F (preF) subunit vaccine has been demonstrated to be one of the most promising approaches, since it elicits highly potent neutralizing antibody responses, and because its sequence is highly conserved between strains. In fact, the first two RSV vaccines to obtain licensure are protein subunit vaccines31-33. Significant progress has been made stabilizing RSV preF, starting with the design and characterization of the Vaccine Research Center’s (VRC / NIH) DS-Cavl molecule 19,34. DS-Cavl elicits substantially higher neutralizing antibody titers than the RSV F protein in its postfusion conformation! 9, 28,35. Several 2nd-generation RSV subunit vaccines are being developed that further improve upon DS-Cavl ’s stability34, 36-42. Current RSV prefusion stabilized first generation vaccines have built upon the success of DS-Cavl and have now achieved full licensure for the elderly and in maternal vaccination31-33,43. These exciting first-generation molecules have obtained high overall efficacy and have also substantially de-risked the clinical development pathway for subunit RSV prefusion vaccines. Nevertheless, ample room for improvement exists. Pfizer’s maternal vaccine does not substantially outperform the protection afforded by monoclonal antibody administration in infants; and GSK’s leading vaccine for the elderly does not adequately protect in the 80+ elderly or the frail where they achieve only 34 and 14% efficacy, respectively and lack statistical significance. These immunosenescent populations are precisely the most likely to be hospitalized and have poor clinical outcomes and vaccines therefore need to elicit potent responses in these groups. This remains an important unmet medical need. Using influenza vaccines formulated for the elderly as an example, surmounting immunosenescence is achievable by modulation of the dose and / or adjuvant44,45. Applying these approaches to the most potent immunogen has the greatest chance of success. Herein we report the application of targeted dityrosine crosslinking to improve the efficacy of a fusion protein-based subunit vaccine. We have harnessed the structural rigidity that dityrosine (DT) bonds can impart to generate an RSV preF subunit vaccine that more stably holds its prefusion conformation.

[0239] Our prefusion F molecule, DT-preF, comprises two, targeted dityrosine crosslinks that lock the preF conformation to elicit Ab responses focused on neutralizing epitopes 29,46. Since dityrosine bonds only form between tyrosine sidechains in close proximity, we have engineered our DT-preF molecule through structure-based design and site-directed mutagenesis to stabilize two of the most highly prefusion specific epitopes (Site 0, and the Site IV / V interface), as described further in PCT / US2014 / 048086 and PCT / US18 / 45463. The dityrosine bonds formed in DT-preF are not present in the DS-Cavl molecule. The previously described DS-2 molecule targeted a disulfide bond to the Site IV / V interface and improved upon the potency of DS-Cavl approximately 4-fold but expresses poorly and has not progressed into clinical development as a subunit vaccine. Dityrosine induced thermostability stabilizes the prefusion conformation of DT-preF both in vitro and in vivo. Our data demonstrate that DT-preF elicits 1 IX higher neutralizing Ab titers than those elicited by DS-Cavl. and can overcome immunosenescence in a mouse model using a high dose and the well tolerated alum adjuvant. DT-preF is also highly potent in cotton rats and elicits high neutralizing antibody titers against RSV A and B strains and achieves sterile lungs upon challenge with the WT RSV Long strain. These preclinical data suggest that dityrosine stabilized DT-preF has clear advantages that will fill the remaining gaps in the protection afforded by first generation RSV vaccines.

[0240] Immunogen design based on dityrosine crosslinking.

[0241] RSV F protein is expressed as FO, a single polypeptide with two furin cleavage sites. The RSV F protein has between 5 and 6 N-linked glycosylation sites on the precursor molecule 47. Proteolytic maturation of the F trimer removes 27 amino acids, yielding the Fl and F2 cleavage products, which together form a protomer of the F trimer. The mature protomer contains 3 N-linked glycans (2 on the F2 subunit and 1 on the Fl subunit)48. We engineered Tyrosine (Tyr) substitutions in the soluble F glycoprotein to preserve protective epitopes recognized by prefusion-specific neutralizing Abs 19. We targeted these substitutions based on the crystal structure of the RSV F protein complexed with the prefusion-specific D25 monoclonal antibody (mAb), and the DT bonding designs were screened in the previously described Cavl background in order to build on a framework sufficiently stable for analysis (RSV F A2)19. Soluble forms of these variants were designed for intra and / or inter- molecular crosslinking and expressed by transient transfection in HEK293T cells and screened for expression and antigenicity by ELISA (Fig. 1A and Fig. IB). Motavizumab, a mAb that has a high affinity for both pre- and postfusion F, was used to measure total protein expression. Prefusion antigenicity was measured using mAbs D25, AM14, and MPE8 to map Site 0, the site IV / V interface, and site III binding, respectively (Fig. IB). A few selected dityrosine mutations that were localized to critical epitopes for neutralization and demonstrated good levels of antigenicity were also tested in the DS-Cavl background (Fig. 1C). For further characterization, the top 14 variants and WT / Cavl / DS-Cavl controls were screened for antigenicity and fluorescence in supernatants of transfected 293T cells (Fig. 1C and Fig.lD). Since dityrosine bonds are fluorescent with unique excitation and emission maxima (ex320 / em405). screening by this method enables the detection of both intra and inter-protomeric (which may also be referred to as intra and inter-molecular) dityrosine bonds. After the crosslinking reaction, DT bonds were detected by fluorimetry (Fig. ID). As shown in Fig. ID, using a representation of samples normalized for protein expression, several hits were identified in the fluorescence intensity screen. While many of the designed constructs demonstrated significant binding to conformation-specific antibodies in the absence of crosslinking, we prioritized crosslinks that would preserve epitopes that elicit the most potently neutralizing prefusion-specific antibodies, namely Site 0-stabilized by the K226Y mutation pairing with endogenous Y198, and the AM 14 binding site (IV / V) interface. Given the utility of AM14 as the most prefusion-specific mAb known for RSV F, we targeted several crosslinks precisely in the AM14 binding site but sought to maintain some level of AM14 binding post-crosslinking46. The variant designed with ty rosine substitutions at Valine 185, Lysine 226, and Asparagine 428, termed preFC (Fig. 2A, bottom right), which is a descendent of Cavl (Fig. 2A, top), and related to DS-Cavl (Fig. 2A, bottom left) through the shared use of the Cavl mutations, possessed near WT expression levels and a favorable AM 14 binding profile before and after crosslinking (Fig. 2C and Fig.2D). This molecule consists of 2 engineered dityrosine bonds: V185Y pairing with N428Y, and K226Y pairing with the endogenous tyrosine at Y198 (Fig 2a). These crosslinks stabilize the antigenic site IV / V interface (AM14 binding region) and prefusion-specific Site 0 (5C4 binding region), respectively (Fig. 2A and Fig. 2B)28,46,49. preFC has 3 additional point mutations that targeted the Foldon region designed to further enhance stability- these are L512V, L513V, and Y519F. We further characterized DT-preF binding properties with purified proteins as compared to DS-Cavl using antibodies that map to key epitopes including Site 0 (D25), Site II (Motavizumab), and Site III (MPE8) (Fig. 8). Additionally, we characterized the fluorescence properties of purified DT-preF relative to protein concentration under native conditions and can demonstrate linearity in the range of 19-152 pg / mL (Fig. 8).

[0242] Dityrosine crosslinking is highly specific for engineered sites.

[0243] To map the location of the observed dityrosine bond, we submitted the protein for LC-MS- MS. To simplify the analysis, purified crosslinked and uncrosslinked samples were submitted for comparison. Unique peptides were identified in the crosslinked sample, and these were selected and sequenced by tandem MS. The intermolecular crosslink V 185 Y - N428Y was identified in the mass spectrum following comparative peptide fragment analysis and the sequence of crosslinked peptides was confirmed by tandem mass spectrometry (Fig. 3A-C). The intramolecular bond was not able to be identified in the mass spectrometry analysis. We characterized the protein further by amino acid analysis and analytical SEC under denatunng conditions. Amino acid analysis confirmed dityrosine’s presence in the sample and betw een 4 and 5 dityrosine bonds w ere present on average per molecule (Fig. 4A). Since only 3 intermolecular bonds can form per trimer, these data suggest that additional dityrosine bonds form in the crosslinked protein. We further characterized our crosslinked molecule by analytical size exclusion chromatography under denaturing conditions with in-line fluorimetry and compared it with uncrosslinked preFC (Fig. 4B, left bottom and top, respectively). Separation of the monomeric and multimeric peaks under denaturing conditions and observation of the fluorescence associated with both peaks demonstrates that an intramolecular bond is forming in the crosslinked molecule that is not observable by mass spectrometry (Fig. 4B, right top and bottom). In support of this finding, we subjected both DS-Cavl and the Cavl control molecules to the crosslinking reaction conditions. As shown in Fig. 9A, only the preFC exposed to these conditions resulted in intermolecular bond formation as apparent by the MW shifts observed under denaturing conditions. The Cavl protein represents the closest control to our molecule since preFC only differs from Cavl by 3 mutations to tyrosine to allow crosslink formation and the 3 stabilizing point mutations near the Foldon domain. Therefore, we confirmed the lack of significant MW shifting on crosslinked and purified C AV 1 protein using SDS-PAGE with Coomassie staining and western blot analysis (Fig. 9B). Since intramolecular bonds would not be apparent by gel shift, we further subjected the crosslinked and purified Cavl protein to denaturing HPLC analysis and can demonstrate that fluorescence is absent in the monomeric peak as compared with a similarly crosslinked and purified DT-preF protein subjected to the crosslinking reaction (Fig. 9B and Fig. 2C).

[0244] Dityrosine bonding of preFC confers stability improvements that translate into prolonged shelf life.

[0245] In order to demonstrate the stability imparted by the DT crosslink, we subjected our DT-preF molecule to differential scanning fluorimetty using the Prometheus Panta instrument. As described elsewhere herein and demonstrated in Fig. 10A, the crosslinked molecule exhibited a melting temperature increase of 25°C as compared with the uncrosslinked preFC molecule. We next tested the stability of our DT-preF molecule in vitro at 4°C in a time-course experiment. We thawed purified DT-preF and DS-Cavl, and normalized protein concentration and buffer composition. Proteins were then incubated at 4°C for 3 and 5 weeks, and compared to Motavizumab-normalized, freshly thawed (day 0) protein on the day of each ELISA (Fig. 5A). Notably, while DT-preF’s binding to AM14 remained stable at weeks 3 and 5, DS-Cavl’s binding continued to fall and reached an 82% loss overall by week 5 (Fig. 5A). Based on these results, we expanded this analysis in terms of time frame and epitopes probed to include D25 (Site 0), and also developed a highly prefusion specific sandwich ELISA that utilizes both 5C4 (Site 0) and AMI 4 (Site IV / V) antibodies to measure the prefusion conformation. As apparent in Fig. 10B, DT-preF maintains the prefusion conformation even out to 11 weeks of storage at 4°C using both ELISA assays. While DS- Cavl maintains binding to D25 over this period, the 5C4 / AM14 sandwich ELISA we have established clearly demonstrates significant loss of the prefusion conformation during 4°C incubation over the same time period. This result demonstrated marked 4°C stability improvement for the DT-preF molecule in vitro so we tested the impact of this result in murine immunogenicity experiments. We injected BALB / c mice intramuscularly with freshly thawed DT-preF and DS-Cavl immunogens and identical immunogens after 4 weeks of cold storage formulated on AdvaxSM 50-52. Advax was chosen for this study since at the time we were trying to elicit more Th-balanced responses for their potential safety advantage. Neutralization assays performed with the mouse serum showed a statistically significant loss of potency (57.6%) for the DS-Cavl protein but not the DT-preF after 4 weeks of incubation at 4°C (Fig. 5B and Fig. 5C). Hence, dityrosine crosslinking of DT-preF resulted in significantly improved stability and shelf life.

[0246] DT-preF is highly potent in mice.

[0247] Hypothesizing that improved stability results in better affinity maturation and improved potency, we compared DT-preF and DS-Cavl formulated with the Th2-skewing adjuvant alum in mice in order to maximize serum antibody responses. Neutralizing Ab titers (NTs, expressed as reciprocal IC50’s) of mouse sera demonstrated that DT-preF elicited 11. 1-fold higher geometric mean NTs than DS-Cavl (Fig. 6A) using a traditional prime: boost regimen. It was important to determine whether these enhanced responses were due to a larger overall anti-F antibody response, or whether the DT-preF immunogen elicited a betterquality response, with a higher percentage of the total antibodies eliciting more potent viral neutralization. Fig. 6B shows that only a 1.7-fold increase in total anti-prefusion F binding titers were detected, demonstrating that DT-preF elicited better quality responses since a larger percentage of the elicited antibodies neutralize the virus.

[0248] A high-dose DT-preF vaccine formulated on alum overcomes immunosenescence in aged mice.

[0249] The elderly represent a crucial patient population at risk for severe complications from RSV infection and specifically the 80+ age bracket and / or the frail are at the greatest risk. Immune responses in these individuals are often referred to as immunosenescent in that the overall responses to immune challenge are blunted due to age related factors. Overcoming this immunosenescence is critical to protecting this high-risk group. Several strategies exist to overcome immunosenescence via the dose, regimen, and / or adjuvant administered. We hypothesized that the improved potency of our molecule on alum would overcome immunosenescence simply with a higher dose avoiding harsher adjuvant and / or dosing requirements. As observed in Fig. 6E, while an equivalent dose given to old mice did not overcome immunosenescence, simply increasing the dose by 4.5X elicited equivalent neutralizing antibody responses in young and old mice. Similarly, the high dose group demonstrated increased overall F binding antibodies that did not differ statistically from the young animals (Fig. 6C), and similar B-cell ELISpot responses were also observed in the old high dose group (Fig. 6D). These data demonstrate that the improved potency of the DT- preF molecule enables surmounting of immunosenescence with an equivalent vaccination regimen, with simply a higher dose. These data suggest that DT-preF will not require a less- tolerable adjuvant and / or repeat dosing to overcome immunosenescence.

[0250] DT-preF is highly potent in cotton rats.

[0251] We further tested the potency of our molecule in cotton rats to evaluate the responses, and the level of protection from challenge, that are elicited by low and high doses of DT-preF (2pg and 10 pg doses per injection per animal) on alum. Controls included one group injected with saline (negative control) and one group was infected with live RSV / A / Tracy (positive control). Since animals were RSV naive, a prime: boost regimen was used and animals were challenged on day 35 with 105 pfu / animal of RSV / A / Tracy intranasally. The animals were sacrificed four days after challenge, and their lungs were collected for RSV titer analysis. The viral lung titers of both the 2 pg and 10 pg DT-preF groups of cotton rats were below the limit of detection (1.4 loglO pfu / g), whereas the saline control group had an average of 105 pfu / g lung tissue (Fig. 7B) demonstrating that both doses of Alum-formulated DT-preF provided complete protection. Serum samples were collected on day(s) 0, 21, 35, and 39- to analyze NTs against RSV / A / Tracy and RSV / B / 18537 subty pes; these results demonstrated that mean NTs of both DT-preF immunized groups exceeded those induced by live virus infection against both strains following the boost (Fig. 7C and Fig. 7D).

[0252] Discussion

[0253] The data presented herein show that we have developed a technology to target dityrosine crosslinks into protein structures that can be applied to conformationally stabilize a viral fusion protein-based recombinant subunit vaccine, which in turn improves its potency, shelflife, and cold chain requirements.

[0254] Given the large market and medical need for improved RSV vaccines, several groups are pursuing strategies encompassing multiple vaccine modalities (vector, mRNA, live- attenuated, nanoparticle, and their use in combination) 53-57. The 2023 licensure of GSK and Pfizer’s first-generation subunit vaccines makes this a very exciting time in RSV vaccinology as first- in-class products will significantly improve the RSV public health crisis58,59. In RSV, subunit vaccines have demonstrated superb safety and efficacy, but the instability of these RSV subunit vaccines remains a key challenge for vaccine potency and delivery. We demonstrate that dityrosine bonding not only improves the stability of target molecules, but also significantly improves their potency.

[0255] In RSV, our current immunogen design introduces two dityrosine bonds into the F protein. The engineered inter-protomeric crosslink (which may also be referred to herein as an inter- molecular crosslink) has been identified by LC-MS / MS (Fig. 3); no other crosslinks could be detected. But amino acid analysis confirms that more than one crosslink forms, and analytical size exclusion chromatography under denaturing conditions with in-line fluorimetry demonstrates the presence of an intra-molecular bond (Fig. 4). The absence of nonspecific crosslinks detected by LC-MS / MS provides evidence for the specificity and selectivity of this targeted crosslinking technology. As demonstrated in Fig. 2C and Fig. 9C, under typical reaction conditions, betw een 65-75% of the protein contains all 3 inter- protomeric bonds and completely shifts to the trimeric species. However, the intramolecular bond forms to a lesser extent and reaches a total average of between 4 and 5 bonds per trimer supported by amino acid analysis. This suggests that some of the monomeric species contain the intramolecular bond. This is supported further by the HPLC analysis run under denaturing conditions.

[0256] We demonstrated that our crosslinked RSV preF immunogen has substantially better conformational stability than our comparator, DS-Cavl. Whereas after a 4-week period of storage at 4°C DS-Cavl elicited a roughly 60% drop in neutralizing Ab titers, neutralizing Ab titers elicited by DT-preF were not statistically reduced, demonstrating dramatically improved stability by targeted DT crosslinking. This scenario is particularly applicable to the distribution of a vaccine as it is expected to permit longer term storage of a multi-use vaccine vial, thus facilitating vaccine distribution. Additionally, dityrosine stabilization achieves 1 lx higher neutralizing antibody titers, through the elicitation of only 1.7x greater overall prefusion F binding titers. These data demonstrate that DT-preF elicits a higher percentage of neutralizing antibodies and thus a better-quality antibody response. The quality of the antibody response is particularly important when addressing safety of the immunogen since a high level of non-neutralizing antibodies were associated with VED in the initial FI-RSV vaccination trials24,25.

[0257] A trend can be observed in the literature which suggests that engineering additional crosslinks to a prefusion pneumo and / or paramyxovirus subunit vaccine immunogen results in the elicitation of higher neutralizing antibody titers upon vaccination. This was shown in the case of the DS-2 molecule described by Joyce et al.42, where the successful incorporation of an additional disulfide bond to DS-Cavl achieved 4X greater neutralizing antibody titers. Similarly, Ou et al. reported a triple disulfide-bonded metapneumovirus F protein that achieved 10-fold higher titers than the single disulfide-bonded version of the protein60. There may, however, be a virus or protein-specific upper limit to potency improvements as incorporation of an additional disulfide bond in an otherwise single-disulfide bond stabilized parainfluenza virus type 3 fusion protein did not further improve neutralization titers61,62. Potency improvements are therefore more likely dependent on the ability of the additional crosslink to maintain the most authentic immunological shape rather than the numerical addition of crosslinks per se.

[0258] Overcoming immunosenescence in the 80+ and frail represents a critical unmet need in the current RSV first generation repertoire. While several levers can be used to surmount the immunosenescence challenge, starting with an immunogen with the highest potency provides an additional lever unavailable to the other immunogens. DT-preF’s high potency overcomes immunosenescence in a mouse model solely with an increased dose on alum, an adjuvant known to be well tolerated. This demonstrates that DT-preF may achieve improved protection in these most critical human patient populations.

[0259] Materials & Methods

[0260] Cells, plasmids, antibodies, viruses: 293T cells were from an in-house stock originally from the Mount Sinai School of Medicine. 293 Freestyle cells were purchased from Invitrogen (ThermoFisher Cat#R79007) and cultured in 293 Freestyle medium (Gibco®). Hep-2 cells were purchased directly from ATCC (with a HeLa contamination disclaimer) (ATCC, Cat#CCL-23). Composite CHO cells expressing preFC were generated and cloned under contract with Abzena Ltd. preFC was cloned into the pcDNA 3. 1 Zeo plasmid and used for transient transfections. Motavizumab, AM 14, 5C4 anti-F primary antibodies w ere gifts from Jason McClellan (UT Austin) and Barney Graham, or purchased from Creative BioLabs (Shirley, NY). D25 and MPE8 were provided by Jason McClellan and Barney Graham. The anti-mouse and human secondary antibodies were purchased from GE Healthcare Life Sciences and Jackson ImmunoResearch Laboratories, Inc. respectively. HRP Rat anti-mouse IgG2a (BD Pharmingen) w as used in the conformational sandwich ELISA. Biotin-SP (long spacer) Affinipure goat anti-mouse IgG. Fc gamma fragment specific (Jackson Immunochemicals) was used in the ELTSPOT assays. The RSV-luciferase virus was obtained from Dr. Martin Moore (Emory University).

[0261] Protein Expression and Purification: Proteins were expressed by transient transfection of adherent HEK 293T (screening), suspension HEK293F cells (production), or from a recombinant stable CHO cell line produced by contract from Abzena Ltd (San Diego) expressing a tag-free version of preFC(production). . 293F Transfection was done at a cell density of 1x106 cells / mL in IL vented Erlenmeyer flasks with 1 :3 DNA / PEI complex while 293T cells for secondary (fluorescence) screening were transfected in a 96-well format with 250ng of DNA, 1.5 uL PEI, and 3.8 X 10A5 cells / well. 293 cell supernatants were harvested after 4 to 5 days of expression and CHO cell culture supernatants were harvested from shake flasks over 14 days of expression. 293F and CHO supernatants were centrifuged 2 x (500 g and3100g). passed over a cell strainer to remove any large debris, and punfied by affinity chromatography over Strep-Tactin® Superflow® resin (IBA), or Capto- Adhere resin (Cytiva), respectively. 293F protein was eluted in a modified Strep-Tactin elution buffer (2.5mM of d-Desthiobiotin (Sigma Aldrich), 1 mM EDTA (Sigma Aldrich), 1:20 of lOOmM Na Phosphate pH 8.4, in lx PBS (LifeTech)) while CHO expressed protein was eluted in a Citrate and L-arginine containing sodium phosphate buffer (pH 7.5). Eluted proteins were concentrated using Tangential Flow Filtration (TFF) (Minimate TFF Capsule with 50 kDa Omega Membrane, Pall Life Sciences) followed by Biorad, or total protein ELISA protein assays. Post DT crosslinking, in the case of 293-expressed DT-preF as well as Strep-Tactin purified DS-Cavl, further purification w as done as follows: Imidazole at a final concentration of 20mM was added to the reaction. To purify DT-preF from the crosslinking enzyme, ARP. the protein was purified over 5mL HisTrap column (GE Life Sciences), using wash and elution steps composed of crosslinking buffer containing 150mM salt plus 50mM and 300mM imidazole respectively. The proteins were further isolated using size exclusion chromatography (SEC). HiLoad Superdex 200 pg columns (GE Life Sciences) with PBS or 10%Sucrose / NTE as the elution buffers. DT-preF purified in lx PBS was concentrated using Amicon Ultra-15 centrifugal filter units with an Ultracel-50 membrane (Sigma). CHO- expressed protein was purified after crosslinking by hydrophobic interaction chromatography using Buty l FF Sepharose resin (Cytiva) and used directly in the elution buffer for immunogenicity studies (10 mM Na-phosphate, 75mM NaCl. pH7.0) or formulated as described in the stability study section.

[0262] When needed, in order to remove the affinity7tags, 1 unit of biotinylated thrombin (Millipore / Sigma) was added to each ImL of concentrated SEC eluate (0.5-1 mg / mL) and incubated for 20hrs at 4°C. After the cleavage reaction, thrombin was removed according to the manufacturer’s instructions followed by addition of 0.5uL Strep Tactin® resin per pg of protein cleaved, to bind / sequester the cleaved tags. Finally, the sample was spun in a tabletop centrifuge over a Pierce spin column (LifeTech) at 500 g for 5 minutes to remove the beads and the protein concentration was measured by Ninhydrin assay63. The proteins were flash frozen in liquid N2 and stored at -80 degrees Celsius until further experimentation.

[0263] ELISA-analysis: The initial expression-based screening of variants was done in a 96-well format ELISA, using supernatants from transiently transfected HEK293T cells. The cells were seeded in a 96-well tissue culture plate (VWR) at a density of 2.5 x 104 cells / well- on day 0 followed by overnight incubation. The plasmid DNA for the expression of designed variants was transfected 1:3 with Truefect Max (United Biosystem). Supernatants were collected on day 5 and coated on Ni-NTA His Sorb plates (Qiagen). Motavizumab. AM14. MPE8. and D25 were used as the primary antibodies, at concentrations of 0.5 pg / mL, 1 pg / mL, 0.5 pg / mL, and 0.5 pg / mL, respectively in PBS with 20mM Imidazole. Postincubation and extensive washing, the plates were incubated with anti-human secondary antibody (Peroxidase AffiniPure F(ab')2 Fragment Goat Anti-Human IgG, Jackson Immunochemicals. 1:2500) in PBS with 20mM Imidazole. The plates were finally washed again, developed with 1-step TMB ELISA substrate solution (Thermo) and read in a NOVOStar plate reader.

[0264] Antibody responses in serum were quantified by standard ELISA methods, with DS-Cavl immobilized on the plate as the capture antigen. HRP -labeled anti-mouse IgG (GE Healthcare) served as the secondary antibody and One-step TMB (Invitrogen) was the developing reagent. Binding titers are show n as the reciprocal of the serum dilution resulting in an OD450 value of 0.75 to lie within the linear range of all serum binding curves.

[0265] Antibody responses in aged animals were conducted by standard ELISA methods with DT- preF immobilized on the plate as the capture antigen. Sera from different timepoints were tested (one day before vaccination for pre- vaccination values (tO), boosted 21 days (t21) and 35 days (t35) after prime / boost). 96-well plates were coated with 2 pg / ml of DT-preF for 1 hr, at RT, washed and blocked with of 1% BSA for 30 min, at 37° C, after washing, sera was diluted at 1:20000 and added for 2 hrs at RT, wells were washed and anti-mouse IgG- HRP conjugated (Jackson 115-036-062) was added for 1 hr at RT, after washing, TMB substrate reagent (BD 555214) w as added to all wells. Plates w ere read for absorbance at 450 nm (BIO-RAD Benchmark Plus microplate spectrophotometer).

[0266] SDS-PAGE analysis: During preliminary screening, inter-protomer crosslinks in Streptactin- purified variants were assessed using SDS-PAGE gel shift. Uncrosslinked and crosslinked protein samples were mixed 1 :4 with NuPAGE 4X LDS sample buffer (LifeTech). TCEP (Sigma- Aldrich) was used 1 : 10 as the reducing agent. Samples were then cooked at 95°C for 5m. After cooling, samples were loaded and run in concentration-compatible volumes on a 4- 12% Bolt mini gel (1mm x 12-wells; LifeTech), followed by staining with Quick Coomassie Stain (Anatrace).

[0267] DT-Crosslinking Reaction / Fluorescence analysis: For screening, 293T transfected cell supernatants w ere clarified by centrifugation, diluted 1: 1 with PBS, normalized for total protein by Motavizumab ELISA, and crosslinked through the addition of Arthromyces ramosus peroxidase (ARP) at 11 pg / mL. The reaction was initiated with the addition of 0.00036% hydrogen peroxide (H2O2; Sigma Aldrich) at room temperature. Fluorescence intensity measurements were taken prior to the initiation of the reaction and post 15 minutes of crosslinking in a Novostar plate reader with an excitation w avelength of 320 nm and an emission wavelength of 405nm. For large scale protein preparation, the reaction was conducted similarly in 30 mL volumes in Falcon tubes or using a DASGIP parallel bioreactor system in a IL BioBlu 1c single-use vessel at 39°C-43.0°C. Crosslinking was conducted to specification using either ARP enzyme or a Nickel-peptide complex as the catalyst and hydrogen peroxide or magnesium monoperoxyphthalate as the oxidant. 150 mM NaCl was added to the crosslinking buffer and the reactions proceeded for 5-50 minutes.

[0268] Mass Spectrometry' Analysis: Endopeptidase try psin (modified, sequencing grade) was purchased from Promega (Madison, WI). Chymotry psin was obtained from Roche (Indianapolis, IN). The Quadrupole ion trap mass spectrometer (LTQ XL) used in the proteomic analysis was manufactured by Thermo (Palo Alto, CA). After sample denaturation, reduction, and alkylation, samples were solution digested for LC / MS / MS sequencing with the following digestive enzymes: trypsin, chymotrypsin, and pepsin. The digested peptide mixture was analyzed by an LC-ESI-MS / MS system, in which an Agilent 1100 Binary pump high-performance liquid chromatography (HPLC) system was used to run a 75-micrometer inner diameter reversed phase Cl 8 column which was on-line coupled with an ion trap mass spectrometer. The solvents used for HPLC were solvent A 98% H2O, 2% acetonitrile and solvent B 10% H2O, 90% acetonitrile, both contain 0.025% TFA. A combination linear / step gradient elution was performed between 2 and 90% solvent B over 180 minutes and the analysis time was 200 minutes. Peaks were manually assigned using Prottech’s in-house software (proprietary), while data for presentation was prepared with Thermo XCalibur Qual Browser 3.1 Release. Mass spectrometry' data for the crosslinked and uncrosslinked proteins have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD048897 [https : / / www. ebi. ac. uk / pri de / archi ve / proj ects / PXD048897] .

[0269] Amino Acid Analysis: Crosslinked protein, a chemically synthesized di tyrosine standard (Chemshuttle). and commercially purchased tyrosine standard (Sigma) were shipped frozen to the University of California, Davis molecular structure facility. A precise volume / mass of protein was transferred, dried, and hydrolyzed in 6N HCL, 1% Phenol, 110°C, 24hr, in vacuo. Samples were then dissolved in a precise volume of sodium diluent (Pickering Labs) containing 40nmol / mL NorLeucine. 50ul was injected on a Concise (AminoSep Beckman Style Na+ column, part AAA-99-6312) strong cation exchange column using a Na-based Hitachi 8800. An amino acid standards solution for protein hydrolysate (Sigma, A-9906) was used to determine response factors, and thus calibrate quantities for all amino acids. Each injection also contains norleucine as an internal standard to allow correction of the results for variations in sample volume and chromatography variables. Data was reviewed by two staff members for accuracy.

[0270] Analytical HPLC-size exclusion chromatography: Crosslinked and uncrosslinked protein samples were treated with Remove-iT PNGase F (New England Biolabs) according to the manufacturer’s instructions, followed by denaturation with SDS. The denatured protein was reduced and alkylated with the G-biosciences FOCUS™ protein reduction-alky lation kit according to the manufacturer’s instructions (St. Louis, MO). Samples were run on an Agilent 1260 Infinity II HPLC system using two LW-403 4D (Shodex) analytical sizeexclusion columns in tandem at 45 degrees Celsius with a mobile phase of 0.2% SDS, 50 mM NaH2PO4, 200mM NaCl (pH 5.5).

[0271] Stability Assay / ELISA: DT-preF and DS-Cavl purified proteins were thawed on ice followed by dilution to 100 pg / mL (Ni-NTA) or 41.5 pg / mL (sandwich / polysorp) with a final 5% sucrose buffer concentration. PMSF at a final ImM concentration was added and the proteins were stored at 4°C until the stability ELISA time points indicated. To confirm equal protein in all samples at the onset of the study.

[0272] Motavizumab ELISA was performed. Proteins from the same stocks were freshly thawed on ice at each timepoint followed by equivalent dilution and PMSF treatment to serve as "Fresh’' control samples. The freshly thawed and 4°C stored samples were compared for antigenicity on microtiter 96-well Ni-NTA HisSorb plates (Qiagen) or on 96-well Immuno Polysorp plates (Thermo Fisher Scientific) for total protein ELISA and microtiter 96-well Ni-NTA HisSorb plates (Qiagen) for AM14 conformational ELISA or 96-well high binding EIA / RIA plates for 5C4 / AM14 conformational sandwich ELISA. The antigen was coated on plates for 2h at RT or at 37°C for Ih 40m for total protein and AM14 ELISA and overnight at 4°C on AMI 4 coated plates for the conformational sandwich ELISA. The plates were then probed with primary antibodies Motavizumab (at 0.5 pg / mL), AM14 (1 pg / mL, Ni-NTA ELISA) or 5C4 (2.5 pg / mL. 5C4 / AM14 sandwich ELISA) or D25 10 pg / mL by incubation for Ih at RT or 37°C Ih, respectively. HRP-conjugated secondary antibodies (Amersham ECL HRP Conjugated Antibodies 1ml sheep anti Mouse, Peroxidase AffiniPure F(ab’)2 Fragment Goat Anti -Human IgG, Fey fragment specific and HRP Rat Anti -Mouse IgG2a) were then added for a Ih RT or 37°C incubation, respectively. The assay was developed using HRP colorimetric substrate 3,3', 5,5'-tetramethylbenzidine (TMB) substrate. Plates were read for absorbance at 450nm after sulfuric acid addition.

[0273] Murine Vaccinations: Animals were handled according to the IACUC protocols at the University of Miami (Protocol # 19-013-LF) and SUNY Downstate (Protocol # 16-105-12), respectively. For the stability study in Fig. 5B, 6-8 week-old, female CB6F1 / J mice and the adult vaccination study in Fig. 6A and Fig. 6B, 15-week-old, female CB6F1 / J mice were vaccinated by IM injection into the hindlimb (50pl) with either 10 pg (stability) or 2.3 pg (potency) of DT-preF or DS-Cavl formulated as indicated (AdvaxSM Img / mouse (Vaxine pty), 2% Alhydrogel (Brenntag, 100 pg / mouse). Animals were primed on day 0, boosted on day 21, and serum was collected on day 35 for determination of neutralization titers / prefusion F binding titers. For the aged animal vaccinations, 17 months old male and female BALB / cJ mice were primed (day 0) and boosted on day 21 via IM injection (50pl) into their right hindlimb with vehicle (PBS), or DT-preF alone (lOpg, low dose, or 45 ug, high dose) formulated on Alhydrogel as above. Mice were bled one day before vaccination for prevaccination values (tO), at 21 days (t21) prime only. All mice were sacrificed at 35 days (t35) after prime / boost. There was a total of 12 or 13 animals in each group.

[0274] Neutralization assay: Heat inactivated serum is diluted 1 :50-1:800 (depending on the expected potency of the serum) in phenol-free MEM supplemented with glutamax, 5% FBS, and Penicillin / Streptomycin and serially diluted in a 3-fold series (50 uL final volume). RSV-Renilla Euciferase virus was then diluted in the same medium as above to 2 XI 04 pfu / mL and 50 uL is added to each well (10,000 pfu / well) with virus only controls. Virus and serum were incubated at 37 degrees Celsius, 5% CO2, for 2 hrs. Hep-2 cells (ATCC) are then trypsinized, counted, and diluted to 1 x 106 cells / mL in the same medium and 25 uL is added to each well (2.5 X 104 cells / well). The cells are then incubated at 37 degrees Celsius, 5% CO2 for 60-72 hrs and luciferase activity is quantified using the Renilla-glo luciferase assay system (Promega) according to the manufacturer’s instructions and read in aNovostar plate reader. Data is analyzed using nonlinear regression to calculate IC50 concentrations of each serum dilution curve (GraphPad Prism). B-cell ELISPOT analysis: 96-well plates were coated with DT-preF (2 pg / ml), o / n 4°C, washed, blocked with 1% BSA and then incubated at 37°C for 12-18 hrs with culturegenerated memory B cells from Day 35 spleens, with a 2- fold serial dilution starting at 2-4 x 106. Plates were then washed and incubated with Biotin-goat anti-Mouse IgG Fc specific (Jackson 115-065-071), Ihr RT. Plates were then washed. streptavidin-HRP (Jackson 016- 030-084) was added, and the reaction was developed with AEC substrate (BD 551951). Plates were scanned and analyzed with a CTL ELISpot Scanner.

[0275] Cotton Rat studies: To determine neutralization titers elicited by injection of DT-preF vs. a live virus infection control in the cotton rat model of RSV challenge, a prime-boost immunization scheme was utilized. Experiments were performed utilizing NIH and United States Department of Agriculture guidelines. The Public Health Service Policy on Humane Care and Use of Laboratory Animals, and experimental protocols approved by the Baylor College of Medicine’s Investigational Animal Care and Use Committee (IACUC; Protocol # AN-2307). 105 PFU / rat of virus (RSV / A / Tracy for Fig. 7) was intranasally administered to lightly anesthetized animals in the live RSV groups only on dayO, and for all animals on the day of challenge (day 35 or 49).

[0276] 5 cotton rats / group (roughly equivalent maleTemale ratio) were injected intramuscularly with DT-preF at 2 pg or 10 pg plus Alhydrogel 2% (ALH; Brenntag). The second vaccination (boost) was given on day 21 followed by intranasal challenge on day 35. On day 39 (4 days post challenge), animals were euthanized, and the lungs were harvested for determination of viral lung titers and histopathology according to established protocols. On all indicated days (day 0, day21, day 35 and day 39) blood was obtained for serum analysis including microneutralization (Nt) assays for serum neutralizing antibodies to RSV / A / Tracy and RSV / B / 18537 performed with HEp-2 cells following heat-inactivation at 56°C for 30 min. Serial two-fold dilutions in duplicates starting at 3-log2 were performed to determine the neutralizing antibody (Ab) titer for each sample. The neutralizing antibody titer is defined as the serum dilution at which >50 % reduction in viral cytopathic effect (CPE) is observed. As an internal standard, Palivizumab was included at 40 pg / mL.

[0277] Direct Binding ELISA Assay: Purified DS-Cavl and DT-preF antigens were coated on 96- Well Flat-Bottom Immuno PolySorp Plates (Thermo Fisher) at a starting concentration of 0.5ug / mL or l ug / mL depending on the primary’ antibody used for probing. The plates were incubated at 37°C for Hi 40m or overnight at 4°C. Post- incubation and blocking, the plates were washed and incubated with Motavizumab at 5 ug / mL. D25 at 1 Oug / mL, or MPE8 at 1 Oug / mL, in 1 % BSA, as the primary antibodies at 37°C for Ih. Post-incubation and extensive washing, the plates were incubated with anti-human secondary antibody (Peroxidase AffiniPure F(ab')2 Fragment Goat Anti -Human IgG, Jackson Immunochemicals) in 1 % BSA at RT for Ih for the Motavizumab plate and 37°C for ih for the D25 and MPE8 plates. The plates were finally washed extensively again, developed with I -step TMB ELISA substrate solution (Thermo), stopped with Sulfuric Acid solution and read in a NOVOStar plate reader at 450nm wavelength.

[0278] Pure DT-preF Fluorescence Analysis: Crosslinked protein purified as described herein was serially diluted in PBS in a black 96-well fluorescence plate. To maximize DT-specific fluorescence signal, 0.1 M NaOH was added to each well to a final concentration of 6.25 mM. Plates were read using fluorescence intensity measurement mode on a Novostar (BMG Labtech) plate reader with the PMT / gain set to 4000. Plates were read in triplicate and nonlinear regression data analysis was performed using GraphPad PRISM version 10.1.1.

[0279] Nano Differential Scanning Fluorimetry (nanoDSF): For DSF measurements, the Prometheus Panta instrument as used which measures changes in tryptophan fluorescence intensity and / or the ratio of tryptophan emission at 350 and 330 nm during unfolding of the protein. Tm was determined by taking the 2nd derivative of the F350 / F330 ratios during the thermal shifts from folded to unfolded states of the protein. For analysis, samples were thawed at RT and run at a ramp rate of 1.5 °C / min from 20 °C to 95 °C and fluorescence signals at 330 and 350 nm were collected for 500 ms per capillary in each round. Approximately 375 data points were collected.

[0280] Mass Spectrometry’ Analysis: The LC Elution gradient parameters and instrument information for the mass spectrometry analysis are as follows: MHPLC: Agilent 1100 Binary pump; LC gradient: Total run time 200 min; Buffer A: 98%H2O 2% Acetonitrile with 0.025% TFA; Buffer B: 90% Acetonitrile, 10% H2O 0.025% TFA.

[0281] Statistical analyses: For neutralization titer analysis, groups of animals were compared on the distribution of the neutralization titer outcome using the Mann-Whitney test (Fig. 6A) or two- sample t-test (Fig. 6E). Groups consisted of 10 animals except one animal that was excluded from the DT-preF group in the potency study since it was found dead before the onset of vaccinations. GraphPad Prism and Excel were used to conduct statistical analysis. P-values are two-sided and are considered significant at a one-sided 0.05 level of significance. For the F binding and B-cell ELISPOT analyses, mean comparisons were performed by a 2-tailed paired Student's t test using GraphPad Prism software. The F Binding and ELISPOT analysis was performed at an earlier timepoint after 3 control and 6 aged animal data had been accumulated. Full neutralization titers were performed with male and female animals totaling 12 or 13 per group. Cotton Rat analysis was performed with male and female animals (5 per group). Data was analyzed using a two-sided student’s t-test with a p<0.05 level of statistical significance.

[0282] Example 2

[0283] Incomplete DT Crosslinking leads to Vaccine Compositions with Improved Potency

[0284] While up to 6 DT crosslinks can form to connect the two engineered DT crosslinking sites (one betw een the mutated K226Y residue and the endogenous Y198 residue and one between the mutated VI 85Y residue and the mutated N428Y residue) in the 3 trimers of the preFC molecule, as described above in Example 1, our data showed that in the compositions tested in Example 1, only 65-75% of the RSV F protein contained all 3 inter-protomeric (also referred to as intermolecular) DT bonds. In other studies, we have shown that about 25-35% of the RSV F molecules in our compositions have no inter-protomeric (also referred to as intermolecular) DT bonds. Our data also shows that the intramolecular bond forms to a lesser extent, and that there is a total of between 4 and 5 DT bonds per trimer on average. Of note, this is the case even though the compositions contain only multimeric (trimeric) molecules (as described above, multimeric (trimeric) molecules the compositions where purified following DT crosslinking based on size). Thus, it is not necessary that all 6 of the possible DT bonds described above (3 possible K226Y - to Y 198 bonds, and 3 possible V185Y - to N428Y bonds) form for the RSV DT-preF molecule to be sufficiently stabilized in its trimeric, pre-fusion conformation and be effective as a vaccine immunogen. Indeed, and surprisingly, data from additional experiments that we performed showed that vaccine compositions in which less that all of these 6 possible DT bonds form are superior to those in which all 6 DT bonds form, such that incomplete crosslinking is actually preferable. Fig. 12 provides data showing neutralization titers achieved following administration of RSV F molecules / compositions having differing levels of DT crosslinking (marked as “low,” “medium,” and “high” levels of DT crosslinking in Fig. 12). The data in Fig. 12 shows that neutralization titers are dramatically increased in the “medium” DT cross linking group (mean neutralization titer of 29,602) as compared to the “low ” and “high” DT crosslinking groups (having mean neutralization titers of 14,351 and 12,068, respectively). The degree of DT crosslinking can be controlled by adjusting the DT crosslinking reaction conditions until the desired DT crosslinking profile is achieved, for example to achieve a final composition in which about 25-35% of the RSV F molecules have no inter-protomeric (also referred to as intermolecular) DT bonds, and / or a final composition in w hich there is a total of about 4-5 DT bonds per trimer on average.

[0285] Reference List

[0286] 1 Andersen. S. O. The Crosslinks in Resilin Identified as Dityrosine and Trityrosine. Biochim Biophys Acta 93, 213-215, doi: 10.1016 / 0304-4165(64)90289-2 (1964).

[0287] 2 Downie, J. W., Labella, F. S. & West, M. An insoluble, dityrosine-containing protein from uterus. Biochim Biophys Acta 263, 604-609, doi: 10.1016 / 0005-2795(72)90041-4 (1972).

[0288] 3 LaBella, F., Keeley, F., Vivian, S. & Thornhill, D. Evidence for dityrosine in elastin. Biochem Biophys Res Commun 26, 748-753, doi:10. 1016 / s0006-291x(67)80137-2 (1967).

[0289] 4 Totsune, H.. Nakano, M. & Inaba, H. Chemiluminescence from bamboo shoot cut. Biochem Biophys Res Commun 194, 1025-1029, doi:10.1006 / bbrc, 1993.1924 (1993). 5 van Eldijk, M. B., McGann. C. L., Krick, K. L. & van Hest, J. C. Elastomeric polypeptides. Top Curr Chem 310, 71 -1 16, doi: 10.1007 / 128_201 1 205 (2012).

[0290] 6 Whittaker, J. L., Dutta, N. K., Elvin, C. M. & Choudhury, N. R. Fabrication of highly elastic resilin / silk fibroin based hydrogel by rapid photo-crosslinking reaction. J Mater Chem B 3, 6576-6579, doi: 10.1039 / c5tb00970g (2015).

[0291] 7 Malencik, D. A. & Anderson, S. R. Dityrosine formation in calmodulin: crosslinking and polymerization catalyzed by Arthromyces peroxidase. Biochemistry 35, 4375-4386, doi: 10.1021 / bi9526037 (1996).

[0292] 8 Malencik, D. A., Sprouse, J. F., Swanson, C. A. & Anderson, S. R. Dityrosine: preparation, isolation, and analysis. Anal Biochem 242. 202-213. doi: 10. 1006 / abio. 1996.0454 (1996).

[0293] 9 Elvin, C. M. et al. Synthesis and properties of crosslinked recombinant pro-resilin. Nature 437, 999-1002, doi: 10.1038 / nature04085 (2005).

[0294] 10 Gill, G., Richter-Rusli, A. A., Ghosh, M., Burrows, C. J. & Rokita, S. E. Nickel-dependent oxidative crosslinking of a protein. Chem Res Toxicol 10, 302-309, doi: 10.1021 / tx960170i (1997).

[0295] 11 Partlow, B. P. et al. Highly tunable elastomeric silk biomaterials. Adv Funct Mater 24, 4615-4624, doi: 10.1002 / adfm.201400526 (2014).

[0296] 12Boguta, G. & Dancewicz. A. M. Radiolytic and enzymatic dimerization of tyrosyl residues in insulin, ribonuclease, papain and collagen. Int J Radiat Biol Relat Stud Phys Chem Med 43, 249-265, doi: 10. 1080 / 09553008314550301 (1983).

[0297] 13 Brown, K. C., Yu, Z., Burlingame, A. L. & Craik, C. S. Determining protein-protein interactions by oxidative crosslinking of a glycine-glycine-histidine fusion protein. Biochemistry 37. 4397-4406, doi: 10.1021 / bi9728046 (1998).

[0298] 14 Horowitz, E. D., Finn, M. G. & Asokan, A. Tyrosine crosslinking reveals interfacial dynamics in adeno-associated viral capsids during infection. ACS Chem Biol 7, 1059-1066, doi: 10.1021 / cb3000265 (2012).

[0299] 15 Marshall, C., Hoffman, A., Errico, JP., Marshall, P. Stabilized Proteins. USA patent (2006).

[0300] 16 Marshall, e. a. Conformationally Stabilized RSV Pre-fusion F Proteins. USA patent.

[0301] 17 Marshall, C. Conformationally Specific Viral Immunogens. USA patent (2018).

[0302] 18 Dombkowski, A. A., Sultana, K. Z. & Craig, D. B. Protein disulfide engineering. FEBS Lett 588, 206-212, doi: 10. 1016 / j.febslet.2013.11.024 (2014).

[0303] 19 McLellan. J. S. et al. Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus. Science 342, 592-598, doi: 10. 1126 / science. 1243283 (2013).

[0304] 20 Chatzis, O. et al. Burden of severe RSV disease among immunocompromised children and adults: a 10 year retrospective study. BMC Infect Dis 18, 111, doi: 10.1186 / s 12879-018-3002- 3 (2018).

[0305] 21 Nair, H. et al. Global burden of acute lower respiratory' infections due to respiratory' syncytial virus in young children: a systematic review and meta-analysis. Lancet 375, 1545- 1555, doi: 10.1016 / S0140-6736(10)60206-1 (2010). 22 Walsh, E. E. Respiratory Syncytial Virus Infection: An Illness for All Ages. Clin Chest Med 38, 29-36, doi: 10.1016 / j.ccm.201 .1 1.010 (2017).

[0306] 23 Kim, H. W. et al. Respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine. Am J Epidemiol 89, 422-434, doi: 10.1093 / oxfordjoumals.aje.al20955 (1969).

[0307] 24 Knudson, C. J., Hartwig, S. M., Meyerholz, D. K. & Varga, S. M. RSV vaccine-enhanced disease is orchestrated by the combined actions of distinct CD4 T cell subsets. PLoS Pathog 11, el004757, doi : 10.1371 / joumal.ppat.1004757 (2015).

[0308] 25 Polack, F. P. et al. A role for immune complexes in enhanced respiratory syncytial vims disease. J Exp Med 196. 859-865, doi: 10.1084 / jem.20020781 (2002).

[0309] 26 Anderson, L. J. et al. Strategic priorities for respiratory syncytial virus (RSV) vaccine development. Vaccine 31 Suppl 2, B209-215, doi:10.1016 / j.vaccine.2012.11.106 (2013).

[0310] 27 McLellan, J. S. Neutralizing epitopes on the respiratory- syncytial virus fusion glycoprotein. Curr Opin Virol 11, 70-75, doi: 10. 1016 / j.coviro.2015.03.002 (2015).

[0311] 28Ngwuta, J. O. et al. Prefusion F-specific antibodies determine the magnitude of RSV neutralizing activity in human sera. Sci Transl Med 7. 309ral62, doi : 10. 1126 / scitranslmed. aac4241 (2015).

[0312] 29Rossey, I., McLellan, J. S., Saelens, X. & Schepens, B. Clinical Potential of Prefusion RSV F-specific Antibodies. Trends Microbiol 26. 209-219, doi: 10. 1016 / j.tim.2017.09.009 (2018).

[0313] 30 Simoes, E. A. F. et al. Past, Present and Future Approaches to the Prevention and Treatment of Respiratory' Syncytial Virus Infection in Children. Infect Dis Ther 7, 87-120, doi:10.1007 / s40121-018-0188-z (2018).

[0314] 31 Kampmann, B., Radley, D. & Munjal, I. Bivalent Prefusion F Vaccine in Pregnancy to Prevent RSV Illness in Infants. Reply. N Engl J Med 389, 1053-1055, doi: 10.1056 / NEJMc2307729 (2023)’

[0315] 32Papi, A. et al. Respiratory Syncytial Virus Prefusion F Protein Vaccine in Older Adults. N Engl J Med 388, 595-608, doi:10.1056 / NEJMoa2209604 (2023).

[0316] 33 Schmoele-Thoma, B. et al. Vaccine Efficacy in Adults in a Respiratory Syncytial Virus Challenge Study. N Engl J Med 386, 2377-2386, doi: 10. 1056 / NEJMoa2116154 (2022).

[0317] 34 Aliprantis, A. O. et al. A phase 1. randomized, placebo-controlled study to evaluate the safety and immunogenicity of an mRNA-based RSV prefusion F protein vaccine in healthy younger and older adults. Hum Vaccin Immunother 17. 1248-1261. doi: 10. 1080 / 21645515.2020. 1829899 (2021).

[0318] 35 Capella, C. et al. Prefusion F, Postfusion F, G Antibodies, and Disease Severity in Infants and Young Children With Acute Respiratory Syncytial Virus Infection. J Infect Dis 216, 1398-1406, doi: 10.1093 / infdis / jix489 (2017).

[0319] 36Krarup, A. et al. A highly stable prefusion RSV F vaccine derived from structural analysis of the fusion mechanism. Nat Commun 6, 8143, doi: 10. 1038 / ncomms9143 (2015).

[0320] 37Ye, C., Dormitzer, P., Gribenko, A., Handke, L., Prasad, A., Qiu, X., Ruppen, M., Song, Xi,. Swanson, K., Kodali, S., Xu, X., Efferen, K., Cai, P., Tompkins, K., Pilar Nunez, L. .

[0321] RSV F Protein Mutants. USA patent (2017). 38 Zhang, B. et al. Protection of calves by a prefusion-stabilized bovine RSV F vaccine. NPJ Vaccines 2, 7, doi: 10.1038 / s41541 -017-0005-9 (2017).

[0322] 39 Zhang, L. et al. Design and characterization of a fusion glycoprotein vaccine for Respiratory’ Syncytial Virus with improved stability. Vaccine 36, 8119-8130. doi:10.1016 / j.vaccine.2018.10.032 (2018).

[0323] 40 Chappell, K. J., Watterson, D., Young, P.R. Chimeric Molecules and Uses Thereof. U.S.A, patent (2018).

[0324] 41 Watterson, D. et al. Preclinical development of a molecular clamp-stabilised subunit vaccine for severe acute respiratory syndrome coronavirus 2. Clin Transl Immunology’ 10, el269, doi: 10.1002 / cti2.1269 (2021).

[0325] 42 Joyce, M. G. et al. Iterative structure-based improvement of a fusion-glycoprotein vaccine against RSV. Nat Struct Mol Biol 23, 811-820, doi: 10.1038 / nsmb.3267 (2016).

[0326] 43 Walsh, E. E. et al. Efficacy and Safety of a Bivalent RSV Prefusion F Vaccine in Older Adults. N Engl J Med 388, 1465-1477, doi: 10.10 6 / NEJMoa2213836 (2023).

[0327] 44 Coleman, B. L., Sanderson, R., Haag, M. D. M. & McGovern, I. Effectiveness of the MF59-adjuvanted trivalent or quadrivalent seasonal influenza vaccine among adults 65 years of age or older, a systematic review and meta-analysis. Influenza Other Respir Viruses 15, 813-823, doi: 10. 111 l / irv.12871 (2021).

[0328] 45 Lee. J. K. H. et al. Efficacy and effectiveness of high-dose influenza vaccine in older adults by circulating strain and antigenic match: An updated systematic review and meta- analysis. Vaccine 39 Suppl 1, A24-A35. doi: 10. 1016 / j.vaccine.2020.09.004 (2021).

[0329] 46 Gilman, M. S. et al. Characterization of a Prefusion-Specific Antibody That Recognizes a Quaternary’, Cleavage-Dependent Epitope on the RSV Fusion Glycoprotein. PLoS Pathog 11, el005035, doi: 10. 1371 / joumal.ppat.1005035 (2015).

[0330] 47 Leemans, A. et al. Characterization of the role of N-glycosylation sites in the respiratory svncytial virus fusion protein in virus replication, syncytium formation and antigenicity'. Virus Res 266, 58-68, doi: 10.1016 / j.virusres.2019.04.006 (2019).

[0331] 48 McLellan, J. S., Ray, W. C. & Peeples, M. E. Structure and function of respiratory' syncytial virus surface glycoproteins. Curr Top Microbiol Immunol 372, 83-104. doi : 10. 1007 / 978-3-642-38919- 1 _4 (2013).

[0332] 49 Kwakkenbos, M. J. et al. Generation of stable monoclonal antibody -producing B cell receptor-positive human memory B cells by genetic programming. Nat Med 16, 123-128, doi: 10.1038 / nm.2071 (2010).

[0333] 50 Eichinger, K. M. et al. Prefusion RSV F Immunization Elicits Th2-Mediated Lung Pathology in Mice When Formulated With a Th2 (but Not a Thl / Th2-Balanced) Adjuvant Despite Complete Viral Protection. Front Immunol 11, 1673, doi:10.3389 / funmu.2020.01673 (2020).

[0334] 51 Eichinger, K. M. et al. Maternal immunization with adjuvanted RSV prefusion F protein effectively protects offspring from RSV challenge and alters innate and T cell immunity7. Vaccine 38, 7885-7891, doi: 10.1016 / j.vaccine.2020.10.065 (2020).

[0335] 52 Kosanovich, J. L. et al. Formulation of the prefusion RSV F protein with a Thl / Th2- balanced adjuvant provides complete protection without Th2-skewed immunity7in RSV- experienced young mice. Vaccine 38, 6357-6362, doi: 10. 1016 / j.vaccine.2020.08.023 (2020). 53 Le Nouen, C. et al. Genetic stability of genome-scale deoptimized RNA virus vaccine candidates under selective pressure. Proc Natl Acad Sci U S A 1 14, E386-E395, doi: 10.1073 / pnas. 1619242114 (2017).

[0336] 54 Marcandalli, J. et al. Induction of Potent Neutralizing Antibody Responses by a Designed Protein Nanoparticle Vaccine for Respiratory Syncytial Virus. Cell 176, 1420-1431 el417, doi:10.1016 / j.cell.2019.01.046 (2019).

[0337] 55 Mazur, N. I. et al. Respiratory syncytial virus prevention within reach: the vaccine and monoclonal antibody landscape. Lancet Infect Dis 23, e2-e21, doi: 10.1016 / S1473- 3099(22)00291-2 (2023).

[0338] 56 Stobart, C. C. et al. A live RSV vaccine with engineered thermostability is immunogenic in cotton rats despite high attenuation. Nat Commun 7, 13916, doi: 10. 1038 / ncommsl3916 (2016).

[0339] 57 Stuart, A. S. V. et al. Phase l / 2a Safety and Immunogenicity of an Adenovirus 26 Vector Respiratory Syncytial Virus (RSV) Vaccine Encoding Prefusion F in Adults 18-50 Years and RSV-Seropositive Children 12-24 Months. J Infect Dis 227, 71-82, doi: 10. 1093 / infdis / jiac407 (2022).

[0340] 58 Papi, A. et al. Respiratory Syncytial Virus Prefusion F Protein Vaccine in Older Adults. N Engl J Med 388, 595-608, doi: 10.1056 / NEJMoa2209604 (2023).

[0341] 59 Sahni, L. C. et al. Sustained Within-season Vaccine Effectiveness Against Influenza- associated Hospitalization in Children: Evidence From the New Vaccine Surveillance Network, 2015-2016 Through 2019-2020. Clin Infect Dis 76, el031-el039, doi: 10.1093 / cid / ciac577 (2023).

[0342] 60 Ou, L. et al. Structure-based design of a single-chain triple-disulfide-stabilized fusion- gly coprotein trimer that elicits high-titer neutralizing responses against human metapneumovirus. PLoS Pathog 19, el011584, doi: 10. 1371 / joumal.ppat. 1011584 (2023).

[0343] 61 Stewart-Jones, G. B. E. et al. Structure-based design of a quadrivalent fusion glycoprotein vaccine for human parainfluenza vims types 1-4. Proc Natl Acad Sci U S A 115, 12265- 12270, doi: 10.1073 / pnas. l811980115 (2018).

[0344] 62 Stewart-Jones, G. B. E. et al. Interprotomer disulfide-stabilized variants of the human metapneumovirus fusion glycoprotein induce high titer-neutralizing responses. Proc Natl Acad Sci U S A 118, doi: 10.1073 / pnas.2106196118 (2021).

[0345] 63 Starcher, B. A ninhydrin-based assay to quantitate the total protein content of tissue samples. Anal Biochem 292. 125-129, doi: 10. 1006 / abio.2001.5050 (2001).

Claims

CLAIMSWe claim:

1. A vaccine composition comprising soluble, trimeric, RSV F molecules di-tyrosine crosslinked in the pre-fusion (pre-F) conformation, wherein each monomer in the trimeric RSV F molecules comprises: an Fl domain, an F2 domain, and a trimerization domain, and wherein each monomer in the trimeric RSV F molecules comprises at least two introduced to-tyrosine mutations, and wherein the di-tyrosine crosslinks connect paired tyrosine residues in the RSV F molecules, wherein at least one tyrosine residue in each pair is an introduced to-tyrosine mutation, and wherein (a) about 25-35% of the RSV F molecules in the composition have no intermolecular di-tyrosine crosslinks, and (b) about 65-75% of the RSV F molecules in the composition have at least 2 intermolecular di-tyrosine crosslinks.

2. A vaccine composition according to claim 1, wherein the average number of dityrosine crosslinks between paired tyrosine residues is 4-5 per trimer.

3. A vaccine composition according to claim 1 , wherein the average number of dityrosine crosslinks between paired tyrosine residues is from about 3.5 to about 4.4 per trimer.

4. A vaccine composition according to claim 1, wherein the average number of dityrosine crosslinks between paired tyrosine residues is from about 4.5 to about 5.4 per trimer.

5. A vaccine composition according to any of the preceding claims, wherein each monomer in the trimeric RSVF molecule comprises a to-tyrosine mutation at two or more of ammo acid residues: 77, 88, 97, 147, 150, 155, 159, 183, 185, 187, 220, 222, 223, 226, 255, 427, 428, and 469, or at or at amino acid residues that correspond to these amino acid residues as determined by alignment to, and using the amino acid numbering of, SEQ ID NO.1.

6. A vaccine composition according to any of the preceding claims, wherein the trimeric RSVF molecule comprises di-tyrosine crosslinks between at least two of the following paired tyrosine residues: 147 and 286 198 and 220; 198 and 222; 198 and 223; 198 and 226; 33 and 469; 77 and 222; 88 and 255; 97 and 159; 183 and 427; 185and 427; 185 and 428; and 187 and 427, or at or at amino acid residues that correspond to these amino acid residues as determined by alignment to. and using the amino acid numbering of, SEQ ID NO. 1.

7. A vaccine composition according to any of the preceding claims, wherein the trimeric RSVF molecule comprises a tyrosine residue at each of amino acid positions 185, 198, 226 and 428.

8. A vaccine composition according to any of the preceding claims, comprising: (a) trimeric RSV F molecules that have an intermolecular di-tyrosine crosslink connecting a tyrosine at amino acid residuel85 of one monomer within the trimer with a tyrosine at amino acid residue 428 of a different monomer within the trimer, and (b) trimeric RSV F molecules that have an intramolecular di-tyrosine crosslink connecting a tyrosine at amino acid residue 226 with a tyrosine at amino acid residue 198.

9. The vaccine composition according to claim 7 or claim 8, wherein: (a) about 25-35% of the RSV molecules in the composition have no intermolecular di-tyrosine crosslinks connecting a tyrosine at amino acid residue 185 of one monomer within the trimer with a ty rosine at amino acid residue 428 of a different monomer withing the trimer, and about 65-75% of the RSV molecules in the composition have at least 2 intermolecular di-tyrosine crosslinks, wherein the at least 2 intermolecular di-tyrosine crosslinks connect a tyrosine at amino acid residue 185 of one monomer within the trimer with a ty rosine at amino acid residue 428 of a different monomer withing the trimer.

10. The vaccine composition of any of the preceding claims, wherein each monomer in the soluble, mature, trimeric RSVF molecules comprises: (a) an F2 polypeptide consisting of amino acid residues 26-109 of SEQ ID NO 81, and (b) an Fl polypeptide consisting of amino acid residues 137-513 of SEQ ID NO. 81.

11. The vaccine composition of any of the preceding claims, further comprising an adjuvant selected from the group consisting of alum, CpG, alum+CpG, and Advax.

12. The vaccine composition of any of the preceding claims, wherein the trimerization domain comprises: a foldon domain, a GCN4 domain, a T4 fibrinitin domain, a human C-propeptide of al (I) collagen, or a 6-helical bundle domain from the post-fusion form of a retroviral fusion protein, Syncytin-1, Syncytin-2. HERV-K ENV, or ERV3.

13. The vaccine composition of any of the preceding claims, wherein the trimerization domain comprises the foldon domain of SEQ ID NO. 93.

14. A method of vaccinating a subject against RSV infection, wherein the method comprises administering to the subject an effective amount of a vaccine composition according to any of the preceding claims.

15. The method of claim 13, wherein the subject is a human subject.

16. The method of claim 14, wherein the subject is a non-elderly human adult subject.

17. The method of claim 15. wherein the amount of the RSVF molecules administered is from about 1 mcg to about 2 mgs.

18. The method of claim 14, wherein the subject is an elderly human subject.

19. The method of claim 17. wherein the amount of the RSVF molecules administered is from about 20 mcgs to about 10 mgs.

20. The method of claim 17, wherein the amount of the RSVF molecules administered is about 4.5 times the amount administered to a non-elderly adult human subject.

21. The method of any of claims 17-19, wherein the subject is at least 75 years old.

22. The method of any of claims 17-19, wherein the subject is at least 80 years old.

23. The method of any of claims 17-19, wherein the subject is at least 85 years old.

24. The method of any of claims 17-19, wherein the subject is at least 90 years old.

25. The method of any of claims 13-33, wherein the vaccination method results in protection of greater than 40% against disease caused by RSV infection.

26. The method of any of claims 13-33, wherein the vaccination method results in protection of greater than 50% against disease caused by RSV infection.

27. The method of any of claims 13-33, wherein the vaccination method results in protection of greater than 60% against disease caused by RSV infection.

28. The method of any of claims 13-33, wherein the vaccination method results in protection of greater than 70% against disease caused by RSV infection.

29. The method of any of claims 13-33, wherein the vaccination method results in protection of greater than 80% against disease caused by RSV infection.

30. The method of any of claims 13-33, wherein the vaccination method results in protection of greater than 90% against disease caused by RSV infection.

31. The method of any of claims 13-33, wherein the vaccination method results in protection of greater than 95% against disease caused by RSV infection.

32. A method of vaccinating an elderly subject against RSV infection, wherein the method comprises administering to the subj ect an effective amount of a vaccine composition comprising soluble, trimeric, RSV F molecules di-tyrosine crosslinked in the pre-fusion (pre-F) conformation, wherein each monomer in the trimeric RSV F molecules comprises: an Fl domain, an F2 domain, and a trimerization domain, and wherein each monomer in the trimeric RSV F molecules comprises at least two introduced to-tyrosine mutations, and wherein the di-tyrosine crosslinks connect paired tyrosine residues in the RSV F molecules, wherein at least one tyrosine residue in each pair is an introduced to-tyrosine mutation.

33. The method of claim 32, wherein the subject is a human subject.

34. The method of claim 33, wherein the amount of the RSVF molecules administered is from about 20 mcgs to about 10 mgs.

35. The method of claim 33, wherein the amount of the RSVF molecules administered is about 4.5 times the amount administered to a non-elderly adult human subject.

36. The method of any of claims 32-35, wherein the subject is at least 75 years old.

37. The method of any of claims 32-35, wherein the subject is at least 80 years old.

38. The method of any of claims 32-35, wherein the subject is at least 85 years old.

39. The method of any of claims 32-35, wherein the subject is at least 90 years old.

40. The method of any of claims 32-39, wherein the vaccination method results in protection of greater than 40% against disease caused by RSV infection.

41. The method of any of claims 32-39, wherein the vaccination method results in protection of greater than 50% against disease caused by RSV infection.

42. The method of any of claims 32-39, wherein the vaccination method results in protection of greater than 60% against disease caused by RSV infection.

43. The method of any of claims 32-39, wherein the vaccination method results in protection of greater than 70% against disease caused by RSV infection.

44. The method of any of claims 32-39, wherein the vaccination method results in protection of greater than 80% against disease caused by RSV infection.

45. The method of any of claims 32-39, wherein the vaccination method results in protection of greater than 90% against disease caused by RSV infection.

46. The method of any of claims 32-39, wherein the vaccination method results in protection of greater than 95% against disease caused by RSV infection.

47. The method of any one of claims 32-46, wherein the48. The method of any one of claims 32-47, wherein each monomer in the trimeric RSVF molecules in the vaccine comprises a lo-tyrosine mutation at two or more of amino acid residues: 77, 88, 97, 147, 150, 155, 159, 183, 185, 187, 220, 222, 223, 226, 255, 427, 428, and 469, or at or at amino acid residues that correspond to these amino acid residues as determined by alignment to, and using the amino acid numbering of, SEQ ID NO. 1.

49. The method of any one of claims 32-48, wherein the trimeric RSVF molecules in the vaccine composition comprise di-tyrosine crosslinks between at least two of the following paired tyrosine residues: 147 and 286; 198 and 220; 198 and 222; 198 and 223; 198 and 226; 33 and 469; 77 and 222; 88 and 255; 97 and 159; 183 and 427; 185 and 427; 185 and 428; and 187 and 427. or at or at amino acid residues that correspond to these amino acid residues as determined by alignment to, and using the amino acid numbering of, SEQ ID NO. 1.

50. The method of any one of claims 32-49, wherein the trimeric RSVF molecules in the vaccine composition comprise a tyrosine residue at each of amino acid positions 185, 198, 226 and 428.

51. The method of any one of claims 32-50, wherein the trimeric RSVF molecules in the vaccine composition comprise (a) an intermolecular di-tyrosine crosslink connecting a tyrosine at amino acid residuel 85 of one monomer within the trimer with a tyrosine atamino acid residue 428 of a different monomer within the trimer, and ( b) an intramolecular di-tyrosine crosslink connecting a tyrosine at amino acid residue 226 with a tvrosine at amino acid residue 198.

52. The method of any one of claims 32-51, wherein each monomer in the trimeric RSVF molecules in the vaccine composition comprises: (a) an F2 polypeptide consisting of amino acid residues 26-109 of SEQ ID NO 81, and (b) an Fl polypeptide consisting of amino acid residues 137-513 of SEQ ID NO. 81.

53. The method of any one of claims 32-52, wherein the vaccine composition further comprises an adjuvant selected from the group consisting of alum, CpG, alum+CpG, and Advax.

54. The method of any one of claims 32-53, wherein the trimerization domain comprises: a foldon domain, a GCN4 domain, a T4 fibrinitin domain, a human C-propeptide of al (I) collagen, or a 6-helical bundle domain from the post-fusion form of a retroviral fusion protein, Syncytin-1, Syncytin-2, HERV-K ENV, or ERV3.

55. The method of any one of claims 32-54, wherein the trimerization domain comprises the foldon domain of SEQ ID NO. 93.

Images