Engineered paramyxovirus soluble fusion (F) proteins and related vaccines

Abstract

The present invention provides engineered soluble F proteins of paramyxoviruses, such as respiratory syncytial virus (RSV), human metapneumovirus (hMPV), and human parainfluenza virus (hPIV). These engineered proteins are stabilized by specific modifications in the wild-type soluble F sequence, such as substitutions in the 023 chain and / or the introduction of engineered disulfide bonds in the 0 hairpin in the F1 subunit. The present invention also provides a nanoparticle vaccine comprising an engineered soluble F immunogen displayed on a self-assembled nanoparticle. The invention also provides methods of using such vaccine compositions in a variety of therapeutic applications, e.g., methods for preventing or treating viral infections, such as RSV, MPV, and PIV infections.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent application claims the benefit of priority to U.S. Provisional Patent Application Nos. 63 / 480,261 (filed January 17, 2023; currently pending) and 63 / 488,985 (filed March 8, 2023; currently pending). The entire disclosures of the above priority applications are incorporated herein by reference in their entirety and for all purposes. Background Art

[0003] Respiratory syncytial virus (RSV), human metapneumovirus (hMPV), and parainfluenza virus (PIV) are enveloped, non-segmented, negative-sense, single-stranded RNA viruses belonging to the family Paramyxoviridae. Among them, RSV has been extensively studied. The RSV genome encodes three envelope glycoproteins and eight nonstructural proteins (NS1, NS2, N, P, M, M2-1, M2-2, and L). The three envelope glycoproteins are the attachment (G) protein, the fusion (F) protein, and the small hydrophobic (SH) protein. F and G are crucial for RSV infectivity and pathogenicity and carry multiple antigenic determinants that are recognized by host neutralizing antibodies (NAbs). As highlighted in a 2015-2016 report from the World Health Organization and the Gates Foundation, as well as a global survey, RSV is a common cause of acute lower respiratory infection (ALRI) in newborns and infants and constitutes a major health burden in developing countries. RSV causes acute respiratory infections, resulting in an estimated 66,000 to 200,000 deaths and 3.5 million hospitalizations in children under five years of age worldwide.

[0004] Over the past decade, a series of advances have been made in RSV vaccine development. First, structural insights into the F protein in both its pre- and post-fusion states and the neutralization of RSV by F-specific NAbs have been achieved. The F protein mediates viral entry and is a primary target for vaccine development. Multiple antigenic sites (AS) on the F protein are recognized by NAbs. Co-crystallization of NAb D25 with F yielded the first atomic structure of pre-fusion F and revealed a novel antigenic site near the apex of the trimer. This site is composed of residues 62 to 69 of F2 and the solvent-exposed portion of F (α4-helix). This structure enables the design of prefusion stabilizing mutations and the modification of other NAbs such as AM1429 and Structural analysis of specific 5C4. Second, strategies based on both epitopes and F proteins have been explored in RSV vaccine development. In early studies, immunogen design by 'epitope transplantation' was shown to be used for the RSV Motavizumab epitope, which led to a proof-of-concept study of an RSV epitope vaccine. A variety of experimental designs with different sets of mutations have been proposed to stabilize the pre-fusion F structure. Third, recent human vaccine trials have revealed the importance of pre-fusion F in NAb elicitation. Compared to the failed post-fusion F vaccine, a rationally designed pre-fusion F trimer (DS-Cav1) showed a 10-fold higher serum NAb response. However, despite these advances, there are still many problems with the current RSV pre-fusion design. For example, after D25 purification, DS-Cav1 showed a poor expression profile with a large number of aggregates and other F forms (F species). Similarly, another major vaccine candidate SC-TM has almost no trimer yield. In negative stain EM analysis, while DS-Cav1 appeared completely monomeric, SC-TM showed closed pre-fusion trimers mixed with post-fusion trimers. A further optimized DS-Cav1 design, termed sc9-10 DS-Cav1, contains inter-protomer disulfide bonds to lock F in a fully closed trimeric conformation. However, such inter-protomer disulfide bonds disrupt the folding of the F-nanoparticle (NP) protein and the assembly of F-presenting NPs, leading to low yields and protein aggregation. In addition, sc9-10 DS-Cav1 contains many mutations generated by random mutagenesis that may or may not be essential for the structure and function of this construct as a vaccine antigen.

[0005] There remains a pressing need in the art for better and more effective vaccines against paramyxoviruses, particularly RSV. The present invention addresses this and other unmet needs in the art. Summary of the Invention

[0006] In one aspect, the present invention provides engineered immunogenic proteins derived from or modified by the fusion (F) protein of a paramyxovirus (e.g., RSV). They comprise a modified soluble F sequence having one or more modifications relative to the wild-type soluble F sequence of a paramyxovirus. Generally speaking, the engineered soluble F protein of the present invention comprises (1) replacement of two or more negatively charged residues around the β23 chain (D486 to A490) with polar or hydrophobic residues, (2) deletion of the P27 peptide (E110 to R136), and (3) an engineered protomer intradisulfide bond located within the F1 subunit or connecting F2 to the F1 subunit. Unless otherwise indicated, the amino acid numbering of the various sequence modifications described herein is based on human RSV A2 strain F protein (UniProt ID P03420). Some engineered soluble F proteins derive from RSV. In some engineered soluble RSV F proteins, the two or more negatively charged residues around the β23 strand are D486 and E487. In some of these embodiments, the replacement around the β23 strand comprises D486N / E487Q or D486L / E487L. In some engineered soluble F proteins of the invention, the engineered disulfide bond is S155C / S290C, S62C / K196C, or E60C / K196C.

[0007] In addition to the above modifications (1) to (3), some engineered soluble RSV F proteins of the invention may further comprise a linker portion that replaces: (1) the furin cleavage site; or (2) the unstructured F2 C-terminus (Q98 to R109) and a portion of the fusion peptide (FP) N-terminus (F137 to V157). In some of these embodiments, the replaced F2 C-terminus comprises residues N104 to R109 ( 104 NNRARR 109; SEQ ID NO:31). In some of these embodiments, the portion of the N-terminus of the fusion peptide (FP) that is replaced comprises F137 to S146. Some engineered soluble F proteins of the present invention further comprise a replacement of residue S215. In some of these embodiments, residue S215 is replaced by P. Some engineered soluble F proteins of the present invention further comprise a replacement of residue E92. In some of these embodiments, residue E92 is replaced by D, Q, other short polar residues, or hydrophobic residues. Some engineered soluble F proteins of the present invention further comprise a replacement of V185P. Some engineered soluble F proteins of the present invention further comprise a replacement of S46G, K462Q, or both. Some engineered soluble F proteins of the present invention further comprise engineered intraprotomer disulfide bonds S180C / S186C or A177C / T189C in the β3 / β4 hairpin. Some engineered soluble F proteins of the present invention may also include engineered inter-protomer disulfide bonds A149C / Y458C. In various embodiments, the engineered soluble RSV F proteins of the present invention may have an amino acid sequence as set forth in any one of SEQ ID NOs: 17 to 23, a conservatively modified variant thereof, or a substantially identical sequence thereof. In some embodiments, the engineered soluble F proteins of the present invention may also include an N-terminal leader sequence. In some embodiments, the engineered soluble F proteins of the present invention may also include a C-terminal foldon motif.

[0008] In a related aspect, the present invention provides nanoparticle vaccines comprising an engineered soluble F protein as described herein, which is displayed on the surface of self-assembling nanoparticles. In some of these embodiments, the self-assembling nanoparticles comprise a trimer sequence, and the C-terminus of the engineered soluble F protein is fused to the N-terminus of the subunit sequence of the nanoparticles. In some embodiments, the self-assembling nanoparticles used in the nanoparticle vaccines of the present invention are I3-01 variants. In some of these embodiments, the subunit sequence of the I3-01 variant comprises SEQ ID NO: 25 (I3-01v9b) or SEQ ID NO: 26 (I3-01v9c).

[0009] In another aspect, the present invention provides polynucleotide sequences encoding the engineered soluble F proteins or nanoparticle vaccines described herein. In another aspect, the present invention provides pharmaceutical compositions comprising the nanoparticle vaccines or polynucleotide sequences described herein and a pharmaceutically acceptable carrier. In another aspect, the present invention provides methods for preventing or treating paramyxovirus infection in a subject. These methods require administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein. Some of these treatment methods are directed to treating or preventing RSV infection.

[0010] In another aspect, the present invention provides different classes of engineered or redesigned immunogenic polypeptides derived from or modified from the fusion protein (F) of a paramyxovirus. These redesigned soluble F immunogens also comprise altered soluble F sequences having one or more modifications relative to the wild-type soluble F sequence of a paramyxovirus. In some of these embodiments, the modifications comprise intraprotomer engineered disulfide bonds linking the β3 / β4 hairpins in the F1 subunit of the soluble F sequence or the corresponding hairpins that form the β sheet amino acid pairs. Unless otherwise indicated, the numbering of the hairpins in the immunogen is based on respiratory syncytial virus (RSV).

[0011] Some of these different classes of engineered soluble F immunogens are derived from the wild-type F sequence of human RSV. In these embodiments, an engineered disulfide bond is introduced between the replacement residues S180C / S186C or A177C / T189C in the β3 / β4 hairpin. The amino acid numbering in these embodiments is based on the human RSV A2 strain with UniProt ID P03420. In some of these embodiments, the wild-type soluble F sequence used is shown in SEQ ID NO: 1 or is a variant or substantially identical sequence thereof through conservative modifications. In some embodiments, the modification relative to the wild-type soluble F sequence also includes a mutation at the C-terminal end of the unstructured F2 subunit. For example, a redesigned RSV soluble F immunogen can include a truncation and / or P102A replacement of residues 104 to 109 (NNRARR) (SEQ ID NO: 31) at the C-terminal end of the unstructured F2. In some embodiments, the modifications relative to the wild-type soluble F sequence further comprise (1) replacing the processed active peptide (P27) (residues E110 to R136) and the N-terminus of the fusion peptide (residues F137 to S146) with a (GS)n linker sequence, wherein n is any integer from 1 to 5, and / or (2) amino acid substitutions I379V and M447V. In some exemplary embodiments, the redesigned RSV soluble F immunogen has an amino acid sequence as set forth in any one of SEQ ID NOs: 36 to 43, or a conservatively modified variant thereof.

[0012] Some of these different classes of engineered soluble F immunogens are derived from the wild-type F sequence of human metapneumovirus (hMPV). In some of these embodiments, an engineered disulfide bond is introduced between the replacement residues A147C / A159C in the β3 / β4 hairpin. The amino acid numbering in these embodiments is based on the hMPV strain CAN97-83 with UniProt ID Q6WB98. In some of these embodiments, the wild-type soluble F sequence used is shown in SEQ ID NO:44 or SEQ ID NO:45 or is a conservatively modified variant or substantially identical sequence thereof. In some embodiments, the modification relative to the wild-type soluble F sequence further comprises a mutation at the C-terminus of the unstructured F2 subunit. In some of these embodiments, the mutation at the C-terminus of the unstructured F2 is a substitution of the unstructured F2 C-terminus DQLAREEQIENP (SEQ ID NO:60) and the cleavage site RQSR (SEQ ID NO:49) with a (GS)n linker sequence, where n is any integer from 1 to 5. In some exemplary embodiments, the redesigned hMPV soluble F immunogen has the amino acid sequence set forth in SEQ ID NO: 46 or SEQ ID NO: 47, or a conservatively modified variant thereof.

[0013] Some of these different classes of engineered soluble F immunogens are derived from soluble F sequences of parainfluenza virus (hPIV). In some of these embodiments, an engineered disulfide bond is introduced between the replacement residues Q159C / A171C in the β1 / β2 hairpin. The amino acid numbering in these embodiments is based on the recombinant hPIV3 / hPIV1 virus with UniProt ID O55888. In some of these embodiments, the wild-type soluble F sequence used is shown in SEQ ID NO: 48 or is a conservatively modified variant or substantially identical sequence thereof. In some embodiments, the modification relative to the wild-type soluble F sequence also includes a mutation at the C-terminus of the F2 subunit. In some of these embodiments, the mutation at the F2 C-terminus is to replace the C-terminal sequence NQESNENTDP (SEQ ID NO: 50) and the cleavage site RTER (SEQ ID NO: 51) with a (GS) n linker sequence, where n is any integer from 1 to 6. In some exemplary embodiments, the redesigned hPIV soluble F immunogen has the amino acid sequence set forth in SEQ ID NO: 61 or SEQ ID NO: 62, or a conservatively modified variant thereof.

[0014] In another aspect, the invention provides engineered or redesigned immunogenic polypeptides derived from the fusion protein (F) of human respiratory syncytial virus (hRSV). In some embodiments, the redesigned hRSV immunogen comprises a modified RSV soluble F sequence that is modified by at least one of the following mutations relative to the wild-type hRSV soluble F sequence: (1) a deletion of the P27 peptide (residue E110 to R136), (2) modification of the unstructured F2 subunit C-terminus (residue Q98 to R109), and (3) a truncation of the fusogenic peptide N-terminus (residue F137 to V157). In these embodiments, amino acid numbering is based on the human RSV A2 strain with UniProt ID P03420. In some of these embodiments, the wild-type RSV soluble F sequence used is shown in SEQ ID NO: 1 or is a conservatively modified variant or substantially identical sequence. In some of these embodiments, the modification of the unstructured F2 C-terminus is (1) truncation of residues 104 to 109 (NNRARR) (SEQ ID NO: 31) and / or (2) substitution of P102A. In some embodiments, the truncation of the fusion peptide N-terminus is deletion of residues F137 to S146.

[0015] In some embodiments, the modifications in the redesigned RSV soluble F immunogen may further comprise (1) a (GS)n linker between the F2 and F1 subunits in the altered soluble RSV sequence, wherein n is any integer from 1 to 6, and / or (2) at least one substitution selected from I379V and M447V, relative to the wild-type soluble F sequence. In some of these embodiments, the linker comprises the sequence GSGS (SEQ ID NO: 27) or GSGSGSGS (SEQ ID NO: 28). In some exemplary embodiments, the redesigned hRSV soluble F immunogen has the amino acid sequence set forth in SEQ ID NO: 34 or SEQ ID NO: 35, or a conservatively modified variant thereof.

[0016] In some embodiments, the modifications in the redesigned RSV soluble F immunogens can further comprise an engineered disulfide bond connecting the β-sheet-forming amino acid pairs in the β3 / β4 hairpin in the F1 subunit relative to the wild-type soluble F sequence. In some of these embodiments, the engineered disulfide bond is introduced between the replacement residues S180C / S186C or A177C / T189C. Some specific examples of these redesigned hRSV soluble F immunogens have the amino acid sequences set forth in any one of SEQ ID NOs: 36, 38, 40, and 42, or conservatively modified variants thereof.

[0017] In some embodiments, the modifications in the redesigned RSV soluble F immunogens may further comprise replacement of one or two amino acid residues between β strands β3 and β4 relative to the wild-type soluble F sequence. In some of these embodiments, the amino acid replacements are S182G and N183P. Some specific examples of these redesigned hRSV soluble F immunogens have the amino acid sequences set forth in any one of SEQ ID NOs: 37, 39, 41, and 43, or conservatively modified variants thereof.

[0018] In addition to the various sequence modifications or mutations described above, some redesigned soluble F immunogens of the present invention may also contain a trimerization motif at the C-terminus. In some of these embodiments, the trimerization motif used is foldon or viral capsid protein SHP.

[0019] In another aspect, the present invention provides a paramyxovirus vaccine composition comprising a redesigned soluble F immunogen described herein displayed on the surface of self-assembling nanoparticles. In some embodiments, the self-assembling nanoparticles comprise a trimer sequence, and the C-terminus of the immunogen polypeptide is fused to the N-terminus of the subunit sequence of the nanoparticle. In another aspect, the present invention provides a pharmaceutical composition comprising a redesigned soluble F immunogen or nanoparticle vaccine described herein, and a pharmaceutically acceptable carrier. In another aspect, the present invention provides a polynucleotide sequence encoding a redesigned soluble F immunogen described herein, or a subunit sequence encoding a vaccine composition displaying a redesigned soluble F immunogen described herein.

[0020] A further understanding of the nature and advantages of the present invention may be obtained by reference to the remaining portions of the specification and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Analysis of the sources of RSV F metastability. (A) Amino acid sequence and secondary structure alignment of RSV F before and after fusion, SEQ ID NO:63 (Figure S3 from McLellan et al., Science 2013, 340:1113-1117). Two potential sources of metastability, the β3 / β4 hairpin and the β23 strand, are circled in dashed boxes. (B) β3 / β4 hairpin (SEQ ID NO:53). Left: Pre- and post-fusion F; Right: Zoomed views of the β3 / β4 strands before and after fusion (potential mutation sites are labeled). (C) β23 strand (SEQ ID NO:64). Left: Side view of β23 in the pre-fusion F trimer structure; Right: Top view of β23 in the pre-fusion F trimer structure (top) and zoomed view of β23 near the three-fold axis (bottom).

[0022] Figure 2 Rational design of I3-01v9b / c for optimized display of trimeric antigens on nanoparticle surfaces. (A) Structural model of I3-01v9a (SEQ ID NO:24), which features an extended N-terminal helix. (B) Schematic diagram of the design process for I3-01v9b / c (SEQ ID NO:25). (C) nsEM analysis of the EBOV GP-I3-01v9b trimer. Top: 2D classification; Bottom: Side and top views of the 3D model.

[0023] Figure 3 Design and negative stain EM analysis of nanoparticles displaying RSV prefusion F trimers. (A) Structural modeling of RSV prefusion F trimers on three 1c-SApNPs (including ferritin 24mer and two 60mer E2p and I3-01v9b). (B) EM analysis of DS-Cav1 on three 1c-SApNPs. (C) EM analysis of sc9-10 DS-Cav1 on three 1c-SApNPs. (D) V2-Ext-P2DB6-D-L2 and NQ on FR and NQ on I3-01v9b 1c-SApNPs. (E) V2-Ext-P2DB6-GDQ-L2 and NQ on FR and NQ on I3-01v9b 1c-SApNPs. For (D) and (E), magnified views of F-FR nanoparticles with closed pre-F trimers on their surfaces are shown.

[0024] Figure 4 Negative stain EM images of RSV F trimers before fusion on nanoparticle platforms. (A) EM images of DS-Cav1, sc9-10 DS-cav1, and SC-TM RSV F displayed on ferritin nanoparticles. DS-Cav1-FR5 failed to properly form nanoparticles; sc9-10-DS-Cav-FR5 formed nanoparticles, but the F trimer appeared open on the ferritin particle surface (one such particle is marked with a red box); SC-TM-FR also failed to properly form nanoparticles. (B) EM images of the two main F trimer designs on ferritin and E2p particles. Column 1: V2-Ext-SSGP on ferritin with a 10GS linker and purified by D25 and MPE8; Column 2: V2-Ext-AT on ferritin with a 5GS linker and purified by D25 and MPE8; Column 3: V2-Ext-AT on E2p 60-mer with a locking domain (LD4) and with LD4 and the T cell epitope PADRE. All newly designed F trimers have a well-formed closed prefusion conformation on the nanoparticle surface (one such V2-Ext-AT-FR5 particle is highlighted). DETAILED DESCRIPTION

[0025] I. Overview

[0026] The present invention relates to the RSV F of the present invention.As Class I virus fusion protein, RSV F has intrinsic metastability, and compared with other Class I fusion proteins such as HIV-1 envelope glycoprotein (Env), it can show with different forms.First, before fusion, RSV F is very unstable, and is easy to make its conformational change to fusion post-state, and this is a kind of phenomenon that has been widely studied in this area.Secondly, before RSV merges, F trimer is easy to dissociate into monomer or become open trimer, unless it is locked by disulfide bond between protomer, this will have a negative effect on F trimer before the fusion of multivalent display on nanoparticle by gene fusion method.

[0027] The present invention is partly derived from research conducted by the inventors, which aims to rationally design new, stable RSV pre-fusion F trimers by minimizing F metastability. The inventors first examined the expression, purification and structure of three known pre-fusion RSV F designs, DS-Cav1, SC-TM and sc9-10 DS-Cav1. It was observed that DS-Cav1 and SC-TM produced a large amount of aggregates and non-trimeric F forms, while sc9-10 DS-Cav1 showed a single trimer peak with high yield and high purity. In negative staining EM analysis, it was found that DS-Cav1 and SC-TM were monomers and monomer / trimer mixtures, respectively, while sc9-10 DS-Cav1 showed a highly purified closed pre-fusion F trimer. When displayed on protein nanoparticles (NPs), all three pre-fusion F designs showed poor performance with low yield and low purity. At the core of the present invention, the inventors have explored new mutations that can significantly reduce the metastability of the pre-fusion F conformation, for example, the RSV F mutant protein in the pre-fusion conformation achieved by engineered disulfide bonds (e.g., S155C / S290C exemplified herein). To this end, the inventors have found that mutations in negatively charged residue pairs (e.g., D486 and E487) in the β23 chain (D486 to A490) can greatly stabilize F in a closed trimeric conformation before fusion.

[0028] The inventors also examined other mutations in the "basic" prefusion F structure (the mutations proposed to be introduced into the β23 chain) that could further improve the antigenic spectrum of the engineered F protein. In addition to the intraprotomer disulfide bond in the β23 chain that locks the RSV F protein in the prefusion conformation, these additional mutations include: a cleavage site linker that replaces the unstructured F2 C-terminus, the P27 peptide, and the fusion peptide; an S215P mutation; and an E92D mutation. This F construct is referred to as "V2-Ext-PDB6-D." It was found that the construct V2-Ext-PDB6-D containing this set of mutations produced high yields and high purity of prefusion F, with only a small fraction of closed trimers. Using this pre-fusion F basic design, the inventors further investigated the V185P mutation in the β3 / β4 hairpin (K176 to S190), as well as two types of mutations in the negatively charged residue pair D486 and E487 in the β23 chain (D486 to A490). The three β23 chains form a repulsive interaction around the three-fold axis directly above the α10 coiled coil. Although the V185P mutation may destabilize the post-fusion F structure, the inventors speculated that the D486-E487 pair would cause the pre-fusion F trimer to open to facilitate cell entry, and thus polar or hydrophobic mutations in these two residues could stabilize the pre-fusion F in a closed trimeric conformation. In fact, negative stain EM analysis of multiple constructs confirmed this hypothesis. Subsequently, the inventors introduced two mutations, S46G and K465Q, into the first basic design to improve the folding of the pre-fusion F. This second basic design was called "V2-Ext-PDB6-GDQ" and was used to test the V185P mutation and the D486-E487 pair of mutations. Although S46G and K462Q did improve the folding of prefusion F, they also reduced the proportion of closed prefusion F trimers. Both V2-Ext-PDB6-D and V2-Ext-PDB6-GDQ derivatives can be developed as soluble trimer vaccines or displayed on single-component self-assembling protein nanoparticles (1c-SApNP) as virus-like particles (VLP) vaccines. Since the F proteins of other paramyxoviruses (such as hMPV and PIV) are structurally similar to RSV F, the same design strategy can be applied to other members of the Paramyxoviridae family.

[0029] In other studies, the present inventors explored different minimal mutation sets that can be introduced into the paramyxovirus fusion (F) glycoprotein to generate stabilized immunogens. As described in detail herein, these paramyxovirus F protein trimer immunogens were redesigned by introducing structural modifications in the soluble F sequence that stabilize the F trimer in the pre-fusion state. Some redesigned soluble F immunogens were stabilized by introducing engineered disulfide bonds in the β hairpin of the F1 subunit. Some redesigned soluble F immunogens were stabilized by introducing other new mutations in the soluble F sequence. The present inventors further displayed the redesigned F trimer immunogens on self-assembling nanoparticles as VLP-type vaccines and developed an industrial production method for the immunogen and a tag-free antigen-specific purification method. Since the hMPV and PIV F proteins are structurally similar to RSV F, the same stabilization design verified by RSV F was also used to redesign hMPV and PIV3 vaccine immunogens. These studies provide a general design strategy for vaccine design based on paramyxovirus pre-fusion F.

[0030] Thus, the present invention provides paramyxovirus immunogens and vaccine compositions according to the design strategies described herein. The present invention also provides related polynucleotide sequences, expression vectors, and pharmaceutical compositions. Unless otherwise indicated herein, the vaccine immunogens, encoding polynucleotides, expression vectors, and host cells of the present invention, as well as related therapeutic applications, can be produced or performed according to the procedures exemplified herein or conventional practices known in the art. See, for example,

[0031] Methods in Enzymology, Volume 289: Solid-Phase Peptide Synthesis, JNAbelson, MISimon, GBFields (Editors), Academic Press; 1st edition (1997) (ISBN-13:978-0121821906); USPat. No. 4,965,343, and 5,849,954; Sambrook et al. al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, (3 rded., 2000); Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1986); or Methods in Enzymology: Guide to Molecular Cloning Techniques Vol.152, SLBerger and ARKimmerl Eds., Academic Press Inc., San Diego, USA (1987); Current Protocols in Protein Science (CPPS) (John E.Coligan, et.al., ed., John Wiley and Sons, Inc.), Current Protocols in Cell Biology (CPCB) (Juan S.Bonifacino et.al.ed., John Wileyand Sons, Inc.), and Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005), Animal Cell Culture Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, Issue, 1998). The following sections provide additional guidance for practicing the compositions and methods of the present invention.

[0032] II. definition

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide a general definition of many of the terms used in this invention for those skilled in the art: Academic Press

[0034] Dictionary of Science and Technology,Morris(Ed.),Academic Press(1 st ed., 1992); Oxford Dictionary of Biochemistry and Molecular Biology, Smith et al. (Eds.), Oxford University Press (revised ed., 2000); Encyclopaedic Dictionary of Chemistry, Kumar (Ed.), Anmol Publications Pvt. Ltd. (2002); Dictionary of Microbiology and Molecular Biology, Singleton et al. al.(Eds.),John Wiley&Sons(3 rd ed., 2002); Dictionary of Chemistry, Hunt (Ed.), Routledge (1 st ed., 1999); Dictionary of Pharmaceutical Medicine, Nahler (Ed.), Springer-Verlag Telos (1994); Dictionary of Organic Chemistry, Kumar and Anandand (Eds.), Anmol Publications Pvt. Ltd. (2002); and A Dictionary of Biology (Oxford Paperback Reference), Martin and Hine (Eds.), Oxford University Press(4 th ed., 2000).

[0035] Further explanation of some of these terms as they apply specifically to the present invention is provided herein.

[0036] As used herein, unless the context clearly indicates otherwise, nouns not qualified by quantifiers refer to both the singular and the plural. For example, "Env-derived trimer" can refer to both single and multiple Env-derived trimer molecules and can be considered equivalent to the phrase "at least one Env-derived trimer".

[0037] As used herein, the terms "antigen" or "immunogen" are used interchangeably and refer to a substance, typically a protein, that is capable of inducing an immune response in a subject. The term also refers to a protein that is immunologically active in the sense that it is capable of eliciting a humoral and / or cellular immune response against the protein upon administration to a subject (either directly or by administering to a subject a nucleotide sequence or vector encoding the protein). Unless otherwise indicated, the term "vaccine immunogen" is used interchangeably with "protein antigen" or "immunogenic polypeptide."

[0038] The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to specific nucleic acid sequences, conservatively modified variants refers to those nucleic acids that encode identical or substantially identical amino acid sequences, or, in the case where the nucleic acid does not encode an amino acid sequence, to substantially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. With respect to polypeptide sequences, "conservatively modified variants" refers to variants having conservative amino acid substitutions, i.e., amino acid residues are replaced with other amino acid residues having similarly charged side chains. Families of amino acid residues having similarly charged side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0039] Epitope refers to an antigenic determinant. These are specific chemical groups or peptide sequences on a molecule that are antigenic, such that they elicit a specific immune response. For example, an epitope is a region of an antigen that responds to B and / or T cells. An epitope can be formed by contiguous amino acids or non-contiguous amino acids juxtaposed by the tertiary folding of a protein.

[0040] The effective amount of vaccine or other medicament is enough to produce the response of expectation, for example, alleviates or eliminates the sign or symptom of illness or disease (for example bronchiolitis or pneumonia).For example, this can be the amount necessary for suppressing viral replication or measurably changing the external symptoms of viral infection. Generally speaking, this amount will be enough to measurably suppress the duplication or infectivity of virus (for example, hRSV).When being applied to object, usually spendable dosage will reach the target tissue concentration that has been shown to realize external suppression of viral replication. In some embodiments, " effective amount " is the amount of one or more symptoms and / or potential cause (for example, for treating RSV infection) of treating (including preventing) any illness or disease. In some embodiments, effective amount is therapeutically effective amount. In some embodiments, effective amount is the amount of one or more signs or symptoms preventing specific disease or illness from occurring (for example, one or more signs or symptoms related to bronchiolitis infection).

[0041] Unless otherwise indicated, fusion protein is a recombinant protein comprising the amino acid sequence from at least two unrelated proteins, which have been linked together by peptide bonds to form a single protein. Therefore, it does not encompass the naturally occurring paramyxovirus surface antigens referred to as fusion (F) proteins as described herein. Unrelated amino acid sequences can be directly connected to each other, or they can be connected using a linker sequence. As used herein, if the amino acid sequence of a protein in its natural environment (e.g., in a cell) is generally found to be not linked together by peptide bonds, the protein is unrelated. For example, the amino acid sequence of a bacterial enzyme (e.g., Bacillus stearothermophilus (B.stearothermophilus) dihydrolipoyl acyltransferase (dihydrolipoyl acyltransferase) (E2p)) and the amino acid sequence of a soluble paramyxovirus F glycoprotein are generally not found to be linked together by peptide bonds.

[0042] An immunogen is a protein or portion thereof that is capable of inducing an immune response in a mammal (e.g., a mammal infected with or at risk of infection by a pathogen). Administration of an immunogen can result in protective immunity and / or active immunity against the pathogen of interest.

[0043] An immunogenic composition refers to a composition comprising an immunogenic polypeptide that induces a measurable CTL response against a virus expressing the immunogenic polypeptide, or induces a measurable B cell response (eg, production of antibodies) against the immunogenic polypeptide.

[0044] The sequence identity or similarity between two or more nucleic acid sequences or two or more amino acid sequences is represented by the identity or similarity between the sequences.Sequence identity can be measured with percentage identity; The higher the percentage, the more identical the sequence.When two sequences are compared and aligned to obtain maximum correspondence in a comparison window or a specified region, if the two sequences have the same amino acid residues or nucleotides of a specified percentage (that is, 60% identity is present in a specified region or when not specified over the entire sequence, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identity), as measured using one of the following sequence comparison algorithms or by artificial alignment and visual inspection, the two sequences are "substantially identical". Optionally, identity is present in a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably in a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[0045] Homologs or orthologs of nucleic acid or amino acid sequences have a relatively high degree of sequence identity / similarity when aligned using standard methods. Sequence alignment methods for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv.

[0046] Appl. Math. 2: 482, 1981; Needleman & Wunsch, J. Mol. Biol. 48: 443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85: 2444, 1988; Higgins & Sharp, Gene, 73: 237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al. Computer Apps. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994.

[0047] Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.

[0048] The term "subject" refers to any animal classified as a mammal, such as humans and non-human mammals. Examples of non-human animals include dogs, cats, cows, horses, sheep, pigs, goats, rabbits, etc. Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably herein. Preferably, the subject is a human.

[0049] The terms "treat" or "alleviate" include administering a compound or agent to a subject to prevent or delay the onset of symptoms, complications, or biochemical markers of a disease (e.g., hRSV infection), alleviate symptoms, or prevent or inhibit further development of a disease, condition, or disorder. Subjects in need of treatment include those already suffering from the disease or disorder and those at risk of developing the disease. Treatment can be prophylactic (preventing or delaying the onset of the disease, or preventing the manifestation of its clinical or subclinical symptoms), or therapeutic suppression or alleviation of symptoms after disease manifestation.

[0050] Vaccine refers to a pharmaceutical composition that causes a prophylactic or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Generally speaking, vaccines cause an antigen-specific immune response against pathogens (e.g., viral pathogens) antigens or against cellular components associated with a pathological condition. Vaccines may include polynucleotides (e.g., nucleic acids encoding disclosed antigens), peptides or polypeptides (e.g., disclosed antigens), viruses, cells, or one or more cellular components. In some embodiments of the invention, vaccines or vaccine immunogens or vaccine compositions are expressed from fusion constructs and self-assembled into nanoparticles that display immunogenic polypeptides or proteins on their surfaces.

[0051] Virus-like particles (VLPs) refer to non-replicative viral capsids derived from any one of a variety of viruses. VLPs are typically composed of one or more viral proteins, such as, but not limited to, those proteins referred to as capsid proteins, coat proteins, shell proteins, surface proteins, and / or envelope proteins, or particle-forming polypeptides derived from these proteins. After recombinant expression of proteins in an appropriate expression system, VLPs can form spontaneously. Methods for producing specific VLPs are known in the art. After recombinant expression of viral proteins, the presence of VLPs can be detected using conventional techniques known in the art (e.g., by electron microscopy, biophysical characterization, etc.). See, for example, Baker et al. (1991) Biophys. J. 60: 1445-1456; and Hagensee et al. (1994) J. Virol. 68: 4503-4505. For example, VLPs can be separated by density gradient centrifugation and / or identified by characteristic density bands. Alternatively, cryo-electron microscopy can be performed on the vitrified aqueous sample of the VLP preparation in question, and images can be recorded under appropriate exposure conditions.

[0052] Self-assembling nanoparticles refer to spherical protein shells with a diameter of tens of nanometers and well-defined surface geometry, which are formed by identical copies of non-viral proteins that can be automatically assembled into nanoparticles with an appearance similar to VLP. A notable example of self-assembling nanoparticles is engineered protein I3-01 (Hsia et al., Nature 535,136-139,2016) and variants derived therefrom, including I3-01v9b and I3-01v9c exemplified herein. Other examples include ferritin (FR), which is conserved between species and forms 24 aggressiveness, as well as Bacillus stearothermophilus dihydrolipoyl acyltransferase (E2p), Aquifex aeolicus dioxotetrahydropteridine synthase (lumazine synthase, LS) and Thermotoga maritima encapsulin, which all form 60 aggressiveness. After protein recombinant expression in an appropriate expression system, self-assembling nanoparticles can form spontaneously. Nanoparticle production, detection, and characterization methods can be performed using the same techniques developed for VLPs.

[0053] III. Paramyxoviruses and fusion (F) glycoprotein

[0054] The present invention provides novel engineered immunogenic proteins and vaccine compositions comprising modified soluble F glycoprotein sequences of paramyxoviruses. Paramyxovirus F proteins are homotrimers. They possess a hydrophobic fusion peptide (FP), two heptad repeat regions (HRA and HRB), which are anchored to the surface by a single-pass transmembrane domain (TM), and contain a C-terminal cytoplasmic tail. Taking RSV as an example, the F gene of paramyxoviruses encodes a type I integral membrane protein synthesized from an inactive 574-amino acid precursor, F0. Three F0 monomers assemble into a trimer, which is activated by a furin-like host protease as it passes through the Golgi apparatus. This protease cleaves once after amino acids 109 and 136, producing three polypeptides. The N-terminal and C-terminal cleavage products are the F2 and F1 subunits, respectively (named in order of size), and are covalently linked to each other by two disulfide bonds. The intervening 27-amino acid peptide, P27, contains two or three N-linked glycans but dissociates upon cleavage. The F2 subunit contains two N-linked glycans, while the larger F1 subunit contains a single N-linked glycosylation site. Unlike other glycans, this F1 glycan is essential for the protein to induce membrane fusion.

[0055] In general techniques, the wild-type soluble F sequence of a paramyxovirus refers to the complete extracellular domain of the fusion glycoprotein (F) of a paramyxovirus. Taking the RSV F protein as an example, the soluble F sequence (amino acids 1 to 529) generally comprises, from its N-terminus to the C-terminus: a leader sequence, followed by the F2 subunit, a processed active peptide (P27) peptide, and a portion of the extracellular domain of the F1 subunit, the N-terminus of which contains a fusion peptide (FP). The deletion of the wild-type soluble F sequence at the C-terminus produces an F construct, which is referred to as Fd (amino acids 1 to 513), which has been used for structural determination of the RSV F protein in pre- and post-fusion conformations. Therefore, the term "wild-type soluble F" as used herein may refer to Fd, and in some embodiments, may also be extended by adding more amino acids to the C-terminus until it contains the full-length extracellular domain portion of the F1 subunit. Unless otherwise indicated, the amino acid numbering of the various components of the RSV soluble F sequence is based on the human RSV A2 strain with accession number P03420 (McLellan et al., J. Virol. 85:7788–96, 2011). In some embodiments, the wild-type soluble RSV F sequence from which the engineered soluble F immunogens of the present invention are derived is shown in SEQ ID NO: 1. Similarly, the amino acid numbering in the soluble F sequences of other paramyxoviruses is also based on the specific virus strains and / or secondary structures described herein.

[0056] hRSV A2 strain "wild type" soluble F sequence (SEQ ID NO: 1):

[0057]

[0058]

[0059] As used herein, the unstructured F2 C-terminus of paramyxovirus F glycoprotein refers to the segment of the amino acid sequence at the C-terminus of its F2 subunit, which is flexible and therefore not visible in the three-dimensional structure of the F protein before fusion. Many paramyxoviruses have unstructured F2 C-termini. For example, based on the crystal structures of the F constructs before fusion of a variety of RSVs that have been determined, it was found that the C-terminus of F2 is always unstructured. The crystal structure of the F construct before hMPV fusion shows that the C-terminus of the F2 of hMPV is unstructured. Similarly, the EM structure of the F construct before PIV3 fusion has been resolved, which shows that the C-terminus of the F2 of PIV3 is unstructured.

[0060] IV. Engineered paramyxovirus soluble F immunogen

[0061] The present invention provides engineered (redesigned or modified) soluble F sequences of paramyxoviruses that can be used to produce vaccine compositions. Redesigned soluble F trimer immunogens or proteins are stabilized by introducing modifications into wild-type soluble F sequences of paramyxoviruses. Some specific wild-type soluble F sequences of specific hRSV strains are exemplified herein. Due to the functional similarity and sequence homology between different strains of a given paramyxovirus, redesigned soluble F immunogens derived from other known paramyxovirus F protein ortholog sequences can also be generated according to the redesign strategy described herein. Many known paramyxovirus orthologs or homologs F protein sequences have been described in the literature. See, e.g., Collins et al., Proc. Natl. Acad. Sci. USA 81:7683-7, 1984; Hause et al., PLoS ONE 12:e0175792, 2017; Chang et al., Viruses 4:613-636, 2012; and Amanda et al., J. Virol. 81:8303–8314, 2007. As described in detail herein, the engineered soluble F proteins of the present invention comprise one or more specific stabilizing mutations in the corresponding wild-type soluble F sequence. These mutations include (a) replacement of two or more negatively charged residues around the β23 chain, as exemplified herein for RSV, (b) deletion of the P27 peptide, (c) disulfide bonds within the engineered protomer, which are located within the F1 subunit or connect F2 and F1 subunits, and (d) disulfide bonds between the engineered protomer, as exemplified herein for RSV. In some embodiments, the engineered soluble F protein of the present invention may include any 2 (e.g., a and d) or 3 (e.g., a, c, and d) combinations of these mutations. In some embodiments, the engineered soluble F protein may include all 4 of these mutations.

[0062] In some embodiments, relative to its wild-type corresponding sequence, the engineered soluble F protein comprises the replacement of two or more negatively charged residues around the β23 chain with polar or hydrophobic residues. As used herein, the negatively charged residues around the β23 chain refer to a negatively charged segment (stretch) centered around β23 or around it. It can include 1 to 2 residues of the β23 chain plus upstream and downstream. In addition to these replacements, the engineered soluble F sequence of the present invention generally also has the disappearance of a processed active peptide (P27), and / or includes an engineered and stabilized disulfide bond, which is present in the Fs subunit or connects F2 and F1 subunits, and locks the protein in a pre-fusion conformation. Using prototype human RSV A2 strain F protein (UniProt ID P03420) as a reference, the P27 peptide corresponds to residues E110 to R136, and the negatively charged segment covers residues at and around β23 chains D486 to A490. It is noteworthy that residue 485 is Ser (S) in human RSV, but the corresponding residue in human MPV is Glu (E453). This residue is also included as part of the negatively charged segment around β23, which forms a repulsive interaction around the trimer axis. In some embodiments, the engineered soluble F protein of the present invention has two residues, D486 and E487, in the β23 chain, which are replaced by polar or hydrophobic residues. In some embodiments of other paramyxoviruses such as hMPV, the immediately upstream residue (E453) of the β23 chain may also be replaced by polar or hydrophobic residues. In some embodiments, the engineered disulfide bond is S155C / S290C, which is present in the F1 subunit. In some other embodiments, the engineered disulfide bond is S62C / K196C or E60C / K196C connecting the F2 and F1 subunits.

[0063] In addition to the above modifications, some engineered soluble F proteins of the present invention may comprise one or more additional mutations compared to their wild-type counterparts. In some embodiments, they have inserted a linker portion that replaces the furin cleavage site. In some other embodiments, a linker portion is inserted to replace the unstructured F2 C-terminus and a portion of the fusion peptide (FP) N-terminus. Similarly, using the human RSV A2 strain as a reference, the substituted F2 C-terminus corresponds to residues N104 to R109 (NNRARR; SEQ ID NO: 31). The substituted N-terminus (F137 to V157) of the fusion peptide (FP) comprises residues F137 to S146. In some preferred embodiments, the replacement linker portion is a GS-rich linker, such as (GS)n, where n can be any integer from 1 to about 5. In various embodiments, the linker comprises the sequence GSGS (SEQ ID NO: 27) or GSGSGSGS (SEQ ID NO: 28).

[0064] In some embodiments, the engineered soluble F protein of the present invention may further comprise a replacement of the residue corresponding to S215 in the F glycoprotein of the RSV A2 strain. In some embodiments, the engineered soluble F protein of the present invention may further comprise a replacement of the residue corresponding to E92 in the F glycoprotein of the RSV A2 strain. For example, residue E92 in the soluble F protein may be replaced with: D; Q; or other short polar residues; or in some cases, hydrophobic residues, such as L and I, to form hydrophobic interactions with adjacent protomers. In some embodiments, the engineered soluble F protein of the present invention may further comprise a replacement at residues S46 and K462. In some embodiments, the replacement is S46G / K462Q. In some further embodiments, the engineered soluble F protein of the present invention comprises an engineered disulfide bond connecting the amino acid pairs that form the β sheet in the β3 / β4 hairpin or corresponding hairpin in the F1 subunit. As shown herein, the function of this engineered disulfide bond is to further reduce the metastability of the pre-fusion soluble F sequence and improve its stability. It is noteworthy that the β3 / β4 hairpin in RSV and MPV has its counterpart in the β1 / β2 hairpin in PIV. In some of these embodiments, the disulfide bond is formed by replacing A177C / T189C, where the amino acid numbering is based on the human RSV A2 strain.

[0065] Some specific examples of engineered RSV soluble F sequences or immunogens are shown in SEQ ID NOs: 17 to 23. In addition to these exemplified sequences, the engineered RSV soluble F immunogens of the invention also include sequences that are conservatively modified variants of these sequences or substantially identical sequences.

[0066] In addition to the modifications in the wild-type soluble F sequence as described above, some engineered soluble F antigens or immunogenic proteins of the present invention may include an N-terminal leader sequence (or "signal peptide"). In some of these embodiments, the N-terminal leader includes the sequence MELLILKANAITTILTAVTFCFASG (SEQ ID NO: 2) as exemplified herein. In some embodiments, the engineered soluble F protein of the present invention may also include one or more C-terminal structural motifs that promote trimerization. For example, the engineered protein may include the C-terminal foldon motif GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 7), as exemplified herein. In some of these embodiments, a restriction site, such as "AS" exemplified herein, may be appended to the N-terminus of the foldon motif.

[0067] V. Engineered paramyxovirus soluble F trimers with different mutation sets

[0068] In addition to the modifications introduced into the soluble F trimer as described above, the present invention also provides engineered paramyxovirus soluble F immunogens comprising different sets of mutations relative to the wild-type F sequence. Similarly, for these additional engineered paramyxovirus soluble F immunogens, some specific wild-type soluble F sequences of specific hRSV, hMPV, and hPIV3 strains are exemplified herein. Due to the functional similarity and sequence homology between different strains of a given paramyxovirus, redesigned soluble F immunogens derived from other known paramyxovirus F protein ortholog sequences can also be generated according to the redesign strategy described herein. Many known paramyxovirus orthologs or homologs F protein sequences have been described in the literature. See, e.g., Collins et al., Proc. Natl. Acad. Sci. USA 81:7683-7, 1984; Hause et al., PLoS ONE 12:e0175792, 2017; Chang et al., Viruses 4:613-636, 2012; and Amanda et al., J. Virol. 81:8303–8314, 2007.

[0069] As described in detail herein (e.g., Examples 8 to 14), some of these additional redesigned soluble F immunogens of the invention comprise engineered disulfide bonds linking pairs of amino acid residues that form the β sheet in the β3 / β4 hairpin (or corresponding hairpin) in the F1 subunit of the soluble F sequence. Some additional redesigned soluble F proteins comprise sets of mutations that stabilize the F trimer in a prefusion state. These include mutations at the C-terminus of F2, deletion of the P27 peptide, and mutations at the N-terminus of the fusion peptide in the F1 subunit. Some additional redesigned soluble F proteins may comprise engineered disulfide bonds, as well as one or more of these mutations.

[0070] In one aspect, the present invention provides engineered or redesigned immunogen proteins or polypeptides derived from the fusion glycoprotein (F) of any paramyxovirus. These immunogens comprise altered soluble F sequences that are modified relative to the wild-type soluble F sequences of paramyxoviruses. These modifications comprise engineered disulfide bonds that connect the β3 / β4 hairpins in the F1 subunit of the paramyxovirus F protein or the amino acid pairs forming the β sheet in the corresponding hairpins. The hairpins are the β3 / β4 hairpins in RSV and MPV. In PIV, the corresponding hairpins are β1 / β2 hairpins. Some of these immunogens are derived from the wild-type soluble F sequence of RSV (e.g., human RSV (hRSV)). In some of these embodiments, the engineered disulfide bonds are produced by the amino acid replacements S180C / S186C in the β3 / β4 hairpins. In some other embodiments, the engineered disulfide bonds are produced by the amino acid replacements A177C / T189C in the hairpins. The amino acid numbering in the redesigned RSV F immunogens of the present invention is based on the F glycoprotein sequence of the human RSV A2 strain, which has UniProt ID number P03420. In some embodiments, the wild-type soluble RSV F sequence from which the redesigned immunogen is derived is shown in SEQ ID NO: 1.

[0071] In some redesigned RSV soluble F immunogens of the present invention, in addition to the engineered disulfide bonds, the modification of the wild-type sequence also includes a mutation at the C-terminus of the unstructured F2 subunit. For example, the redesigned soluble F sequence may include a truncation of the unstructured F2 C-terminus. As a specific illustration, residues 104 to 109 (NNRARR) (SEQ ID NO: 31) at the F2 C-terminus may be deleted. In addition or as an alternative, amino acid substitutions at the unstructured F2 C-terminus may also be introduced into the redesigned soluble F sequence. For example, some redesigned RSV soluble F immunogens of the present invention may include a P102A substitution at the F2 C-terminus.

[0072] In some redesigned RSV soluble F immunogens of the present invention, modifications to the wild-type sequence may include one or more other mutations. These include, for example, replacing the processed active peptide (P27) (residues E110 to R136) and the N-terminus of the fusion peptide (e.g., residues F137 to S146) with a short GS linker sequence. In various embodiments, the GS linker may have the sequence formula (GS) n , wherein n is any integer from 1 to about 5. Additional modifications may also comprise additional substitutions in the F1 subunit. These include, for example, substitutions I379V and M447V, as exemplified herein using the wild-type soluble F sequence shown in SEQ ID NO: 1.

[0073] Some specific examples of redesigned RSV soluble F sequences or immunogens are shown in SEQ ID NOs: 36 to 43. In addition to these exemplified sequences, the redesigned RSV soluble F immunogens of the present invention also include sequences that are conservatively modified variants of these sequences or substantially identical sequences.

[0074] Some redesigned soluble F immunogens comprising engineered disulfide bonds are derived from the wild-type soluble F sequence of a metapneumovirus (e.g., hMPV). In some of these embodiments, the engineered disulfide bond is generated by the amino acid substitution A147C / A159C in the β3 / β4 hairpin. The amino acid numbering is based on the crystal structure (PDB ID: 5WB0) and the UniProt definition of the hMPV strain CAN97-83 with ID Q6WB98. In some embodiments, the wild-type soluble MPV F sequence from which the redesigned immunogen is derived is shown in SEQ ID NO: 44 or SEQ ID NO: 45. These two sequences are based on hMPV isolate TN03.03.19, whose GenBank ID is AEZ52364.

[0075] In addition to engineered disulfide bonds, modifications to the wild-type sequence in some redesigned hMPV soluble F immunogens of the present invention also include mutations in the C-terminus of the unstructured F2 subunit. In some of these embodiments, the mutation in the C-terminus of the unstructured F2 is a deletion of the unstructured F2 C-terminus DQLAREEQIENP (SEQ ID NO: 60) and the cleavage site RQSR (SEQ ID NO: 49). In addition, the deleted sequence can be replaced with the shorter (GS)n linker sequence described above. Some specific examples of redesigned hMPV soluble F immunogens of the present invention are shown in SEQ ID NOs: 46 and 47. In addition to these exemplified sequences, the redesigned hMPV soluble F immunogens of the present invention also include sequences that are conservatively modified variants of these sequences or substantially identical sequences.

[0076] Some additional redesigned soluble F immunogens containing engineered disulfide bonds are derived from wild-type soluble F sequences of human parainfluenza viruses. These include, for example, human parainfluenza viruses 1 to 5 (hPIV1 to 5). Taking hPIV3 as an example, some redesigned hPIV3 soluble immunogens have engineered disulfide bonds generated by the amino acid substitutions Q159C / A171C in the β1 / β2 hairpin. The amino acid numbering is based on the cryo-EM structure (PDB ID: 6MJZ) and the UniProt definition of the recombinant PIV3 / PIV1 virus with ID (O55888). In some embodiments, the wild-type soluble MPV F sequence from which the redesigned immunogen is derived is shown in SEQ ID NO: 48. This sequence is from the F protein of the hPIV3 strain "HPIV3 / USA / 629-D01959 / 2007", which has GenBank ID AGW51052. Due to the substantial structural similarities between different PIVs (eg, hPIV3 and hPIV5 as exemplified herein), the redesign strategies exemplified herein for hPIV3 can be readily applied to other PIVs.

[0077] In addition to engineered disulfide bonds, in some redesigned PIV soluble F immunogens of the present invention, modifications to the wild-type sequence may also include mutations at the C-terminus of the F2 subunit. In some of these embodiments, the mutations at the F2 C-terminus are deletions of NQESNENTDP (SEQ ID NO: 50) and the cleavage site RTER (SEQ ID NO: 51). In addition, the deleted sequences can be replaced with the shorter (GS)n linker sequence described above. Some specific examples of the redesigned hMPV soluble F immunogens of the present invention are shown in SEQ ID NOs: 61 and 62. In addition to these exemplified sequences, the redesigned PIV soluble F immunogens of the present invention also include sequences that are conservatively modified variants of these sequences or sequences that are substantially identical.

[0078] In another aspect, the invention provides an engineered or redesigned RSV soluble F immunogen or protein, which is stabilized by a specific modification group of a wild-type RSV soluble F sequence. Using amino acid numbering based on human RSV A2 strain (UniProtID P03420), in these redesigned immunogens, the modification of the wild-type soluble RSV F sequence comprises the disappearance of (1) P27 peptide (residue E110 to R136), (2) modification of the unstructured F2 subunit C-terminus (residue Q98 to R109), and (3) brachymemma of the fusogenic peptide N-terminus (e.g., residue F137 to V157). In some embodiments, the wild-type soluble RSV F sequence from which the redesigned immunogen is derived is shown in SEQ ID NO:1.

[0079] In some of these immunogen proteins, the modification of the unstructured F2 C-terminus is a truncation of residues 104 to 109 (NNRARR; SEQ ID NO: 31) and a P102A replacement. In some embodiments, the truncation of the fusion peptide N-terminus is a deletion of residues F137 to S146. In some embodiments, the redesigned RSV soluble F immunogen polypeptide comprises an inserted (GS)n linker between F2 and F1. In the linker formula, n can be any integer from 1 to about 5. In various embodiments, the linker comprises the sequence GSGS (SEQ ID NO: 27) or GSGSGSGS (SEQ ID NO: 28). In some embodiments, the redesigned RSV soluble F immunogen polypeptide comprises the amino acid replacements I379V and / or M447V. Exemplary redesigned RSV soluble F immunogen sequences are shown in SEQ ID NO: 34 or SEQ ID NO: 35. In addition to these exemplified sequences, the redesigned RSV soluble F immunogens of the present invention also include sequences that are conservatively modified variants of these sequences or substantially identical sequences.

[0080] In addition to the specific mutation groups described above, modifications in the redesigned RSV soluble F immunogens of the present invention may also include engineered disulfide bonds relative to the wild-type soluble RSV F sequence. The engineered disulfide bonds connect the amino acid pairs that form the β sheet in the β3 / β4 hairpin in the F1 subunit, and their function is to reduce the metastability of the soluble F sequence before fusion and improve its stability. In some of these embodiments, the engineered disulfide bonds are generated by the amino acid replacements S180C / S186C in the β3 / β4 hairpin. In some other embodiments, the engineered disulfide bonds are generated by the amino acid replacements A177C / T189C in the β3 / β4 hairpin. Some examples of these redesigned RSV soluble F immunogen sequences of the present invention are shown in SEQ ID NOs: 36, 38, 40, and 42. In addition to these exemplified sequences, the redesigned RSV soluble F immunogens of the present invention also include sequences that are conservatively modified variants of these sequences or substantially identical sequences.

[0081] In some other embodiments, the modification in the redesigned RSV soluble F immunogen of the present invention may include the replacement of the amino acid residues between the two β chains β3 and β4. For example, the redesigned immunogen sequence may include the amino acid replacement S182G and / or N183P. Some specific examples of these redesigned RSV soluble F immunogen sequences are shown in SEQ ID NO:37, 39, 41 and 43. In some embodiments, the redesigned RSV soluble F immunogen of the present invention may have a sequence that is a conservatively modified variant of these exemplary sequences or a substantially identical sequence.

[0082] VI. Nanoparticle-display vaccine compositions

[0083] The present invention provides vaccine compositions comprising heterologous scaffolds displaying stable soluble F proteins or immunogens of paramyxoviruses as described herein. In the construction of the vaccines of the present invention, any heterologous scaffold can be used to display the engineered soluble F protein or immunogen. This includes virus-like particles (VLPs), such as bacteriophage Q β VLP and nanoparticle. A variety of nanoparticle platforms can be used to produce vaccine compositions of the present invention. Generally, nanoparticles used for the present invention need to be formed by multiple copies of a single subunit. Nanoparticles are typically spherical in shape and / or have rotational symmetry (e.g., having a triple and quintuple axis), e.g., have the icosahedral structure illustrated herein. In addition or as an alternative, the amino termini of the particle subunits must be exposed to and adjacent to the triple axis, and the intervals between the three amino termini must closely match the intervals between the carboxyl termini of the soluble F protein stabilized by the displayed trimer.

[0084] In various embodiments, the self-assembled nanoparticles employed are about 25 nm in diameter or less (typically assembled from 12, 24, or 60 subunits) and have a triple axis on the particle surface. Such nanoparticles provide a suitable particle platform for producing multivalent vaccines. In some preferred embodiments, paramyxovirus immunogenic proteins or polypeptides can be displayed on self-assembling nanoparticles, such as the self-assembling nanoparticles derived from I3-01 exemplified herein (I3-01v9b and I3-01v9c). Other examples of nanoparticles suitable for the present invention include nanoparticles derived from ferritin (FR) or E2p. Ferritin, as is well known in the art and commonly used, is a globular protein present in all animals, bacteria, and plants. As is well known in the art, its primary function is to control the rate and location of polynuclear Fe(III)2O3 formation by transporting hydrated iron ions and protons to and from the mineralized core. The globular form of ferritin is composed of monomeric subunit proteins (also called monomeric ferritin subunits), which are polypeptides with a molecular weight of approximately 17 to 20 kDa. E2p is a redesigned variant of the dihydrolipoyl acyltransferase from Bacillus stearothermophilus that has been shown to self-assemble into thermostable 60-mer nanoparticles. See, for example, He et al., Nat. Commun. 7:12041, 2016. Similarly, I3-01 is an engineered protein that can self-assemble into ultrastable nanoparticles. See, for example, Hsia et al., Nature 535, 136-139, 2016. Database searches show that I3-01 is engineered from a bacterial enzyme with a known crystal structure (PDBID: 1VLW). The sequences of the subunits of these proteins are known in the art. See, for example, WO2017 / 192434. The art provides more detailed information on the structural and functional properties of various nanoparticle scaffolds and their use in displaying trimeric protein immunogens. See, for example, WO2017 / 192434, WO2019 / 089817, and WO2019 / 241483. In various embodiments, the paramyxovirus vaccine compositions of the present invention can use any of these known nanoparticles, as well as conservatively modified variants thereof or variants having substantially the same (e.g., at least 90%, 95%, or 99% identical) sequences.

[0085] In addition to the nanoparticle sequences described above, many other nanoparticles or VLPs known in the art can also be used in the practice of the present invention. These include, for example, Aquifex aeolicus lumazine synthase, Thermotoga Maritima encapsulin, Myxococcus xanthus encapsulin, bacteriophage Qβ virus particles, Flock House Virus (FHV) particles, ORSAY virus particles, and infectious bursal disease virus (IBDV) particles.

[0086] In addition to the displayed soluble F immunogen, the nanoparticle vaccine composition of the present invention may include additional motifs to obtain better biological or pharmaceutical properties. Additional structural components may serve to promote the display of immunogens on the surface of the nanoparticles, enhance the stability of the displayed immunogens and / or improve the yield and purity of the self-assembling protein vaccine. In these embodiments, one or more linkers (linker sequences, motifs or parts) may be used to connect the various structural components in the construct. An example of an additional structural component is a trimerization motif, such as the foldon described above. In some embodiments, the coding sequence of a polypeptide fragment or motif that serves as an active site for chemical conjugation may be inserted into the construct at an appropriate position. In some other embodiments, additional structural components such as CD4 + T helper epitope or CD8 + T cell epitopes are inserted into the nanoparticle construct at appropriate locations. These include, for example, the PADRE T helper epitopes exemplified herein.

[0087] In other embodiments, the nanoparticle vaccines of the present invention may include a locking domain that stabilizes the nanoparticles. The locking domain encoding sequence may be fused directly or indirectly to the C-terminus of the nanoparticle subunit encoding sequence. The locking domain stabilizes the nanoparticles from the inside, allowing the nanoparticles displaying the paramyxovirus immunogen polypeptide to remain intact during manufacturing, vaccine formulation, and immunization. The nanoparticle vaccine immunogen thus constructed has significantly enhanced stability. Generally, the locking domain suitable for the present invention is a protein subunit that can naturally form a dimer with another protein subunit in solution through non-covalent interactions at the interface. In some preferred embodiments, the two protein subunits can be identical in sequence and form homodimers. In some other embodiments, the two protein subunits can be different proteins or two different domains of a single protein obtained by engineering, which can form heterodimers in solution through non-covalent interactions at the interface. Generally, the locking domain is covalently fused to the nanoparticle subunit to which the immunogen polypeptide is attached. Some examples of specific locking domains and guidance on the use of locking domains (e.g., LD7 or LD4 exemplified herein) in the construction of nanoparticles displaying trimeric immunogens can be found in the art, for example, WO 2019 / 241483. Two specific locking domains, LD4 and LD7, suitable for use in nanoparticle vaccines of the present invention are exemplified herein.

[0088] Locking domain LD4 (SEQ ID NO: 29):

[0089]

[0090] Locking domain LD7 (SEQ ID NO: 30):

[0091]

[0092] Nanoparticles displaying any stable paramyxovirus soluble F protein immunogen described herein (e.g., a stable RSV soluble F trimer immunogen) can be constructed by fusing the subunits of the immunogen polypeptide or multimeric immunogen protein (e.g., a trimer immunogen) to the subunit sequence of the nanoparticle (e.g., E2pI3-01v9b or I3-01v9c subunit sequence as exemplified herein) and other optional or alternative components described herein (e.g., a locking domain or trimerization motif). In order to construct the nanoparticles displaying the fusion vaccine immunogen of the present invention, one or more linker motifs or portions can be used to facilitate the connection of different components and maintain the structural integrity of the different components. Typically, the linker motif comprises a short peptide sequence. In various embodiments, the linker or linker motif can be any flexible peptide that connects two protein domains or motifs without interfering with their function. For example, any of these linkers used in the construct can be a peptide having (G a S b ) n A GC-rich peptide of the sequence, wherein a is an integer from about 1 to 5, b is an integer from about 0 to 2, and n is an integer from about 1 to 5. In some embodiments, the linker used comprises the sequence GSGS (SEQ ID NO: 27) or GSGSGSGS (SEQ ID NO: 28). The detailed procedures for recombinantly producing the vaccine composition of the present invention can be based on the protocols described herein and / or other methods described in the art, for example, He et al., Nat. Comm. 7, 12041, 2016; Kong et al., Nat. Comm. 7, 12040, 2016; He et al., Sci Adv. 4 (11): eaau6769, 2018; WO 2017 / 192434; WO 2019 / 089817 and WO 2019 / 241483.

[0093] VII. Polynucleotides and expression constructs

[0094] The stable paramyxovirus soluble F protein and related vaccine compositions of the present invention are generally produced by first generating an expression construct (i.e., an expression vector) comprising operably linked coding sequences for various structural components described herein. Thus, in some related aspects, the present invention provides substantially purified polynucleotides (DNA or RNA) encoding nanoparticles displaying immunogens described herein (e.g., stable RSV soluble F immunogens), as well as expression vectors (e.g., CMV vectors) comprising such polynucleotides and host cells (e.g., HEK293F and ExpiCHO cell lines exemplified herein) for producing vaccine immunogens. Fusion polypeptides encoded by the polynucleotides or expressed by the vectors are also included in the present invention. As described herein, such polypeptides will self-assemble into nanoparticle vaccines displaying immunogenic polypeptides or proteins on their surfaces.

[0095] Polynucleotides and related vectors can be readily produced by standard molecular biology techniques or the protocols exemplified herein. For example, general protocols for cloning, transfection, transient gene expression, and obtaining stably transfected cell lines are described in the art, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, (3 rd ed., 2000); and Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003). Introducing mutations into polynucleotide sequences by PCR can be performed as described in, for example, PCR Technology: Principles and Applications for DNA Amplification, HA Erlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, CA, 1990; Mattila et al., Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods and Applications 1:17, 1991.

[0096] The selection of a specific vector depends on the intended use of the fusion polypeptide. For example, the selected vector must be able to drive the expression of the fusion polypeptide in the desired cell type, whether the cell type is prokaryotic or eukaryotic. Many vectors contain sequences that allow both eukaryotic expression of operably connected gene sequences and prokaryotic vector replication. The vectors that can be used for the present invention can replicate autonomously, that is, the vector is present outside the chromosome, and its replication is not necessarily directly linked to the replication of the host cell genome. Alternatively, the replication of the vector can be linked to the replication of the host chromosome DNA, for example, the vector can be integrated into the chromosome of the host cell, as achieved by retroviral vectors and in stably transfected cell lines. Both viral-based expression vectors and non-viral expression vectors can be used to produce immunogens in mammalian host cells. Non-viral vectors and systems include plasmids, additional vectors (usually with expression cassettes for expressing proteins or RNA) and human artificial chromosomes (see, for example, Harrington et al., Nat. Genet. 15: 345, 1997). Useful viral vectors include those based on lentivirus or other retroviruses, adenovirus, adeno-associated virus, cytomegalovirus, herpes virus, SV40, papillomavirus, HBP Epstein Barr virus, vaccinia virus, and Semliki Forest virus (SFV). See Brent et al., supra; Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeld et al., Cell 68:143, 1992.

[0097] Depending on the specific vector for expressing fusion polypeptide, a variety of known cells or cell lines can be used for the practice of the present invention.Host cell can be any cell that can introduce the recombinant vector carrying fusion body of the present invention, and wherein allows the carrier that drives fusion polypeptide expression to be used for the present invention.It can be prokaryotic, such as any one in many bacterial strains, or can be eukaryotic, such as yeast or other fungal cells, insect or amphibian cells, or mammalian cells, including such as rodent, monkey or human cells.The cell expressing fusion polypeptide of the present invention can be primary culture cell or can be established cell line.Therefore, except the cell line (such as Chinese hamster ovary celI) exemplified herein, many other host cell lines well known in the art also can be used for the practice of the present invention.These include such as multiple Cos cell lines, HeLa cells, Sf9 cells, HEK293, AtT20, BV2 and N18 cells, myeloma cell lines, transformed B cells and hybridomas.

[0098] Using mammalian tissue cell culture to express polypeptides is generally discussed in, for example, Winnacker, From Genes to Clones, VCH Publishers, NY, NY, 1987. The vector expressing the fusion polypeptide can be introduced into the selected host cell by any of the many suitable methods known to those skilled in the art. In order to introduce the vector encoding the fusion polypeptide into mammalian cells, the method used will depend on the form of the vector. For plasmid vectors, the DNA encoding the fusion polypeptide sequence can be introduced by any of the many transfection methods, including, for example, lipid-mediated transfection ("lipofection"), DEAE-dextran-mediated transfection, electroporation, or calcium phosphate precipitation. These methods are described in detail in, for example, Brent et al. supra. Liposome transfection reagents and methods suitable for transient transfection of a wide variety of transformed and untransformed or primary cells are widely available, making lipofection an attractive method for introducing constructs into eukaryotic cells, especially cultured mammalian cells. For example, LipofectAMINE TM (Life Technologies) or LipoTaxi TM (Stratagene) kits are available. Other companies that provide reagents and methods for liposome transfection include Bio-Rad Laboratories, CLONTECH, Glen Research, Life Technologies, JBL Scientific, MBI Fermentas, PanVera, Promega, Quantum Biotechnologies, Sigma-Aldrich, and Wako Chemicals USA.

[0099] In order to produce recombinant fusion polypeptides in high yield over a long period of time, stable expression is preferred. Instead of using an expression vector containing a viral origin of replication, a sequence encoding a fusion polypeptide and a selection marker controlled by suitable expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) can be used to transform host cells. The selection marker in the recombinant vector confers resistance to selection and allows the cell to stably integrate the vector into its chromosome. Commonly used selection markers include neo (Colberre-Garapin, et al., J. Mol. Biol., 150: 1, 1981), which confers resistance to the aminoglycoside G-418; and hygro (Santerre et al., Gene, 30: 147, 1984), which confers resistance to hygromycin. By appropriate selection, transfected cells can contain an integrated copy of the fusion polypeptide coding sequence.

[0100] VIII. Pharmaceutical compositions and therapeutic applications

[0101] In another aspect, the present invention provides pharmaceutical compositions and related treatment methods using the redesigned paramyxovirus F immunogens and nanoparticle vaccine compositions described herein. In some embodiments, soluble F trimer immunogens of different viruses (e.g., hRSV) can be used to prevent and treat corresponding viral infections. Some embodiments of the present invention relate to the use of vaccines based on hRSV soluble F for preventing or treating RSV infection in human subjects. Some embodiments of the present invention relate to the use of vaccines based on hMPV soluble F for preventing or treating MPV viral infection. Some embodiments of the present invention relate to the use of vaccines based on hPIV soluble F for preventing or treating PIV viral infection.

[0102] In the practice of the various therapeutic methods of the present invention, the corresponding nanoparticle vaccines, immunogenic proteins or polypeptides described herein, or encoding polynucleotides are administered to an object that needs to prevent or treat a disease or condition (e.g., hRSV infection). Typically, the nanoparticle vaccines, immunogenic proteins or encoding polynucleotides disclosed herein are included in a pharmaceutical composition. The pharmaceutical composition can be a therapeutic formulation or a preventive formulation. Typically, the composition can also include one or more pharmaceutically acceptable carriers, and optional other therapeutic ingredients (e.g., antiviral drugs). A variety of pharmaceutically acceptable additives can also be used in the composition.

[0103] Therefore, some pharmaceutical compositions of the present invention are vaccine compositions. For vaccine compositions, suitable adjuvants may also be included. Some examples of suitable adjuvants include, for example, aluminum hydroxide, lecithin, Freund's adjuvant, MPL TM and IL-12. In some embodiments, the vaccine compositions or nanoparticle immunogens disclosed herein (e.g., hRSV vaccine compositions) can be formulated as controlled-release or time-release formulations. This can be achieved in compositions comprising a sustained-release polymer or by microencapsulated delivery systems or bioadhesive gels. A variety of pharmaceutical compositions can be prepared according to standard procedures known in the art. See, for example, Remington's Pharmaceutical Sciences, 19 thEd., Mack Publishing Company, Easton, Pa., 1995; Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978); U.S. Patent Nos. 4,652,441 and 4,917,893; U.S. Patent Nos. 4,677,191 and 4,728,721; and U.S. Patent No. 4,675,189.

[0104] The pharmaceutical compositions of the present invention can be easily used in a variety of therapeutic or preventive applications, for example, for treating hRSV infection or bronchiolitis in a subject, or for eliciting an immune response to hRSV. In multiple embodiments, the vaccine composition can be used to treat or prevent infections caused by pathogens from which the immunogenic polypeptides displayed in the nanoparticle vaccine are derived. Therefore, the vaccine composition of the present invention can be used in a variety of clinical settings to treat or prevent infections caused by a variety of viruses. As an example, the RSV nanoparticle vaccine composition can be administered to a subject to induce an immune response to hRSV, for example, to induce the production of broadly neutralizing antibodies to the virus. For subjects at risk of RSV infection, the vaccine composition of the present invention can be administered to provide preventive protection against viral infection. The therapeutic and preventive applications of vaccines derived from other immunogens described herein can be similarly performed. Depending on the specific subject and condition, the pharmaceutical composition of the present invention can be administered to a subject by a variety of administration methods known to those of ordinary skill in the art, for example, intramuscularly, subcutaneously, intravenously, intra-arterially, intra-articularly, intraperitoneally, or parenterally. Typically, the pharmaceutical composition is administered to an object in need of such treatment for a period of time sufficient to prevent, suppress, and / or improve a selected disease or condition or one or more of its symptoms. For therapeutic applications, the composition should comprise a therapeutically effective amount of the nanoparticle immunogen described herein. For prophylactic applications, the composition should comprise a prophylactic effective amount of the nanoparticle immunogen described herein. The appropriate amount of the immunogen can be determined based on the specific disease to be treated or prevented or the condition, the severity, the age of the object, and other personal attributes of the specific object (e.g., the overall condition of the object's health and the robustness of the object's immune system). The determination of the effective dose is also guided by animal model studies and subsequent human clinical trials, and is guided by an administration regimen that significantly reduces the occurrence or severity of the targeted disease symptoms or condition in the object.

[0105] In some embodiments, the present invention provides immunogenic compositions for the treatment of a disease or condition.For preventive use, immunogenic compositions are provided before any symptom (for example, before infection). The preventive administration of immunogenic compositions is used to prevent or improve any subsequent infection. Therefore, in some embodiments, object to be treated is to have infection (for example, RSV infection) or be in the object in the risk of infection, for example, due to being exposed to or possibly being exposed to virus (for example, RSV). After administering the disclosed therapeutic composition of therapeutically effective amount, the infection (for example, RSV infection) of object can be monitored, the symptom relevant to infection (for example, RSV infection) or the two.

[0106] For therapeutic applications, the immunogenic composition is provided at or after the onset of symptoms of the disease or infection (e.g., after symptoms of infection (e.g., RSV infection) occur or after diagnosis of infection). Thus, the immunogenic composition can be provided before expected exposure to the virus to reduce the severity, duration, or extent of the expected infection and / or related disease symptoms, after exposure to or suspected exposure to the virus, or after the actual infection begins. The pharmaceutical compositions of the present invention can be combined with other agents known in the art for treating or preventing infection with related pathogens (e.g., hRSV infection).

[0107] Nanoparticle vaccine compositions (e.g., hRSV vaccines) or pharmaceutical compositions of the present invention comprising novel structural components as described herein can be provided as components of a kit. Optionally, such kits include additional components, including packaging, instructions, and various other reagents, such as buffers, substrates, antibodies or ligands (e.g., control antibodies or ligands), and detection reagents. Optional instructions can also be provided in the kit.

[0108] Example

[0109] The following examples are provided to illustrate the present invention but not to limit it.

[0110] Example 1 Comparative analysis of existing RSV prefusion F designs

[0111] In this study, compared three known RSV fusion front F designs: DS-Cav1 (McLellan et al, Science 2013, 342: 592-598), SC-TM (Krarup et al., Nat Comm 2015, 6: 8143) and sc9-10DS-Cav1 (Joyce et al., Nat Struct Mol Biol 2016, 23: 811-820). For these three fusion front F designs, generated a construct with an enzymatic site (amino acid " AS ") and a foldon motif connected to the C-terminus. Sequence is as follows.

[0112] MELLILKANAITTILTAVTFCFASG (SEQ ID NO: 2): N-terminal leader.

[0113] QSTPPTNNRARR (SEQ ID NO: 3): Unstructured F2 C-terminus.

[0114] QSTPATNNQAR (SEQ ID NO: 4): F2 C-terminus with mutation.

[0115] ELPRFMNYTLNNAKKTNVTLSKKRKRR (SEQ ID NO:5): P27 peptide.

[0116] FLGFLLGVGS (SEQ ID NO: 6): fusion peptide (FP).

[0117] GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 7): C-terminal foldon.

[0118] A2_DS-Cav1-foldon (PDB ID: 4MMU) (SEQ ID NO: 8)

[0119]

[0120] A2_SC-TM-foldon(PDB ID:5C6B)(SEQ ID NO:9)

[0121]

[0122] A2_sc9-10DS-Cav1-foldon (PDB ID: 5K6I) (SEQ ID NO): 10)

[0123]

[0124] The three constructs were transiently expressed in 25 ml ExpiCHO cells and purified using a D25 antibody column followed by size-exclusion chromatography (SEC) on a Superdex 200 Increase 100 / 300 GL column. Potent neutralizing antibodies (NAb) (McLellan et al, Science 2013, 342: 592-598) will ensure pre-fusion specific RSV F purification. For DS-Cav1, The high-resolution crystal structure showed a football-shaped, closed pre-fusion F trimer conformation. After D25 purification, transient expression of DS-Cav1 in ExpiCHO cells produced reasonable yields. However, the SEC spectrum showed a high aggregation peak (at about 9ml) and a second peak corresponding mainly to trimers. It is worth noting that a visible shoulder was observed to the left of the trimer peak in SEC, indicating the presence of higher-order pre-fusion F species in the DS-Cav1 sample. Negative-stain EM (nsEM) was performed to characterize the trimer fraction (about 12ml). All 2D classes showed monomers or dimers, without any signs of closed pre-fusion trimers. For SC-TM, The high-resolution crystal structure shows a closed pre-fusion trimer similar to DS-Cav1. It is worth noting that SC-TM has extremely low production in ExpiCHO expression, and both trimer peaks and monomer leaks appear in SEC. 2D classification images from nsEM show football-shaped molecules with the characteristics of closed pre-fusion F trimers, as well as monomers and dimers. These 2D classifications were used to construct a 3D EM model that almost perfectly matches the crystal structure and shows unoccupied density at the bottom of the trimer, which corresponds to the C-terminal foldon. However, wedge-shaped molecules corresponding to the post-fusion F trimer were also found in the nsEM images, indicating that SC-TM cannot prevent the pre-to-post conformational transition.

[0125] For sc9-10 DS-Cav1, The high-resolution crystal structure revealed a nearly identical closed prefusion F trimer to that of DS-Cav1 and SC-TM. The sc9-10 DS-Cav1 construct exhibited high yield and purity in SEC, revealing a trimer peak >10-fold and >250-fold higher than that of DS-Cav1 and SC-TM, respectively. In nsEM analysis, nearly all 2D clusters exhibited a "soccer ball" shape, indicating nearly 100% closed prefusion F trimer. A 3D structural model constructed from the EM data confirmed this finding. In summary, DS-Cav1 becomes a monomer in solution, SC-TM expresses prefusion monomers and closed trimers, as well as low yields of postfusion trimers, and sc9-10 DS-Cav1 produces closed prefusion trimers in high yield and purity.

[0126] Example 2 Analysis of the origin of RSV F metastability

[0127] The sequence and structure of RSV F were analyzed to identify potential causes of F metastability. RSV strain A2 (GenBank ID: AAB59858.1, UniProt ID: P03420), previously used to design prefusion F constructs, was used as a template in this study. Briefly, sequence and secondary structure alignment of A2 F in the prefusion and postfusion states revealed several key regions ( Figure 1 , A). Two such regions are β3 / β4 and β23, both of which undergo secondary structural changes and become α-helical in the postfusion conformation.

[0128] For the β3 / β4 segment (K176 to S190), it behaves as a β hairpin in the prefusion state but becomes part of an extended α helix in the postfusion state ( Figure 1 , B). Structural analysis showed that the disulfide bonds S180C / S186C and A177C / T189C (their Cβ-Cβ distances are and ) can stabilize RSV F in the prefusion state (this has been included in the inventor's previous patent application for paramyxovirus). Here, another mutation, V185P, is reported. In the unmutated prefusion F (PDBID: 4JHW), the main chain dihedral angles of V185 are -70.6 (Phi) and 126.4 (Psi), which are very compatible with the main chain dihedral angles of trans-proline: -75 (Phi) and 145 (Psi). Hypothetically, V185P can rigidify the prefusion hairpin structure, but introduce a kink in the postfusion helix, thereby destabilizing the postfusion conformation. Figure 1 , B, right). For the β23 segment (S485 to A490), it forms interactions with the β23 segments of the other two protomers around the three-fold axis and is located above the trimeric coiled-coil formed by the three α10 helices, which keeps the three F protomers in a trimeric conformation ( Figure 1 , C, left). Cross-sectional analysis revealed that the β23 cluster is located at the bottom of the hollow interior of the soccer-shaped RSV prefusion F trimer ( Figure 1 , C, top right). For wild-type RSV prefusion F trimer, this hollow interior is partially filled by the fusion peptides of the three F protomers, but becomes empty once the fusion peptides are removed, such as in the prefusion F design sc9-10DS-Cav1. Further analysis of the β23 cluster revealed an unusual interaction pattern ( Figure 1, C, lower right). More specifically, the short β23 chain contains three negatively charged residues: D486, E487 and D489, which form repulsive charge-charge interactions around the triple trimer axis. It is hypothesized that unfavorable interactions in the β23 cluster can promote the rapid opening of the wild-type pre-fusion F trimer on the RSV virion surface, thereby exposing the fusion peptide and accelerating the pre-to-post conformational change during cell entry. In other words, it is hypothesized that the β23 cluster is the main cause of RSV F metastability. In this study, this hypothesis was tested by mutating D486 and E487 to (1) polar residues (e.g., D486N and E487Q, which can form salt bridges) and (2) hydrophobic residues (e.g., D486L and E487L, which can form hydrophobic clusters). Other mutations for the β23 segment can further improve the stability of the trimer.

[0129] Example 3 Characterization of RSV prefusion F construct based on "V2-Ext-PDB6-D"

[0130] Five soluble F constructs were generated using "V2-Ext-PDB6-D" as basic design, and all constructs included a C-terminal foldon motif (sequence listed below). The first construct was the basic design. The second construct incorporated the V185P mutation into the basic design to examine the effect of the second proline mutation V185P. The third and fourth constructs incorporated mutations D486N / E487Q and D486L / E487L into the second construct, respectively, to examine whether the repulsive charge-charge interaction at removal β23 can improve the stability of the F trimer before RSV fusion. The fifth construct incorporated the A149C / Y458C mutation (called SS4) into the basic design to test the effect of the combination of the disulfide bond between the protomer and the minimum mutation set in the basic design. This disulfide bond is used in sc9-10 DS-Cav1 (Joyce et al., Nat Struct Mol Biol 2016, 23:811-820). Therefore, using a covalent bond, RSV fusion front F is locked in the 5th construct in a closed trimer, providing a "positive control" for the 3rd and 4th constructs, which attempt to retain RSV fusion front F in a closed trimer by the engineered non-covalent interactions at β23.

[0131] A2V2-Ext-PDB6-D-foldon((S215P,DB6=S155-S290,E92D)(SEQ ID NO:11)

[0132]

[0133] GA2V2-Ext-P2DB6-D-foldon(S215P,=S155-S290,E92D,V185P)(SEQ ID NO:12)

[0134]

[0135] A2_V2-Ext-P2DB6-D-NQ (without N-terminal leader and C-terminal foldon) (S215P, DB6 = S155-S290, E92D, V185P, D486N + E487Q) (SEQ ID NO: 17)

[0136]

[0137] A2_V2-Ext-P2DB6-D-L2 (without N-terminal leader and C-terminal foldon) (S215P, DB6=S155-S290, E92D, V185P, D486L+E487L) (SEQ ID NO: 18)

[0138]

[0139] A2V2-Ext-PDB6-D-SS4-foldon(S215P, DB6=S155-S290, E92D, SS4=A149-Y458) (SEQ ID NO: 13)

[0140]

[0141] Five soluble F constructs in the "V2-Ext-PDB6-D" series were transiently expressed in 25 ml ExpiCHO cells and purified using a D25 column, followed by SEC on a Superdex 200Increase100 / 300GL column. For the first construct, or base design A2_V2-Ext-PDB6-D-foldon, a single trimer peak was observed in SEC, with high yield and high purity. However, nsEM analysis of SEC fractions around approximately 12 ml showed predominantly "open" prefusion F trimers, with two classifications showing closed prefusion trimers. The 3D EM models constructed from these two classifications closely matched the prefusion F crystal structure. Therefore, the results indicate that this base design with a minimal set of mutations can produce prefusion F with a small fraction of closed trimers. For the second construct, A2_V2-Ext-P2DB6-D-foldon, a similar spectrum to the base design was observed, suggesting that the second proline mutation, V185P, likely has little effect on protein properties. For the third construct, A2_V2-Ext-P2DB6-D-NQ-foldon, distinct characteristics were observed compared to the second construct, which lacks the D486N / E487Q mutations. More specifically, A2_V2-Ext-P2DB6-D-NQ-foldon produced a single trimer peak with high yield and purity, but exhibited a slight increase in aggregate peaks around approximately 8.5 ml. Notably, nsEM analysis revealed that many 2D clusters, or approximately 73% of the molecules, corresponded to closed prefusion F trimers. A 3D EM model constructed from the EM data matched the prefusion F crystal structure almost perfectly, with some differences around the α10 helix at the base of the trimer. For the 4th construct A2_V2-Ext-P2DB6-D-L2-foldon, it was observed that the overall characteristics were similar to those of the 3rd construct, wherein the ratio of the F trimer before closure fusion was slightly higher, 76% vs. 73%. For the 5th construct A2_V2-Ext-PDB6-D-SS4-foldon, a trimeric peak with high yield and high purity was observed, but compared with the 3rd and 4th constructs, aggregation was further improved. nsEM analysis showed that among all constructs in this series, the highest percentage of F trimer before closure fusion was 88%. The 3DEM model not only matched the crystal structure of the F trimer before fusion, but also showed the density of the C-terminal foldon domain. In short, the results obtained from constructs #3 and #4 supported the following hypothesis: the β23 chain is the main cause of RSV F metastability, and removing the repulsive charge-charge interaction at β23 significantly improved trimer stability, and its level was similar to the disulfide bond between well-placed protomers.All five constructs, especially constructs #3, #4, and #5, which have 73% to 88% closed trimers, can be developed for RSV F trimer vaccine development. The presence of open trimers should not be a major concern, because wild-type prefusion F must exist in equilibrium as "closed" and "open" trimers on the RSV virion surface, and both states can induce neutralizing antibodies to block viral entry.

[0142] The third and fourth constructs were further explored using known RSV antibodies, both of which contain engineered non-covalent mutations at β23. First, the two constructs were explored using the post-fusion specific antibody ADI14359. nsEM analysis showed three types of 2D classification images corresponding to the following: unbound ADI14359 Fab, open pre-fusion F trimer or F monomer without bound Fab, and closed pre-fusion F trimer without bound Fab. In this analysis, no 2D classification was found corresponding to post-fusion F and post-fusion F / ADI14359 complexes. A perfect 3D model of the closed pre-fusion F trimer was constructed, indicating that ADI14359 did not interfere with the two pre-fusion F constructs and did not induce any conformational changes. Then, the two constructs were explored using the pre-fusion specific antibody D25 (which is also the antibody used to purify the pre-fusion F protein in this study). nsEM analysis revealed three types of 2D classification images corresponding to the following: unbound D25 Fab, open prefusion F trimer or F monomer bound to D25, and closed prefusion F trimer bound to D25. A nearly perfect 3D structural model of the closed prefusion F trimer in complex with D25 was constructed from the EM data, providing strong evidence that:

[0143] A2_V2-Ext-P2DB6-D-NQ-foldon and A2_V2-Ext-P2DB6-D-L2-foldon are ideal candidates for vaccine development based on the RSV prefusion F trimer.

[0144] Example 4 Characterization of RSV prefusion F constructs based on the "V2-Ext-PDB6-GDQ" base

[0145] Eight soluble F constructs were generated using "V2-Ext-PDB6-GDQ" as the base design, all of which contained a C-terminal foldon motif (sequences listed below). The first construct was a base design combining V2-Ext-PDB6-D with S46G and K465Q. It was hypothesized that the S46G / K465Q mutations could reduce the aggregation of some "V2-Ext-PDB6-D" derivatives. The second construct incorporated a second proline mutation, V185P, into the base design. The third and fourth constructs incorporated the D486N / E487Q mutations into the base design, but the fourth construct included the V185P mutation. The fifth and sixth constructs incorporated the D486L / E487L mutations into the base design, but the sixth construct included the V185P mutation. The 7th construct incorporates A149C / Y458C sudden change (being called SS4) into basic design.Disulfide bond between this protomer uses (Joyce et al., Nat Struct Mol Biol 2016,23:811-820) in sc9-10 DS-Cav1.Therefore, using covalent bond, RSV is merged before F is locked in the 7th construct in closed tripolymer and provides " positive control " for the 3rd to 6 kinds of constructs, and described 3rd to 6 kinds of constructs attempt to be retained in closed tripolymer before RSV is merged by the through engineering approaches non-covalent interaction at β23 place.The 8th construct is intended to check whether disulfide bond (i.e. A149C / Y458C) between protomer and the polar mutation (i.e. D486N / E487Q) at β23 place can be combined in a construct, to further stabilize the front F tripolymer of fusion.

[0146] A2_V2-Ext-PDB6-GDQ-foldon(S215P, DB6=S155-S290, S46G+E92D+K465Q) (SEQID NO: 14)

[0147]

[0148] A2_V2-Ext-PDB6-GDQ-foldon(S215P, DB6=S155-S290, S46G+E92D+K465Q, V185P) (SEQ ID NO:15)

[0149]

[0150] A2_V2-Ext-PDB6-GDQ-NQ (without N-terminal leader and C-terminal foldon) (S215P, DB6 = S155-S290, S46G + E92D + K465Q,, D486N + E487Q) (SEQ ID NO: 19)

[0151]

[0152] A2_V2-Ext-P2DB6-GDQ-NQ (without N-terminal leader and C-terminal foldon) (S215P, DB6 = S155-S290, S46G + E92D + K464Q, V185P, D486N + E487Q) (SEQ ID NO: 20)

[0153]

[0154] A2_V2-Ext-PDB6-GDQ-L2 (without N-terminal leader and C-terminal foldon) (S215P, DB6 = S155-S290, S46G + E92D + K465Q, D486L + E487L) (SEQ ID NO: 21)

[0155]

[0156] A2_V2-Ext-P2DB6-GDQ-L2 (without N-terminal leader and C-terminal foldon) (S215P, DB6 = S155-S290, S46G + E92D + K465Q, V185P, D486L + E487L) (SEQ ID NO: 22)

[0157]

[0158] A2_V2-Ext-PDB6-GDQ-SS4-foldon(S215P, DB6=S155-S290, S46G+E92D+K465Q, SS4=A149-Y458) (SEQ ID NO: 16)

[0159]

[0160] A2_V2-Ext-P2DB6-GDQ-SS4-NQ (without N-terminal leader and C-terminal foldon) (S215P, DB6 = S155-S290, S46G + E92D + K465Q, V185P, D486N + E487Q, SS4 = A149-Y458) (SEQ ID NO: 23)

[0161]

[0162] Seven kinds of soluble F constructs in " V2-Ext-PDB6-GDQ " series are transiently expressed in 25ml ExpiCHO cells, and D25 posts are used to purify, and SEC is carried out on Superdex 200Increase 100 / 300GL posts subsequently.For the 1st construct or basic design A2_V2-Ext-P2DB6-GDQ-foldon, in SEC, observe the tripolymer peak with high yield and high purity, also observe monomer peak.The nsEM analysis of the SEC fraction at about 11.5ml places demonstrates " open " fusion front F, and does not have the classification corresponding to " closed " tripolymer. Therefore, the result discloses that mutation S46G and K465Q (probably S46G) tend to make equilibrium to " open " conformational shift while keeping RSV F in pre-fusion state.For the 2nd construct A2_V2-Ext-P2DB6-GDQ-foldon, observe a spectrum similar to basic design. Notably, the slightly aberrant trimer peak likely results from the high yield of this construct, a pattern similar to that observed elsewhere. For the third and fourth constructs, A2_V2-Ext-PDB6-GDQ-NQ-foldon and A2_V2-Ext-P2DB6-GDQ-NQ-foldon, SEC profiles revealed substantial trimer yield and purity, without any aggregate peaks. nsEM analysis revealed that the majority of trimers were open, with 12% and 6% of closed trimers observed for the third and fourth constructs, respectively. 3D structural models constructed from the EM data further confirmed that both constructs can form closed prefusion F trimers.

[0163] For the 5th and 6th constructs A2_V2-Ext-PDB6-GDQ-L2-foldon and A2_V2-Ext-P2DB6-GDQ-L2-foldon, high trimer yields and purities were observed in the SEC spectra without any aggregate peaks. nsEM analysis showed that most trimers were open, with approximately 29% of closed trimers observed for these two constructs. The 3D structural model confirmed that these two constructs can form closed pre-fusion F trimers. For the 7th construct, incorporating the inter-protomer disulfide bond into the "V2-Ext-PDB6-GDQ" base resulted in a slight increase in aggregation in the SEC spectrum. nsEM analysis showed that 76% of the molecules were closed pre-fusion F trimers, which was >10% less than when the disulfide bond was incorporated into the V2-Ext-PDB6-D base (88%). Despite this, a nearly perfect 3D structural model was constructed from the EM data. For the eighth construct, incorporating both the interprotomer disulfide bond SS4 and the non-covalent β23 mutation into the "V2-Ext-PDB6-GDQ" base resulted in a further increase in aggregation but did not increase the proportion of closed prefusion trimers in nsEM analysis, with 73% of the molecules corresponding to closed prefusion trimers. A nearly perfect 3D structural model was constructed from the EM data. Overall, the results from this systematic comparison support the hypothesis that the S46G / K465 mutation minimizes aggregation in prefusion F expression. However, an adverse effect associated with the S46G / K465 mutation was noted, namely a decrease in the proportion of "closed" prefusion F trimers. Furthermore, the construct containing the D486N / E487Q (or "NQ") mutations showed greater sensitivity to the S46G / K465 mutations compared to the construct containing the D486L / E487L (or "L2") mutations, suggesting that the salt bridge formed by the polar residues at β23 is less effective than the hydrophobic contacts in holding the prefusion F in a closed trimer.

[0164] Example 5 Characterization of multiple RSV prefusion F constructs by X-ray crystallography

[0165] Eleven F constructs were structurally characterized by X-ray crystallography. First, the crystal structures of six “V2-Ext-PDB6-D” derivatives were determined. The structures of the “V2-Ext-PDB6-D” base design with two different C-terminal domains (1TD0 and foldon with a 5GS linker) were determined. In both cases, the base design exhibited a perfectly closed prefusion F trimer, although it was mostly open in solution. Subsequently, crystal structures of constructs containing the D486L / E487L (“L2”) and D486N / E487Q (“NQ”) mutations were obtained, confirming that the engineered non-covalent interactions at β23 can indeed stabilize the prefusion F trimer as expected. Finally, the crystal structures also confirmed that well-placed interprotomer disulfide bonds can effectively lock the prefusion F in a closed trimeric conformation. Second, the crystal structures of three “V2-Ext-PDB6-GDQ” derivatives were determined. Focus was on constructs containing the D486N / E487Q (NQ) mutations, inter-protomer disulfide bonds, and both. Crystal structures confirmed that the mutations or a combination of the two can be used to stabilize the RSV prefusion F trimer. However, the crystal structure of the "V2-Ext-PDB6-GDQ" base was not obtained. Third, the crystal structures of three constructs containing the disulfide bond A177C / T189C in the β3 / β4 hairpin were determined. The crystal structure confirmed that this disulfide bond can be combined with the minimal "V2-Ext-PDB6" base to stabilize the RSV prefusion F trimer.

[0166] Example 6 Optimization of I3-01v9 nanoparticles for displaying trimeric glycoproteins with a narrow stem

[0167] Previously, the I3-01v9 nanoparticle scaffold was rationally redesigned to optimize the display of monomeric antigens. In I3-01v9a, the N-terminal helix was extended so that its first amino acid is located just above the nanoparticle surface ( Figure 2 , A). Based on I3-01v9a, the N-terminal helix was further redesigned to achieve optimal display of trimeric antigens (e.g., RSV prefusion F trimer) ( Figure 2, B). First, the 11aa N-terminal helix in I3-01v9a was cut into 7aa. Then, a 13aa helix-turn fragment (all alanine) was fused to the 7aa helix of I3-01v9a in such a way that the new N-terminal helix was stacked in the groove of the two helices that are part of the I3-01 core. Next, the I3-01 core helix was mutated several times to remove the steric conflict between the new N-terminal helix and the groove. The helix-turn backbone was relaxed using a computational program called IMO (Zhu et al., Proteins 2006, 65(2):463-79), and the protein structure sampling program CONCOORD was further used to generate 1000 slightly perturbed main-chain conformations. Subsequently, the amino acids of the first 9 aa of the 13 aa fragment were predicted using an ensemble-based protein design program previously used to optimize HIV gp140 and HCV E2 core using the RAPDF scoring function based on Cα and Cβ (the 4 aa turn was set as a "GSGS" (SEQ ID NO: 27) linker).

[0168] The final design, I3-01v9b, was selected by integrating the prediction data, and the second design, I3-01v9c, was made by mutating the flexible corner from "GSGS" (SEQ ID NO: 27) to "GPPS" (SEQ ID NO: 32) to improve its rigidity. After further main chain relaxation, the structural model of I3-01v9b was constructed. The triangular shape makes it suitable for displaying trimeric antigens. In a recent study, a stable Ebola virus (EBOV) GP trimer (GPΔmuc-WL 2 P 4 ) as a test case to verify the I3-01v9b / c design ( Figure 2 , C). Briefly, EBOVGPΔmuc-WL 2 P 4 The I3-01v9b fusion construct was transiently expressed in HEK293F cells and purified by mAb100 antibody column followed by SEC. nsEM analysis identified 2D sorting corresponding to the well-formed GP-I3-01v9b fusion protein ( Figure 2 , C, top). A 3D model was constructed from the EM data, which showed a perfect EBOV GP trimer with a narrow stem displayed on the I3-01v9b trimer ( Figure 2, C, bottom). The subunit sequences of the I3-01v9a, I3-01v9b, and I3-01v9c nanoparticle scaffolds are shown in SEQ ID NOs: 24 to 26, respectively. For each of these sequences, an enzymatic site AS can be attached at the N-terminus for fusion with the antigen to be displayed. A GGGGS (SEQ ID NO: 33) linker can also be inserted after the enzymatic site of I3-01v9a.

[0169] I3-01v9a (for monomeric antigen display) (SEQ ID NO: 24)

[0170]

[0171] I3-01v9b (for trimeric antigen display; wherein the first residue is mutated to G) (SEQ ID NO: 25)

[0172]

[0173] I3-01v9c (for trimeric antigen display; wherein the first residue is mutated to G) (SEQ ID NO: 26)

[0174]

[0175] Example 7 Design and negative stain EM analysis of nanoparticles displaying RSV prefusion F trimer

[0176] In recent studies, RSV prefusion F (DS-Cav1) was displayed on a two-component nanoparticle platform (Marcandalli et al., Cell 2019, 176 (6): 1420-1431.e17) and ferritin 24-mer (Swanson et al., Sci Immunol 2020, 5 (47): eaba6466). However, EM analysis showed that DS-Cav1 tends to be monomeric in solution and may not be suitable for nanoparticle display. In this study, the possibility of known RSV prefusion F display on a variety of nanoparticle platforms was examined, and the inventors' highly optimized prefusion F trimer design for nanoparticle display was subsequently tested. Computational modeling was first performed to design 1c-SApNP ( Figure 3 , A). RSV prefusion F trimer (PDB ID: 4JHW) was directly fitted onto ferritin (FR) 24-mer, and the Cα-RMSD was In the present invention, the 1c-SApNP vaccine of the present invention is the most widely used vaccine of the present invention.And produce the F-FR nanoparticle of 34nm.Based on this calculation, 5GS linker is added into all RSV F-FR constructs between the C-terminal of RSV F and the N-terminal residue D5 of FR.As a result, the diameter of F-5GS-FR will reach about 38nm.RSV fusion front F trimer (PDBID:4JHW) is directly assembled on E2p and I3-01v9b 60 aggressiveness, obtains the big nanoparticle of 45.4nm and 47.5nm respectively.As shown in the previous research of the inventor, compared with little independent antigen, such large display trimeric 1c-SApNP vaccine can induce more potent and lasting immune response.

[0177] We then tested whether DS-Cav1 pre-fusion F could be displayed on three 1c-SApNP platforms ( Figure 3 , B). DS-Cav1-5GS-FR, E2p-LD4-PADRE, and I3-01v9b-LD7-PADRE constructs were transiently expressed in ExpiCHO cells and purified using a D25 antibody column. nsEM analysis showed that FR nanoparticles mixed with "naked" FR nanoparticles had irregular display of F protein, indicating protein misfolding ( Figure 3 , B, left). However, the DS-Cav1-E2p-LD4-PADRE sample showed only aggregates and fragments in the EM image ( Figure 3 , B, middle), the DS-Cav1-I3-01v9b-LD7-PADRE construct showed very low yield, and no nanoparticles were observed in the EM images ( Figure 3 , B, right). Due to extremely low yield, the SC-TM1c-SApNP fusion construct could not generate sufficient sample for nsEM analysis.

[0178] Next, we tested whether sc9-10 DS-Cav1 pre-fusion F could be displayed on three 1c-SApNP platforms ( Figure 3 , C). Due to the presence of inter-protomer disulfide bonds, the sc9-10 DS-Cav11c-SApNP fusion construct showed much lower yield than its DS-Cav1 counterpart. Nevertheless, nsEM analysis showed FR nanoparticles displaying closed prefusion F trimers mixed with partially "naked" FR nanoparticles, suggesting that inter-protomer disulfide bonds may not be formed on the nanoparticle surface ( Figure 3 , C, left). Both E2p and I3-01v9b constructs showed very low yields ( Figure 3 , C, center and right), where some partially formed particles mixed with aggregates were observed for I3-01v9b ( Figure 3 , C, right).

[0179] After testing the previously reported RSV prefusion F design, we attempted to test the “V2-Ext-P2DB6-D-L2 / NQ” design on FR and I3-01v9b1c-SApNP ( Figure 3 , D). Notably, EM images showed that for both “L2” and “NQ” constructs, the FR nanoparticles had closed prefusion F trimers ( Figure 3 , D, left and middle). Well-formed large I3-01v9b nanoparticles were observed with pre-fusion F trimers evenly distributed on their surface ( Figure 3 , D, right). Finally, the “V2-Ext-P2DB6-GDQ-L2 / NQ” design was tested on FR and I3-01v9b 1c-SApNP ( Figure 3 , E). Similar success was achieved in terms of expression yield and structural integrity. Notably, a well-formed layer of pre-fusion F trimers was observed on the surface of I3-01v9 1c-SApNPs ( Figure 3 , E, right).

[0180] Example 8 Additional redesigned RSV prefusion F trimers

[0181] This example describes additional redesigned RSV prefusion F trimers with different minimal sets of mutations to effectively stabilize the prefusion F trimer.

[0182] The F protein sequence of human respiratory syncytial virus A (strain A2) is available from GenBank, ID (P03420). Numbering is based on the UniProt definition of ID (P03420). Soluble F herein (or being called Fd) is defined as M1-L513, and wherein M1-G25 is a signal peptide (referring to SEQ ID NO:1, or A2N-WT). By the following uncut version of soluble F derived from A2-WT: by shortening and mutating the unstructured F2 C-terminal (residue Q98 to R109), by removing " processing active peptide " or P27 (residue E110 to R136) of 27 residues, by removing the N-terminal (residue F137 to S146) of fusion peptide (F137 to V157) and by adding 4 residues to 8 residues GS joint. Add two sudden changes (I379V and M447V) to improve F protein expression. This F construct design is considered the "base design" (SEQ ID NO:34 and SEQ ID NO:35, or A2N-JZ0-V2-Ext and A2N-JZ0-V2-Ext2).

[0183] The uncleaved, prefusion-optimized (UFO) soluble F construct was derived from a "base design" in which specific mutations were incorporated into the "β3 / β4 hairpin" (residues K176 to S190), where this region is hypothesized to be the root cause of RSV F metastability and undergo the greatest conformational changes during the membrane fusion process. Two types of mutations can be introduced into the β3 / β4 hairpin:

[0184] (i) Disulfide bonds between amino acids forming the β-sheet: Two disulfide mutations (S180C / S186C and A177C / T189C) have been experimentally tested because their C β -C β The shortest distances are and This gave rise to the so-called "SS" and "AT" designs.

[0185] (ii) Mutations between the two β strands: A double mutation (S182G / N183P) has been experimentally tested because it effectively disrupts the helical propensity at the turn between β3 and β4, resulting in the so-called "GP" design.

[0186] A total of 8 combinations were tested (Ext vs. Ext2+SS vs. AT+no GP vs. GP):

[0187] SS-based design:

[0188] A2N-JZ0-V2-Ext-SS(SEQ ID NO:36),A2N-JZ0-V2-Ext-SSGP(SEQ ID NO:37),A2N-JZ0-V2-Ext2-SS(SEQ ID NO:38),A2N-JZ0-V2-Ext2-SSGP(SEQ ID NO:39)

[0189] AT-based design:

[0190] A2N-JZ0-V2-Ext-AT (SEQ ID NO: 40), A2N-JZ0-V2-Ext-ATGP (SEQ ID NO: 41), A2N-JZ0-V2-Ext2-AT (SEQ ID NO: 42), A2N-JZ0-V2-Ext2-ATGP (SEQ ID NO: 43)

[0191] A trimerization motif (e.g., foldon and viral capsid protein SHP (PDB: 1TD0)) can be added to the C-terminus of the redesigned F construct with a short GS linker in between to stabilize the trimer and increase the trimer ratio in the total protein yield. A His6-tag can be added to the C-terminus of the trimerization motif to facilitate protein purification by nickel column.

[0192] The C-terminus of the redesigned F construct can be fused to the N-terminus of the nanoparticle-forming subunit, such that when expressed in a suitable cell line, the fusion construct can self-assemble into nanoparticles with the pre-fusion F trimer displayed on the nanoparticle surface.

[0193] RSV F construct sequence (based on A2 strain wild-type sequence):

[0194] MELLILKANAITTILTAVTFCFASG (SEQ ID NO: 2): leader

[0195] QSTPPTNNRARR (SEQ ID NO: 3): Unstructured F2 Ctm

[0196] ELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKV (SEQ ID NO: 52): P27 peptide + fusion peptide (FP)

[0197] KAVVSLSNGVSVLTS (SEQ ID NO: 53): β3 / β4 hairpin region.

[0198] SEQ ID NO: 34(A2N-JZ0-V2-Ext)

[0199]

[0200] SEQ ID NO: 35(A2N-JZ0-V2-Ext2)

[0201]

[0202] SEQ ID NO:36(A2N-JZ0-V2-Ext-SS)

[0203]

[0204] SEQ ID NO:37(A2N-JZ0-V2-Ext-SSGP)

[0205]

[0206] SEQ ID NO:38(A2N-JZ0-V2-Ext2-SS)

[0207]

[0208] SEQIDNO:39(A2N-JZ0-V2-Ext2-SSGP)

[0209]

[0210] SEQ ID NO:40(A2N-JZ0-V2-Ext-AT)

[0211]

[0212] SEQ ID NO:41(A2N-JZ0-V2-Ext-ATGP)

[0213]

[0214] SEQ ID NO:42(A2N-JZ0-V2-Ext2-AT)

[0215]

[0216] EQ ID NO:433(A2N-JZ0-V2-Ext2-ATGP)

[0217]

[0218] Example 9 Vaccine antigen expression and purification

[0219] This example describes the expression and purification of the redesigned prefusion F trimer vaccine antigen as described in Example 8.

[0220] Cell lines: All F trimers were expressed in HEK293F / Expi293F cells and ExpiCHO cells, with ExpiCHO cells showing higher protein yields. All F-displaying nanoparticles were expressed in ExpiCHO cells.

[0221] Purification: After transient expression, based on (1) pre-fusion site- -Specific neutralizing antibody D25 and (2) neutralizing antibody MPE8 that recognizes two protomers of the F trimer, antigen-specific antibody columns were used to purify RSV F-containing antigens from the supernatant. MPE8 binds to both pre-fusion and post-fusion F trimers, but tends to favor the pre-fusion structure. Both D25 and MPE8 antibody columns can effectively purify RSV F trimers and nanoparticles, with D25 showing a higher protein yield. For hMPV and PIV1 to 5F trimers, His-tag purification was performed using a nickel column. An antibody column for tag-free trimer / nanoparticle purification will be developed.

[0222] Example 10 Study on paramyxovirus F metastability using known prefusion F trimers

[0223] This example describes the characterization of prefusion F trimers reported in the literature.

[0224] The present inventors set out to investigate the underlying causes of paramyxovirus F metastability and develop a simple, universal, and effective prefusion F trimer stabilization strategy applicable to all members of the paramyxovirus family and enabling the multivalent display of stable F trimers on self-assembling nanoparticles as virus-like particle (VLP) vaccines. Because RSV has been the most studied in structure-based vaccine design and three representative prefusion F designs are available, RSV was chosen as the focus of the current study, with hMPV and PIV3 also included to validate the F stabilization strategy. Three RSV F designs reported in the literature were first characterized to compare their expression, trimer formation, and purification. The three RSV F designs were DS-Cav1 (McLellan et al., Science 342:592-8, 2013), sc9-10 DS-Cav1 (Joyce et al., Nat Struct Mol Biol 23:811–820, 2016), and SC-TM (Krarup et al., Nat Commun 6:8143, 2015).

[0225] For expression, HEK293F (a transient mammalian cell line) and ExpiCHO (a transient, high-yielding version of the industrial CHO-S cell line) were used to examine the effect of cell line on RSV F trimer yield and purity. For purification methods, methods based on recognition of the prefusion site- The ability of two immunoaffinity columns, D25 and MPE8 that bind two adjacent F subunits, to perform tag-free purification as antibody columns was investigated. The role of the C-terminal trimerization motif in terms of trimer stability was examined using a soluble F construct without the C-terminal motif and two F constructs with the foldon and 1TD0 motifs at their C-termini.

[0226] Three representative RSV pre-fusion F constructs (DS-Cav1, sc9-10 DS-Cav1 and SC-TM) were transiently expressed in HEK293F and ExpiCHO cells. Previous research by the inventors (He et al., Sci Adv 4(11): eaau6769, 2018) showed that HIV-1 gp140 trimers can be expressed in ExpiCHO cells with significantly higher yields and purity than in 293F cells. Based on this discovery, a small volume of 25 ml ExpiCHO cells was used for transfection, followed by purification using D25 and MPE8 antibody columns, while 400 ml and 100 ml 293F cells were used for transfection, followed by D25 and MPE8 purification, respectively. Due to the site of D25 recognition of pre-fusion F The RSV F protein of the present invention is characterized by the use of the Superdex 200 10 / 300 post and the size exclusion chromatography (SEC) of the RSV F protein.And no matter F is in monomer or polymer state (for example, dimer, trimer and aggregation), therefore expect total F protein (and trimer) output that D25 produces is higher than MPE8, and more complete spectrum is provided.Therefore, when evaluating the combination of D25 antibody post and 293F cell, used larger volume, so that the usefulness of the data obtained from this combination is maximized.After transient expression and antibody purification, RSV F protein is characterized by size exclusion chromatography (SEC) using Superdex 200 10 / 300 post.

[0227] The three RSV prefusion F trimers exhibited distinct patterns of design specificity. Overall, the optimized sc9-10 DS-Cav1 design significantly outperformed the original DS-Cav1 and SC-TM designs in nearly every aspect examined in this comparison. Of particular note, sc9-10 DS-Cav1 exhibited the highest trimer yield and purity when fused to a C-terminal trimerization motif, but produced primarily monomers in the absence of any C-terminal trimerization motif, suggesting that the underlying cause of F metastability remains in sc9-10 DS-Cav1. With respect to the effect of cell line on F expression, DS-Cav1 and SC-TM exhibited little to no yield in 293F cells but behaved differently in ExpiCHO cells, where DS-Cav1 displayed measurable expression levels, while SC-TM did not. Therefore, in terms of protein expression, the three pre-fusion F design constructs can be ranked as sc9-10DS-Cav1>>DS-Cav1>>SC-TM, among which ExpiCHO is a more suitable expression system.

[0228] With respect to the antibody columns, DS-Cav1 and sc9-10 DS-Cav1 exhibited rather different patterns: for DS-Cav1, D25 showed slightly lower yields than MPE8 for constructs without a trimerization motif and with foldon, whereas for sc9-10 DS-CaV1, D25 gave significantly higher yields than MPE8, as indicated by the UV280 absorbance values ​​in the SEC profiles.

[0229] In terms of trimer stability, it is evident from the SEC spectra that both DS-Cav1 and sc9-10 DS-Cav1 require a C-terminal trimerization motif to remain as trimers, suggesting that these F designs do not improve trimer formation or that the primary cause of F metastability still exists in these constructs. Finally, in terms of the composition of the F protein, DS-Cav1 and sc9-10 DS-Cav1 also exhibit different patterns: DS-Cav1 shows a higher molecular weight peak at 11 ml, which merges with the trimer peak at 12 ml when foldon is used, but with another C-terminal trimerization motif, 1TD0, the peak of this F species can be clearly seen; in contrast, for sc9-10 DS-Cav1, a single trimer peak at 12 ml is shown when foldon is used, but aggregates are generated when 1TD0 is used. It is also noteworthy that the trimer peak generated by 1TD0 is narrower than that generated by foldon, indicating that the trimer purity of the sc9-10 DS-Cav1 F trimer linked to 1TD0 is higher. Blue native polyacrylamide gel electrophoresis (BN-PAGE) showed that the different F constructs had consistent monomer and trimer bands.

[0230] Example 11 Characterization of the redesigned RSV F trimer

[0231] This example describes the characterization of the inventors' redesigned RSV prefusion F trimer vaccine antigen as described in Example 8.

[0232] Based on the hypothesis that β3 / β4 hairpin is the root cause of RSV F metastability, two disulfide bond mutations were studied, which are used to lock this region in its pre-fusion structure (β hairpin) and prevent it from being converted into a post-fusion structure (extended α helix). These two disulfide bond mutants are referred to as A2N-JZ0-V2-Ext-SS and A2N-JZ0-V2-Ext-AT, or simply referred to as V2-Ext-SS and V2-Ext-AT (SEQ ID NO:36 and SEQ ID NO:40). The F constructs of these three newly designed constructs were characterized using the previously described scheme. Overall, both disulfide bond mutants were superior to the basic design, with V2-Ext-AT being the best performer, producing a significant trimer peak with high yield and without any C-terminal trimerization motif. These results provide the strongest evidence that β3 / β4 hairpin is the root cause of RSV F metastability, and introducing a minimum of a single disulfide bond mutation (if properly introduced) into this region can effectively eliminate metastability. The base design could be expressed in both cell lines, with higher yields obtained from ExpiCHO cells.

[0233] Regarding the antibody column, D25 consistently yielded higher yields than MPE8. Notably, MPE8, which recognizes both F protomers within the trimer, showed no preference for the trimer over the other species within the produced F protein. For example, after D25 purification, a more pronounced trimer peak was observed for V2-Ext (lacking any C-terminal trimerization motif) expressed in ExpiCHO cells. Regarding trimer formation, the trimerization motif fused to the C-terminus of V2-Ext can lead to more difficult trimer folding, e.g., no yield of the V2-Ext-foldon construct, or aggregation, e.g., the presence of a peak (8 to 10 mL) of high molecular weight aggregates corresponding to the V2-Ext-1TD0 construct in ExpiCHO cells. The first mutant (V2-Ext-SS), with a disulfide bond engineered near the β-turn (one residue away), showed significantly increased F protein expression in both cell lines, with ExpiCHO slightly outperforming HEK 293F. Regarding the antibody columns, D25 and MPE8 produced similar SEC profiles for ExpiCHO-produced proteins, but behaved differently for 293F-produced proteins.

[0234] In addition, the disulfide bond mutation is shown to be more effective for trimer stabilization when used with foldon than when used with 1TD0, or more effective than for trimer stabilization in the absence of any C-terminal trimerization motif. The second mutant (V2-Ext-AT) with disulfide bond engineering at the far end of the β3 / β4 hairpin has shown the most desirable properties, although its output is lower than that of the first disulfide bond mutant. In addition to output, the two disulfide bond mutations are also significantly different in their ability to promote trimer folding and in their compatibility with different C-terminal trimerization motifs. Specifically, when the C-terminal trimerization motif is not connected, AT is shown to be much more effective than SS in promoting trimer formation. The AT disulfide bond design also shows that it is more compatible with 1TD0 than with foldon.

[0235] Next, it was studied whether adding a double mutation (GP) to two disulfide bond mutants at the β turn would further destabilize the post-fusion helix and thus stabilize the β3 / β4 hairpin before fusion. Unexpectedly, the GP mutation at the β turn had a completely different effect on the two disulfide bond mutants. The SSGP design showed that the trimer ratio in the produced F protein was greatly improved, and the total yield was significantly reduced. A higher trimer peak was also observed for the V2-Ext-SSGP construct fused to foldon and 1TD0, regardless of the cell line used. On the contrary, GP had a major negative impact on the F construct containing AT. Although the ATGP design showed a lower monomer and aggregate ratio, the total yield was too low to be used for vaccine production in CHO cells. In short, for the design of the pre-fusion F trimer, V2-Ext-SSGP provides a promising alternative to V2-Ext-AT.

[0236] Finally, we investigated whether a longer linker between F2 and F1 could improve the two "best" prefusion F constructs identified to date—V2-Ext-SSGP and V2-Ext-AT. Notably, in the absence of any C-terminal trimerization motif, Ext2 mutations consistently improved trimer ratios. However, the use of a long cleavage site linker was shown to negatively impact trimer ratios or trimer yields for any F construct with a C-terminal trimerization motif. In conclusion, long cleavage site linkers can be used in the absence of a trimerization motif.

[0237] Example 12 Redesigned hMPV and PIV prefusion F trimers

[0238] This example describes the redesign of hMPV and PIV prefusion F trimers with a minimal set of mutations corresponding to the minimal set of mutations used for the RSV F trimer described in Example 8 to effectively stabilize the prefusion F trimer. Further details of the study are described in Example 13.

[0239] The F protein sequence of human metapneumovirus hMPV (isolate "TN03.03.19") was obtained from GenBank, ID AEZ52364. Numbering is based on the crystal structure (PDB ID: 5WB0) and the UniProt definition (IDQ6WB98) of another hMPV strain (strain CAN97-83). Soluble F is defined as M1-T489, where M1-G18 is the signal peptide (see SEQ ID NO: 44, or TN-WT). A shortened version of soluble F (corresponding to Fd of hRSV) is defined as M1-L481, where M1-G18 is the signal peptide (see SEQ ID NO: 45, or TN-WT-cut). The uncleaved, prefusion-optimized (UFO) soluble F construct was designed based on the TN-WT-cut, with specific mutations incorporated into the corresponding "β3 / β4 hairpin" (residues E146 to T160) based on the hypothesis that this region is fundamentally responsible for hMPV F metastability and undergoes the greatest conformational changes during membrane fusion. Disulfide bonds can be introduced between amino acids forming the β sheet to lock the hMPV F structure in the prefusion state. The disulfide-introducing mutations A147C / A159C were tested using two different treatments of the fusion peptide (FP) (see SEQ ID NO:46 and SEQ ID NO:47, or TN-cut-UFO1 and TN-cut-UFO2) to validate the metastability hypothesis and the importance of the "β3 / β4 hairpin" for hMPV F.

[0240] A trimerization motif (e.g., foldon and viral capsid protein SHP (PDB: 1TD0)) can be added to the C-terminus of the redesigned F construct with a short GS linker in between to stabilize the trimer and increase the trimer ratio in the total protein yield. A His6-tag can be added to the C-terminus of the trimerization motif to facilitate protein purification by nickel column.

[0241] The C-terminus of the redesigned F construct can be fused to the N-terminus of the nanoparticle-forming subunit, such that when expressed in a suitable cell line, the fusion construct can self-assemble into nanoparticles with the pre-fusion F trimer displayed on the nanoparticle surface.

[0242] hMPV F construct sequence:

[0243] MSWKVVIIFSLLITPQHG (SEQ ID NO: 54): leader

[0244] DQLAREEQIENPRQSRFVLGAIALGV (SEQ ID NO: 55): Unstructured F2 N-terminus + cleavage site + fusion peptide

[0245] EAVSTLGNGVRVLAT (SEQ ID NO: 56): corresponding β3 / β4 hairpin region

[0246] SEQ ID NO:44 (TN-WT)

[0247]

[0248] SEQ ID NO:45 (TN-WT-cut)

[0249]

[0250] SEQ ID NO:46 (TN-cut-UFO1)

[0251]

[0252] SEQ ID NO:47((TN-cut-UFO2)

[0253]

[0254] PIV3 F: The sequence of the F protein of human parainfluenza virus type 3 (PIV3) (strain "HPIV3 / USA / 629-D01959 / 2007") was obtained from GenBank, ID (AGW51052). Numbering is based on the cryo-EM structure (PDB ID: 6MJZ) and the UniProt definition of the recombinant PIV3 / PIV1 virus (ID (055888)). The soluble F sequence (corresponding to Fd of hRSV) is defined as M1-T484, where M1-C18 is the signal peptide (see SEQ ID NO: 48, or PIV3-WT).

[0255] The uncut pre-fusion optimized (UFO) soluble F construct was designed based on PIV3-WT by removing the unstructured F2 N-terminus and cleavage site and incorporating specific mutations into the "β1 / β2 hairpin" (residues V158 to I172) based on the hypothesis that this region is the root cause of PIV3 F metastability and undergoes the greatest conformational changes during the membrane fusion process. Disulfide bonds can be introduced between the amino acids forming the β sheet in chains V158 to V161 and I169 to I172 to lock the PIV3 F structure in the pre-fusion state. It must be noted that any disulfide bond introduced in Q162 to L168 can lead to structural distortion. The disulfide bond-introducing mutation Q159C / A171C was tested using two different approaches to manipulate the fusion peptide (FP) region (see SEQ ID NO:61 and SEQ ID NO:62, or PIV3-UFO1 and PIV3-UFO2) to verify the metastability hypothesis and the importance of the “β1 / β2 hairpin” for PIV3 F.

[0256] A trimerization motif (e.g., foldon and viral capsid protein SHP (PDB: 1TD0)) can be added to the C-terminus of the redesigned F construct with a short GS linker in between to stabilize the trimer and increase the trimer ratio in the total protein yield. A His6-tag can be added to the C-terminus of the trimerization motif to facilitate protein purification on a nickel column.

[0257] The C-terminus of the redesigned F construct can be fused to the N-terminus of the nanoparticle-forming subunit, such that when expressed in a suitable cell line, the fusion construct can self-assemble into nanoparticles with the pre-fusion F trimer displayed on the nanoparticle surface.

[0258] Other PIVs: Considering the high structural similarity between PIV3, PIV5, and other PIVs, UFO trimer constructs can be designed according to the same principles to modify the β1 / β2 hairpin region.

[0259] PIV3 F construct sequence:

[0260] MLISILSIITTMIMASHC (SEQ ID NO: 57): leader

[0261] GLKLQKDVIVTNQESNENTDPRTERFFGGVIGTIALGV (SEQ ID NO: 58): unstructured F2 N-terminus + cleavage site + fusion peptide VQSVQSSVGNLIVAI (SEQ ID NO: 59): β1 / β2 hairpin, corresponding to the β3 / β4 hairpin region.

[0262] SEQ ID NO: 48 (PIV3-WT)

[0263]

[0264] SEQ ID NO:61 (PIV3-UFO1)

[0265]

[0266] SEQ ID NO:62 (PIV3-UFO2)

[0267]

[0268] Example 13 Characterization of redesigned F trimers of other paramyxoviruses

[0269] After determining that the β3 / β4 hairpin is the root cause of RSV F metastability as described in Examples 8 to 11, the possibility of extending this design concept to other members of the Paramyxovirus family was examined. To examine this possibility, two sets of UFO constructs were generated for hMPV F (SEQ ID NO: 46 and SEQ ID NO: 47) and PIV3 F (SEQ ID NO: 61 and SEQ ID NO: 62), all of which had a C-terminal trimerization motif (1TD0) followed by a His6-tag.

[0270] In preliminary tests, all four constructs were transiently expressed in 250mL 293F cells and purified using nickel column and then SEC was performed. In general, for both hMPV and PIV3, the UFO2 design was significantly superior to the UFO1 design comprising fusogenic peptide (FP). For hMPV, a significant trimer peak (11 to 12mL) was observed for UFO2 (rather than UFO1), consistent with BN-PAGE analysis. For PIV3, the UFO2 construct produced a high trimer peak (11 to 12mL) with a significant aggregate peak (8 to 10mL), while the UFO1 construct mainly produced aggregates in 293F cells, similar to the observed results for the hMPV construct.

[0271] In summary, this comparative analysis confirms that the β3 / β4 and β1 / β2 hairpins are the root cause of F metastability in hMPV and PIV3, respectively. Given the similarities between PIV1 to 5, this result also suggests that the UFO2 design may be applicable to other PIVs.

[0272] Example 14 EM Characterization of Nanoparticles Displaying RSV F Trimer

[0273] Negative-stain electron microscopy (nsEM) analysis was performed to characterize the RSV F trimer-displaying nanoparticles as described in Example 8 ( Figure 4 Two nanoparticle platforms were examined in this study, 24-mer ferritin and 60-mer E2p.

[0274] Using ferritin nanoparticles as a model presentation system, three representative RSV F designs ( Figure 4 , A). A 5-GS linker was inserted between the C-terminus of F and the N-terminus of the ferritin subunit. Among the three designs, sc9-10 DS-Cav1 was the best performer with good nanoparticle formation ( Figure 4 , A, middle), whereas the ferritin fusion constructs of DS-Cav1 and SC-TM failed to form nanoparticles or failed to form native-like F trimers ( Figure 4 , A, left and right). Two redesigned RSV F trimers (V2-Ext-SSGP and V2-Ext-AT) were displayed on ferritin using 10-GS and 5-GS linkers, respectively ( Figure 4 , B, columns 1 and 2). D25 and MPE8 antibody columns can be used to purify nanoparticles ( Figure 4 , B. Rows 1 and 2, columns 1 and 2). EM analysis showed that both redesigned F trimers were well displayed on ferritin nanoparticles, with the shorter (5-GS) linker showing a more visible and complete pre-fusion F spike on the nanoparticle surface. Most importantly, when both the sc9-10 DS-Cav1 trimer and the newly designed pre-fusion F were displayed on ferritin nanoparticles, the newly designed pre-fusion F spike showed visually recognizable differences in shape compared to the sc9-10 DS-Cav1 trimer spike. Specifically, the V2-Ext-AT F trimer spike exhibited a "thumb-like" shape with a solid surface, which is characteristic of a pre-fusion, closed trimer spike, while the sc9-10 DS-Cav1 trimer spike appeared "lollipop-shaped" and hollow, indicating an open conformation ( Figure 4 ,A, Column 2, Box vs. Figure 4, B, 1st row, 2nd column, box). EM data show that the F trimer (without any RSV F metastability characteristics) that is more stable in nature is crucial for the development of RSV F nanoparticle vaccines. In addition, it is obvious that all three existing representative RSV pre-fusion F designs (DS-Cav1, optimized sc9-10DS-Cav1 and SC-TM) are not suitable for nanoparticle display. Finally, it was checked whether the newly designed RSV pre-fusion F trimer can be displayed on large 60-aggressor nanoparticles with locking domain (LD) and built-in T helper epitopes. For this reason, two nanoparticle constructs, V1-Ext-AT-E2p-LD4 and V1-Ext-AT-E2p-LD4-PADRE, were expressed and purified for EM analysis ( Figure 4 , B, column 3). Consistently, well-formed nanoparticles with "thumb-like" arrays of prefusion F trimers on the surface were observed for both constructs, confirming that the newly designed prefusion RSV F trimers can be displayed on a multilayer nanoparticle platform as a vaccine candidate.

[0275] ***

[0276] Thus, the invention has been broadly disclosed and illustrated with reference to the representative embodiments described above. It will be appreciated that various modifications can be made thereto without departing from the spirit and scope of the invention.

[0277] It should also be noted that all publications, sequence accession numbers, patents, and patent applications cited herein are hereby expressly incorporated by reference in their entirety and for all purposes, just as if each were individually so indicated. To the extent that they conflict with definitions in this disclosure, definitions contained in the text incorporated by reference are excluded.

Claims

1. An engineered soluble fusion (F) protein of a paramyxovirus comprising an altered soluble F sequence having modifications relative to the wild-type soluble F sequence of the paramyxovirus, wherein the modifications comprise (1) replacement of two or more negatively charged residues surrounding the β23 strand (D486 to A490) with polar or hydrophobic residues, (2) deletion of the P27 peptide (E110 to R136), and (3) an engineered intraprotomer disulfide bond located within the F1 subunit or connecting the F2 to the F1 subunit; wherein the amino acid numbering is based on the human RSV A2 strain F protein (UniProt ID P03420).

2. The engineered soluble F protein of claim 1, wherein the paramyxovirus is RSV.

3. The engineered soluble F protein of claim 1, wherein the two or more negatively charged residues surrounding the β23 strand are D486 and E487.

4. The engineered soluble F protein of claim 3, wherein the substitutions around the β23 strand comprise D486N / E487Q or D486L / E487L.

5. The engineered soluble F glycoprotein of claim 1, wherein the engineered disulfide bond is S155C / S290C, S62C / K196C, or E60C / K196C.

6. The engineered soluble F protein of claim 1, further comprising a linker portion that replaces: (1) the furin cleavage site; or (2) the unstructured F2 C-terminus (Q98 to R109), and a portion of the fusion peptide (FP) N-terminus (F137 to V157).

7. The engineered soluble F protein of claim 6, wherein the replaced F2 C-terminus comprises residues N104 to R109 ( 104 NNRARR 109 ; SEQ ID NO:31).

8. The engineered soluble F protein of claim 6, wherein the replaced portion of the N-terminus of the fusion peptide (FP) comprises F137 to S146.

9. The engineered soluble F protein of claim 1, further comprising a substitution of residue S215.

10. The engineered soluble F protein of claim 9, wherein residue S215 is replaced by P.

11. The engineered soluble F protein of claim 1 , further comprising a substitution of residue E92.

12. The engineered soluble F protein of claim 11, wherein residue E92 is substituted with D, Q, other short polar residues, or a hydrophobic residue.

13. The engineered soluble F protein of claim 1, further comprising a V185P substitution.

14. The engineered soluble F protein of claim 1, further comprising one or both of S46G and K462Q substitutions.

15. The engineered soluble F protein of claim 1, further comprising an engineered intraprotomer disulfide bond S180C / S186C or A177C / T189C within the β3 / β4 hairpin.

16. The engineered soluble protein of claim 1, further comprising an engineered inter-protomer disulfide bond A149C / Y458C.

17. The engineered soluble F protein of claim 1, comprising the sequence shown in any one of SEQ ID NOs: 17 to 23, a conservatively modified variant thereof, or a substantially identical sequence thereof.

18. An engineered soluble fusion (F) protein of a paramyxovirus comprising a modified altered soluble F sequence relative to the wild-type soluble F sequence of the paramyxovirus, wherein the modification comprises an engineered disulfide bond connecting the β3 / β4 hairpin or corresponding hairpin forming β-sheet amino acid pairs in the F1 subunit, wherein the numbering of the hairpins is based on respiratory syncytial virus (RSV).

19. The engineered soluble F protein of claim 18, wherein the paramyxovirus is human RSV, wherein the engineered disulfide bond is located between the replaced residues S180C / S186C or A177C / T189C in the β3 / β4 hairpin, and wherein the amino acid numbering is based on the human RSV A2 strain with UniProt ID P03420.

20. The engineered soluble F protein of claim 19, wherein the wild-type soluble F sequence is shown in SEQ ID NO: 1, or a conservatively modified variant thereof.

21. The engineered soluble F protein of claim 19, wherein the modification further comprises a mutation at the C-terminus of the unstructured F2 subunit.

22. The engineered soluble F protein of claim 21, wherein the mutations in the unstructured F2 C-terminus comprise a truncation of residues 104 to 109 (NNRARR) (SEQ ID NO: 31) and a P102A substitution.

23. The engineered soluble F protein of claim 19, wherein the modifications further comprise (1) replacing the processed active peptide (P27) (residues E110 to R136) and the N-terminus of the fusion peptide (residues F137 to S146) with a (GS)n linker sequence, wherein n is any integer from 1 to 5, and / or (2) replacing I379V and M447V.

24. The engineered soluble F protein of claim 19, comprising the amino acid sequence shown in any one of SEQ ID NOs: 36 to 43 or a conservatively modified variant thereof.

25. The engineered soluble F protein of claim 18, wherein the paramyxovirus is human metapneumovirus (hMPV), wherein the engineered disulfide bond is located between replaced residues A147C / A159C in the β3 / β4 hairpin, and wherein amino acid numbering is based on hMPV strain CAN97-83 with UniProt ID Q6WB98.

26. The engineered soluble F protein of claim 25, wherein the wild-type soluble F sequence is shown in SEQ ID NO: 44 or SEQ ID NO: 45, or a conservatively modified variant thereof.

27. The engineered soluble F protein of claim 25, wherein the modification further comprises a mutation at the C-terminus of the unstructured F2 subunit.

28. The engineered soluble F protein of claim 27, wherein the mutation of the unstructured F2 C-terminus comprises replacing the unstructured F2 C-terminus DQLAREEQIENP (SEQ ID NO: 60) and the cleavage site RQSR (SEQ ID NO: 49) with a (GS)n linker sequence, wherein n is any integer from 1 to 5.

29. The engineered soluble F protein of claim 25, comprising the amino acid sequence shown in any one of SEQ ID NOs: 46 and 47, or a conservatively modified variant thereof.

30. The engineered soluble F protein of claim 18, wherein the paramyxovirus is human parainfluenza virus (hPIV), wherein the engineered disulfide bond is located between replacement residues Q159C / A171C in the β1 / β2 hairpin, and wherein the amino acid numbering is based on the recombinant hPIV3 / hPIV1 virus with UniProt ID O55888.

31. The engineered soluble F protein of claim 30, wherein the wild-type soluble F sequence is shown in SEQ ID NO: 48, or a conservatively modified variant thereof.

32. The engineered soluble F protein of claim 30, wherein the modification further comprises a mutation at the C-terminus of the F2 subunit.

33. The engineered soluble F protein of claim 32, wherein the mutation at the F2 C-terminus comprises replacing the C-terminal sequence NQESNENTDP (SEQ ID NO: 50) and the cleavage site RTER (SEQ ID NO: 51) with a (GS)n linker sequence, wherein n is any integer from 1 to 6.

34. The engineered soluble F protein of claim 30, comprising the amino acid sequence shown in any one of SEQ ID NOs: 61 and 62, or a conservatively modified variant thereof.

35. An engineered soluble F protein of respiratory syncytial virus (RSV), comprising a soluble RSV F sequence altered by (1) deletion of the P27 peptide (residues E110 to R136), (2) modification of the unstructured F2 subunit C-terminus (residues Q98 to R109), and (3) truncation of the N-terminus of the fusion peptide (residues F137 to V157), wherein the amino acid numbering is based on the human RSV A2 strain with UniProt ID P03420.

36. The engineered soluble F protein of claim 35, wherein modification of the unstructured F2 C-terminus comprises at least one of (1) truncation of residues 104 to 109 (NNRARR) (SEQ ID NO: 31) and (2) a P102A substitution.

37. The engineered soluble F protein of any one of claims 1 to 36, further comprising a C-terminal trimerization motif.

38. The engineered soluble F protein of any one of claims 1 to 37, further comprising an N-terminal leader sequence.

39. A Paramyxovirus immunogenic composition comprising the engineered soluble F protein of any one of claims 1 to 38 displayed on the surface of self-assembling nanoparticles.

40. The immunogenic composition of claim 39, wherein the self-assembling nanoparticle comprises a trimer sequence, and wherein the C-terminus of the immunogenic polypeptide is fused to the N-terminus of the subunit sequence of the nanoparticle.

41. A polynucleotide sequence encoding the immunogenic polypeptide of any one of claims 1 to 38 or the immunogenic composition of claim 39.

42. A pharmaceutical composition comprising the immunogenic polypeptide of any one of claims 1 to 38, the immunogenic composition of claim 39, or the polynucleotide of claim 41, and a pharmaceutically acceptable carrier.

43. A method for preventing or treating a Paramyxovirus infection in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 42.

44. The method of claim 43, wherein the paramyxovirus is RSV, hMPV, or PIV.

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