Pre-fusion stable human parainfluenza virus 3F proteins

Abstract

Provided herein are engineered parainfluenza virus fusion protein (PIV F) polypeptides. In some aspects, the engineered PIV F polypeptides exhibit enhanced conformational stability and / or antigenicity. Also provided are methods of using the engineered PIV F polypeptides as diagnostic agents, using the engineered PIV F polypeptides at screening platforms, and / or using the engineered PIV F polypeptides in vaccine compositions.

Description

[0001] Citation of Related Applications

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 479,127, filed January 9, 2023, the entire contents of which are incorporated herein by reference.

[0003] References to sequence listings

[0004] This application contains a sequence listing XML that has been submitted electronically and is hereby incorporated by reference in its entirety. The sequence listing XML was created on January 5, 2024, is named UTFBP1325WO_ST26.xml, and is 32,897 bytes in size. Background Art 1. Technical Field

[0005] The present disclosure relates generally to the fields of medicine, virology, and immunology. More specifically, the present disclosure relates to engineered parainfluenza virus fusion protein (PIV F) polypeptides and uses thereof.

[0006] 2. Description of Related Technology

[0007] Parainfluenza viruses 1, 2, 3, and 4 (PIV 1-4) cause mild to severe respiratory illness in humans. PIVs are enveloped, single-stranded, negative-sense RNA [ssRNA(-)] viruses, members of the Paramyxoviridae family. Seasonal PIV infection is associated with approximately 40% of childhood hospitalizations for lower respiratory tract infections and approximately 75% of confirmed cases of croup. PIV infection can occur throughout a person's life. Most adults are at low risk for severe illness from PIV infection, but infection in immunocompromised individuals and the elderly can occasionally result in severe or life-threatening lower respiratory tract illness. Although the development of a vaccine against PIV is of public health importance, no vaccine has been licensed to date.

[0008] Long-term surveillance of respiratory viruses in the United States has found that PIV3 is the most common PIV serotype associated with symptomatic disease in children and adults, followed by PIV 1, 2, and 4. The genome of PIV encodes several envelope glycoproteins, one of which is the fusion glycoprotein (F). The F glycoprotein is a fusogenic agent required for viral entry into cells and is also an important target for neutralizing antibody (nAb) infection responses. PIV F proteins have varying degrees of sequence conservation, with identities ranging from 20-50% and similarities ranging from 35-65% among exemplary F proteins of the respirovirus and mumps virus genera. Although PIV vaccines incorporating F subunit antigens are under development, no PIV vaccine or antiviral therapy has been approved for use in humans. Therefore, vaccines that provide long-lasting protection and therapies that avoid mortality and morbidity are desired.

[0009] Therefore, safe and effective immunogenic compositions and therapies are needed to protect against PIV infection and its associated sequelae. Diagnostic reagents are also needed to detect immune responses to PIV, guide the design of F-based PIV vaccines, and support the development of therapeutic or prophylactic antibodies against PIV. Summary of the Invention

[0010] Thus, provided herein are engineered proteins having at least one amino acid substitution relative to the amino acid sequence of a native respiratory or mumps virus F protein (i.e., SEQ ID NOs: 1-7), wherein the engineered protein is stabilized in the prefusion conformation of respiratory or mumps virus F. The engineered protein can specifically bind to a respiratory or mumps virus F protein prefusion-specific antibody.

[0011] In one embodiment, provided herein is a viral or mumps fusion protein (preferably, a parainfluenza virus fusion protein (PIV)) comprising at least 90% sequence identity to amino acids 19-481 of SEQ ID NO: 1 or 2. F)) An engineered protein of an extracellular domain comprising at least one substitution or a group of substitutions selected from the group consisting of: H27C / F437C; H27C / T439C; H27C / P440C; H27C / I443C; H27Y; V28M; V30I; N33C / K295C; G37C / S337C; S41C / P283C; Y48C / I169C; Y48C / I169C / A140Y; L49C / L278C; L49F; L49W; I50C / A171C; I50C / T277C; I50W; S52C / K173C ;S52C / S275C;S52L;L53C / S174C;P55C / V175C;P55C / Q176C;K56C / N155C;I57F;E58C / I183C;G64C / G196C;G64C / G200C;Q67C / L199C;Q67 C / G200C; Y71C / L203C; L86C / V266C; Q89C / A131C; K90Y; I93C / G116C; V94C / G116C; C / G381C; A123P; T124P; S125C / P374C; S125P; S125W; A126P; I128F; I128W; L134C / I267C; A137C / I267C; I144W; L147C / A171C; I151C / A17 1C; A157C / Q176C; A157C / D177C; A157F; V158L; Q159C / A171C; L168Q; V170I; V170M; A171V; K173Q; V175L; V175P; V179L; E182F; E182W; P1 85C / A195C; G191P; G200E; I201F; I201W; A202T; E209W; I213C / I226C; I213C / G230C; G219C / E333C; 236Y; S246V; L256Y; V264F; V264W; V266F; V266W; S275F; S275M; T277F; T277L; T277W; L278F; L278W; V280F;D327C / P344C; A334S; G345M; F346C / T369C; F346C / S370C; N349P; L356F; S361C / T444C; Q362C / N447C; P36 4C / N447C; T366C / V449C; T367R; N380C / G433C; G381C / K431C; G382C / G433C; V384I; T413C / A436C; G433F; I 443W;I443Y;V449C / I454C;V449C / D455C;V449C / I456C;V449C / S457C;A450F;L451P;D452P;I454F;D455K;I456W;S457C / V449C;S457C / I456C;K464C / V449C;S470C / K471C;K471A;K471L;W473A, and wherein the positions are relative to SEQ ID NO: 1 or 2. ;

[0012] The engineered protein may comprise or may further comprise at least one set of paired cysteine ​​substitutions selected from the group consisting of: H27C / F437C; H27C / T439C; H27C / P440C; H27C / I443C; N33C / K295C; G37C / S337C; S41C / P283C; Y48C / I169C; Y48C / I169C / A140Y; L49C / L278C; I50C / A171C; I50C / T277C; S52C / K173C; S52C / S275 C; L53C / S174C; P55C / V175C; P55C / Q176C; K56C / N155C; E58C / I183C; G64C / G196C; G64C / G200C; Q67C / L199C; Q67C / G20 0C; Y71C / L203C; L86C / V266C; Q89C / A131C; I93C / G116C; V94C / G116C; T95C / G116C; / G381C; S125C / P374C; L134C / I267C; A137C / I267C; L147C / A171C; I151C / A171C; A157C / Q176C; A157C / D177C; Q159C / A 171C; P185C / A195C; I213C / I226C; I213C / G230C; G219C / E333C; L228C / V264C; D327C / P344C; F346C / T369C; F346C / S37 The engineered protein may further comprise a cysteine ​​substitution of S186C / A195C. The paired cysteine ​​substitutions preferably form disulfide bonds.

[0013] The engineered protein may comprise or may further comprise at least one cavity filling substitution or a group of cavity filling substitutions selected from the group consisting of: H27Y; V28M; V30I; L49F; L49W; I50W; S52L; I57F; K90Y; A140Y; I144W; A157F; V158L; V170I; V170M; A171V; V175L; V179L; E182F; E182W; G200E; I201F; I201W; L228 F; L228W; R236Y; S246V; L256Y; V264F; V264W; V266F; V266W; S275F; S275M; T277F; T277L; T277W; L278F; L278W; V280F; V280W; R281Y; L282F; L282W; G345M; L356F; V384I; G433F; I443W; I443Y; A450F; I454F; and I456W. Substitutions may form salt bridges within pairs or between individual substitutions and naturally occurring amino acids in the protein.

[0014] The engineered protein may comprise or may further comprise at least one substitution or a group of substitutions selected from the group consisting of: T117P; A123P; T124P; S125P; A126P; V175P; G191P; N349P; L451P; and D452P.

[0015] The engineered protein may comprise or may further comprise at least one substitution or a group of substitutions selected from the group consisting of: S125W; I128F; I128W; E209W; and R236W.

[0016] The engineered protein may comprise or further comprise at least one substitution selected from the group consisting of: K173Q; A202T; A334S; T367R; D455K; K471A; K471L; and W473A.

[0017] The engineered protein may comprise or may further comprise an E at position 108.

[0018] The engineered protein may comprise a combination of at least one engineered disulfide bond and at least one cavity-filling substitution; or a combination of at least one engineered disulfide bond and at least one proline substitution; or a combination of at least one engineered disulfide bond, at least one cavity-filling substitution, and at least one proline substitution.

[0019] The engineered protein may comprise a set of substitutions selected from the group consisting of: G64C / G196C / V28M; G64C / G196C / V175L; G64C / G196C / V158L; G64C / G196C / A123P; G64C / G196C / S125P; G64C / G196C / I201W; G64C / G196C / L282F; G64C / G196C / L228W; G64C / G196C / R281Y; G64C / G196C / L282W; G64C / G196C / N349P; G64C / G196C / T367R; G64C / G196C / K47 1A; A137C / I267C / V28M; A137C / I267C / V175L; A137C / I267C / V158L; A137 C / I267C / A123P; A137C / I267C / S125P; A137C / I267C / I201W; A137C / I267C / L282F; A137C / I267C / L228W; A137C / I267C / R281Y; A137C / I267C / L282W ;A137C / I267C / N349P;A137C / I267C / T367R;A137C / I267C / K471A;L147C / A171C / V28M; L147C / A171C / V175L; L147C / A171C / V158L; L147C / A171C / A 123P; L147C / A171C / S125P; L147C / A171C / I201W; L147C / A171C / L282F; L 147C / A171C / L228W; L147C / A171C / R281Y; L147C / A171C / L282W; L147C / A 171C / N349P; L147C / A171C / T367R; L147C / A171C / K471A; G64C / G196C / A13 7C / I267C / K471A; G64C / G196C / A137C / I267C / V175L; G64C / G196C / A137C / I267C / S125P; G64C / G196C / A137C / I267C / S125P / V175L; G64C / G196C / A1 37C / I267C / T367R; G64C / G196C / A137C / I267C / K471A / S125P; G64C / G196 C / A137C / I267C / K471A / S125P / T367R / V175L; G64C / G196C / A137C / I267C;G64C / G196C / A137C / I267C / K471A / S125P / L282F / V175L;G64C / G196C / L147C / A171C / K471A / S125P / L282F / V175L;G64C / G196C / L147C / A171C / A137C / I267C / K471A / S125P / L282F / V175L;G64C / G196C / L147C / A171C;G64C / G196C / L147C / A171C / V28M / V175L / I201W / L228W / S125P / T367R / K471A;G64C / G196C / A137C / I267C / V28M / V175L / I201W / L228W / S125P / T367R / K471A;G64C / G196C / L147C / A171C / A137C / I267C / V28M / V175L / I201W / L228W / S125P / T367R / K471A;G64C / G196C / I151C / A171C / A137C / I267C;G64C / G196C / I151C / A171C / A137C / I267C / I231C / G230C;G64C / G196C / A137C / I267C / V28M / V175L / I201W / L228W / R281Y;G64C / G196C / A137C / I267C / V28M / V175L / I201W / L282F / L228W / R281Y;I151C / A171C / V449C / S457C;L168Q / G64C / G196C / A137C / I267C / K471A;L168Q / G64C / G196C / A137C / I267C / V175L;L168Q / G64C / G196C / A137C / I267C / S125P;L168Q / G64C / G196C / A137C / I267C / S125P / V175L;L168Q / G64C / G196C / A137C / I267C / T367R;L168Q / G64C / G196C / A137C / I267C / K471A / S125P;L168Q / G64C / G196C / A137C / I267C / K471A / S125P / T367R / V175L;L168Q / G64C / G196C / A137C / I267C;L168Q / G64C / G196C / A137C / I267C / K471A / S125P / L282F / V175L;L168Q / G64C / G196C / I151C / A171C / A137C / I267C;L168Q / G64C / G196C / I151C / A171C / A137C / I267C / I231C / G230C; L168Q / G64C / G19 6C / A137C / I267C / V28M / V175L / I201W / L228W / R281Y; L168Q / G64C / G196C / A137C / I267C / V28M / V175L / I201W / L282F / L228W / R281Y; L168Q / I151C / A171C / V449C / S4 57C; L168Q / I151C / A171C / V449C / S457C / V28M / V175L / R281Y; I151C / A171C / S186 C / A195C / V28M / V175L / R281Y; L168Q / I151C / A171C / L228W / L282F / R821Y / V175L; I151C / A171C / L228W / L282F / R821Y / V175L; I151C / A171C / L228W / L282F / R821Y / V 175L / G200E / I57F; I151C / A171C / L228W / L282F / R821Y / V175L / G200E / I57F / V449C / S457C; I151C / A171C / G200E / I57F; and I151C / A171C / G200E / I57F / V449C / S457C. ;

[0020] The engineered protein may comprise a substitution or group of substitutions selected from any one of the substitutions and groups of substitutions in Tables 1 and 2. Any substitution or group of substitutions may be further combined with the L168Q substitution.

[0021] The respiratory virus or mumps virus fusion protein (preferably, parainfluenza virus fusion protein (PIV F)) extracellular domain of the engineered protein can be a human PIV (hPIV) F extracellular domain. The human PIV F extracellular domain can be an hPIV3 F extracellular domain. The hPIV3 F extracellular domain can comprise a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to amino acids 19-481 of SEQ ID NO: 1 or 2.

[0022] The engineered protein may not contain the cytoplasmic tail of PIV F.

[0023] The engineered protein can be fused to or conjugated to a trimerization domain. The trimerization domain can include a T4 fibrin trimerization domain, a GCN4 domain, a 4J4A domain, or a combination thereof. The trimerization domain can comprise a sequence selected from the group consisting of:

[0024] SKIYHIENEIARI AKIEGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG, LKQIVLRIMEI EARIAKIEGSEFNSLKQIVLRIMEIEARIAKIE, LKQIVLRIMEIEARI AKIEGSLKQIVLRIMEIEARIAKIE and LKQIVLRIMEIEARIAKIEGSLELIKLRIMEIEARIAKIEKDRAIL.

[0025] The engineered protein can be fused to or conjugated to a transmembrane domain. The transmembrane domain can comprise a PIV F protein transmembrane domain. The PIV F protein transmembrane domain can comprise the sequence IIIILIMMIILFIINITIITI. The transmembrane domain can not comprise a PIV F protein transmembrane domain.

[0026] The engineered protein may comprise an N-terminal signal sequence.

[0027] The engineered proteins may exhibit improved solubility or stability compared to native PIV F in its post-fusion conformation. The engineered proteins may be immunogenic.

[0028] In one embodiment, provided herein is an engineered respiratory virus or mumps virus fusion protein (preferably, a parainfluenza virus fusion protein (PIV F)) trimer comprising three engineered proteins disclosed herein. Relative to a trimer of native PIV F protein subunits, the trimer can be stabilized in a pre-fusion conformation. The trimer can comprise at least one engineered disulfide bond between subunits. The trimer can comprise at least one engineered disulfide bond between subunits selected from the group consisting of: I118C / G381C; A119C / G381C; L120C / G381C; S125C / P374C; G219C / E333C; F346C / T369C; F346C / S370C; and V449C / S457C.

[0029] In one embodiment, provided herein are nucleic acid molecules comprising a nucleotide sequence encoding the amino acid sequence of an engineered protein disclosed herein. The nucleic acid molecule may also include a DNA expression vector. The nucleic acid molecule may be mRNA. The nucleic acid molecule may be a self-replicating RNA molecule. The nucleic acid molecule may comprise at least one chemical modification. The at least one chemical modification can be selected from the group consisting of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thiol-1-methyl-1-deaza-pseudouridine, 2-thiol-1-methyl-pseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxy-pseudouridine, 4-thiol-1-methyl-pseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-0-methyluridine.

[0030] In one embodiment, provided herein is a pharmaceutical composition comprising (i) an engineered protein disclosed herein, (ii) an engineered trimer disclosed herein, or (iii) a nucleic acid molecule disclosed herein; and a pharmaceutically acceptable carrier. The pharmaceutical composition may further comprise an adjuvant. The pharmaceutical composition may further comprise another PIV antigen. The pharmaceutical composition may be formulated in cationic lipid nanoparticles. The pharmaceutical composition can be used to treat or prevent parainfluenza virus (PIV) infection or a disease associated with PIV infection in a subject, or to induce an immune response against parainfluenza virus (PIV). The subject may be a mammal, such as a human.

[0031] In one embodiment, provided herein is a method for preventing parainfluenza virus (PIV) infection in a subject or a disease associated with PIV infection or eliciting an immune response in a subject or reducing PIV viral shedding in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition provided herein. The subject can be a mammal, such as a human.

[0032] In one embodiment, provided herein is the use of (i) an engineered protein disclosed herein, (ii) an engineered trimer disclosed herein, or (iii) a nucleic acid molecule disclosed herein; or (iv) a pharmaceutical composition provided herein in the manufacture of a medicament for treating or preventing parainfluenza virus (PIV) infection or a disease associated with PIV infection.

[0033] In one embodiment, provided herein are compositions comprising (i) an engineered protein provided herein or (ii) an engineered trimer provided herein bound to an antibody. The antibody can specifically bind to the PIVF extracellular domain in the prefusion conformation.

[0034] Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration only, as various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The following drawings constitute part of this specification and are included to further demonstrate certain aspects of the present invention. The present invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0036] Figure 1 Sequence identity and similarity matrices for representative respiroviruses and orthomopsviruses. Multiple sequence alignments (Clustal Omega) were analyzed to determine pairwise sequence identity and similarity for a representative set of F proteins from the respirovirus and orthomopsvirus genera. Points in each matrix are colored according to their percentage value from 0% to 100% [scale on the right]. Similarities and identities were scored using the SIAS server hosted by the Immunomedicine Unit of the Complutense University of Madrid, SECRETARIA GENERAL DE CIENCIA, TECNOLOGIA EINNOVACION OF SPAIN [http: / / imed.med.ucm.es / Tools / sias.html].

[0037] Figure 2Clustered sequence alignment of human respiratory virus type 1 and type 3 (also known as HPIV1 and HPIV3) fusion proteins. The sequence identity of the soluble extracellular domain is 44%, and the sequence similarity is >60%. Identical residues at homologous positions are colored with white letters on a black background; similar amino acids are colored with black letters on a gray background. Non-similar residues at homologous sites are colored black against a white background.

[0038] Figure 3 Cryo-EM structure of the PIV3 F base (L168Q) with a C-terminal GCN4 tag in the post-fusion conformation. (Top left) Representative cryo-electron micrographs. (Top right) Representative 2D class averages. (Bottom left) Gold standard Fourier shell correlation plot showing the relationship between GSFSC and angstrom resolution. Resolution values ​​correspond to an FSC cutoff of 0.143. (Bottom right) Coulomb potential map, or Coulomb potential map, of the PIV3 F base trimer in the post-fusion conformation.

[0039] Figure 4 Single substitution SDS-PAGE. PIV3 F protein, purified by affinity chromatography, was analyzed by reducing SDS-PAGE (Coomassie blue staining). The variant ID is indicated at the top of each lane. Integrated band intensities were quantified using ImageJ / Fiji or LicorOdyssey CLx. Molecular weight markers are included on the left side of each gel. For the bottom panel, the affinity chromatography flow-through [FT] and elution [E] are shown.

[0040] Figure 5 Biolayer interferometry quantification of HPIV3 F variant binding to the prefusion-specific antibody PIA174 IgG. Each graph shows the response (nm) versus time (seconds) for PIV3 F variant binding to the AHC tip. The AHC tip was functionalized with PIA174 IgG, which recognizes prefusion PIV3 F.

[0041] Figure 6 Negative-stained electron micrographs of the disulfide variants JM-17 (I50C / A171C) and JM-20 (A137C / I1267C) in their prefusion conformations. Representative 2D class averages of negative-stained EM images of JM-17 [left] and JM-20 [right] are provided at the top of each figure. The box size is indicated below the 2D class and is equal to 230 angstroms. 3D reconstructions of JM-17 [left] and JM-20 [right] in their prefusion conformations are provided at the bottom of each figure.

[0042] Figure 7Figure 4. Biolayer interferometry quantification of relative expression yields of HPIV3 F variants. Each graph shows the response (nm) versus time (seconds) for binding of a HPIV3 F variant to an AHC tip. The AHC tip was functionalized with MF5 IgG, which recognizes the foldon tag. Binding curves were fit to a straight line over the initial portion of the curve corresponding to a 60-second window after immersion in medium containing each variant from a small-scale HEK293F expression culture.

[0043] Figure 8 SDS-PAGE quantification of combinatorial variants containing tandem GCN4 / Foldon tags at the C-terminus. Individual and combinatorial variant IDs are indicated above each lane. Integrated band intensities are plotted below each gel and quantified in Licor OdysseyCLx.

[0044] Figure 9 .Size exclusion chromatograms of PIV3 F variants Each graph shows the relationship between mAU and elution volume for a group of PIV3 F variants. Each sample shown in this graph was transfected and purified in parallel under identical conditions. Briefly, the PIV3F variants were purified by Streptactin Sepharose, then concentrated and snap-frozen in liquid nitrogen. The samples were thawed and analyzed individually by running them through the same Superose 6 size exclusion chromatography (SEC) column. For ease of comparison, the UV trajectory of the base construct sample L168Q is reproduced on each graph.

[0045] Figure 10 Additional SDS-PAGE quantification of combinatorial variants containing tandem GCN4 / Foldon tags at the C-terminus. Integrated band intensities from a panel of gels of individual and combinatorial variants are plotted and quantified in the Licor Odyssey CLx.

[0046] Figure 11 SDS PAGE after size exclusion chromatography of PIV3F combinatorial variants 41 and 43. Large-scale expression (500 mL) of the two combinatorial variants 41 and 43 were first purified by streptavidin affinity chromatography and then by size exclusion chromatography (Superose 6).

[0047] Figure 12 Size exclusion chromatograms of PIV3F combinatorial variants 43, 56, 57, and 58. Superose 6 size exclusion elution curves for a panel of combinatorial variants, where absorbance units at 280 nm [mAU] are plotted against elution volume in milliliters [mL]. In the inset table, the expressed protein yield [mg / mL] and apparent melting temperature (Tm) are indicated next to each variant ID. app , ℃). Tmapp The mutations of the combined variants are listed in Table 2.

[0048] Figures 13A-13B Size exclusion chromatography and cryo-EM of the PIV F variant L168Q+I151C / A171C. (Figure 13A) SEC of three variants: L168Q, L168Q+I151C / A171C, and L186Q+I213C / G230C. The traces show characteristic trimer and trimer-dimer peaks. (Figure 13B) Cryo-EM of the L168Q+I151C / A171C variant. The figure shows 2D class averages, 3D reconstruction and a zoomed-in view of the model in the Coulomb potential map.

[0049] Figure 14 Cryo-EM structure of the PIV3 F base (L168Q) with a tandem GCN4 / Foldon tag at the C-terminus in the prefusion conformation, without any prefusion-specific antibodies. (Top left) Representative cryo-electron micrographs. (Top right) Representative 2D class averages. (Bottom left) Gold standard Fourier shell correlation plot showing GSFSC versus angstrom resolution. Resolution values ​​correspond to an FSC cutoff of 0.143. (Bottom right) Coulomb potential map, or Coulomb potential map, of the PIV3 F base with a C-terminal tandem GCN4 / Foldon tag in the trimer prefusion conformation. The PIV3 F protomer is colored in blue, red, and green. The extended portion of the structure stabilized by the C-terminal tag is colored in gray.

[0050] Figure 15 Cryo-EM structure of PIV3 F combination variant 43 (Combo 43) in the prefusion conformation, in complex with the prefusion-specific antibody PIA174. (Top left) Representative cryo-electron micrograph. (Top right) Representative 2D class averages. (Middle left) Gold standard Fourier shell correlation plot showing the relationship between GSFSC and angstrom resolution. The resolution value corresponds to an FSC cutoff of 0.143. (Middle right) Coulomb potential map, or Coulomb potential map, of PIV3 F variant Combo 43 (I151C / A171C) with C-terminal tandem GCN4 / foldon tags in the trimeric prefusion conformation. The PIV3 F Combo 43 protomer is colored in blue, red, and green. The extended portion of the structure stabilized by the C-terminal tag is colored in gray. (Bottom) Zoomed-in view of the atomic model of all exemplary substitutions within the Coulomb potential map, shown in gray. Atoms are colored according to the substitution type: disulfide bond, proline, and cavity filling.

[0051] Figure 16Cryo-EM of PIV variant I93C / G116C. The figure shows representative micrographs, 2D class averages, and For reconstruction, wild-type PIV F was used as background (Leu at position 168).

[0052] Figure 17 .Biochemical and structural characterization of the PIV3 F variant PB-68, V449C / S457C, which forms inter-protomer disulfide bonds. (Upper left) Non-reducing SDS-PAGE analysis of the PIV3 variant PB-68, V449C / S457C, which operates as a disulfide-linked trimer. A control PIV3 variant is included that does not form inter-protomer disulfide bonds and operates as a single protomer. (Upper right) Representative 2D class averages of the PIV3 variant PB-68 under negative stain electron microscopy. (Lower right) 3D negative stain EM reconstruction of PB-68. (Lower left) Magnified view of the simulated substitution site near the heptad repeat.

[0053] Figures 18A-18B .Sample stability testing of PIV F variant PB-68, namely V449C / S457C. (Figure 18A) Purified V449C / S457C protein was incubated at 4°C for 0, 7 and 30 days and then analyzed by non-reducing SDS-PAGE. (Figure 18B) Separately prepared V449C / S457C protein was incubated at 37°C for 1, 7 and 14 days and then analyzed by reducing and non-reducing SDS-PAGE. The band indicated by the arrow indicates the presence of non-reducing trimers. A small portion of the sample ran at the size expected for the protomer under non-reducing conditions (low molecular weight band, near the 71kDa MW marker), which may also form disulfide bonds within the protomer.

[0054] Figures 19A-19B Four cryo-EM structures of PIV F combo variants 41 and 58, each with two oligomeric states. ( FIG19A ) Representative class averages and Coulomb potential plots for trimers and dimers of trimers of combo variant 41 and ( FIG19B ) combo variant 58. The resolution of each structure is indicated next to the figure.

[0055] Figures 20A-20B Variants adopt a mixture of closed and open conformations at the PIA174 binding site, which can be biased toward closed conformation by substitutions at the central trimer interface. (Figure 20A) Coulomb potential plots of Combo41 for four unique conformations, ranging from open to closed, demonstrating conformational heterogeneity in the central apical region (black arrows). (Figure 20B) Coulomb potential plots of Combo58 in the closed conformation. Despite extensive efforts to classify the central apical region based on conformational heterogeneity (arrows), no classes were observed in the open conformation. Atomic model and zoomed-in view of Combo58 with substitution position 201.

[0056] Figure 21 SDS-PAGE of PIV3 F wild-type, single, and combinatorial variants. Wild-type PIV3 F and several variants (L168Q, I151C / A171C, Combo61, Combo61-0-1, Combo62-0, and Combo62-0-1) were analyzed by reducing and non-reducing SDS-PAGE. Variants containing the V449C / S457C substitutions (Combo61-0-2 and Combo62-0-1) all showed the expected inter-protomer disulfide trimer band.

[0057] Figure 22 Size exclusion chromatograms of PIV3 F wild-type, single, and combined variants. Each graph shows the relationship between mAU and elution volume for a panel of PIV3 F variants. Each sample shown in this graph was transfected and purified in parallel under identical conditions. Briefly, PIV3F variants were purified on Streptactin Sepharose, then concentrated and flash-frozen in liquid nitrogen. The samples were thawed and analyzed individually on the same Superose 6 column. For ease of comparison, the UV trace of a wild-type PIV3 F sample is simulated in each graph.

[0058] Figure 23 Thermal stability analysis of PIV3 F wild-type, single, and combined variants. Purified PIV F proteins, purified in parallel under identical conditions, were analyzed by differential scanning fluorimetry. The y-axis represents the change in fluorescence as a function of temperature, and the x-axis represents temperature. Variants are plotted in groups, with the wild-type DSF trajectory mimicked in each plot for ease of comparison. DETAILED DESCRIPTION

[0059] Provided herein are engineered parainfluenza virus (PIV) fusion (F) proteins having one or more amino acid substitutions that stabilize the PIV F protein in a prefusion conformation. Prefusion PIV F can be used as a vaccine antigen or reagent for detecting and / or isolating antibodies in serum. Prefusion PIV F proteins described herein and nucleic acids encoding the proteins can be used, for example, as potential immunogens in immunogenic compositions or vaccines against PIV, methods for inducing an immune response in a subject, and as diagnostic tools, among other uses.

[0060] I. Native hPIV F

[0061] The envelope glycoprotein of hPIV1, hPIV2, hPIV3 or hPIV4 promotes the fusion of the viral and cellular membranes. In nature, the F protein of hPIV1, hPIV2, hPIV3 and hPIV4 is initially synthesized as a single polypeptide precursor of approximately 550 amino acids in length, called F0. F0 includes an N-terminal signal peptide that directs its localization to the endoplasmic reticulum where it is proteolytically cleaved. The remaining F0 residues oligomerize to form a trimer and can be proteolytically processed by cellular proteases to produce two disulfide-linked fragments, F1 and F2. In hPIV1 F, the cleavage site is located between approximately residues 112 / 113, in hPIV2 F, the cleavage site is located between approximately residues 106 / 107, in hPIV3 F, the cleavage site is located between approximately residues 109 / 110, and in hPIV4 F, the cleavage site is located between approximately residues 103 / 104. The smaller of these fragments, F2, is derived from the N-terminal portion of the F0 precursor (hPIV1, approximately residues 22-113; hPIV2, approximately residues 22-106; hPIV3, approximately residues 19-109; hPIV4, approximately residues 21-103). The larger of these fragments, F1, comprises the C-terminal portion of the F0 precursor (hPIV1, approximately residues 114-555; hPIV2, approximately residues 107-551; hPIV3, approximately residues 110-539; hPIV4, approximately residues 104-544), including the extracellular / luminal region (hPIV1, approximately residues 114-497; hPIV2, approximately residues 107-493; hPIV3, approximately residues 110-493; hPIV4, approximately residues 104-486), the transmembrane domain (hPIV1, approximately residues 498-518; hPIV2, approximately residues 494-514; hPIV3, approximately residues 494-514; hPIV4, approximately residues 487-507), and a C-terminal cytoplasmic tail. The extracellular portion of the hPIV F protein is the hPIV F extracellular domain, which includes the F2 protein and the F1 extracellular domain.

[0062] The hPIV F protein shows remarkable sequence conservation among hPIV subtypes and other members of the Respirovirus and Orthomopesvirus genera ( Figure 1). Given this conservation, one of ordinary skill in the art can readily compare the amino acid positions of different hPIV F proteins of the same subtype, or with the amino acid positions of the F proteins of other members of the Respirovirus and Orthomopsovirus genera. Unless the context indicates otherwise, the numbering of the amino acid substitutions disclosed herein is with reference to SEQ ID NO: 1 (GenBank AGW51052.1) or 2 (SWISS-PROT: P06828.2) of hPIV3 (also known as human respiratory virus 3) F, unless otherwise indicated.

[0063] Therefore, the term PIV F polypeptide as used herein should be understood to refer to native PIV F polypeptides from any PIV strain (not limited to human PIV3 strains), as well as any F protein from other members of the Respirovirus and Orthomopsvirus genera. Depending on the actual sequence alignment, the actual residue position numbering of the F protein from other strains may need to be adjusted. Additional viral F proteins to which the disclosed amino acid substitutions can be applied include, for example, hPIV1F (also known as human respiratory virus 1) (GenBank BAS30410.1; SEQ ID NO:3), hPIV2 (also known as human mumps virus 2) F (GenBank AAA46842.1; SEQ ID NO:4), hPIV4 (also known as human mumps virus 4) F (GenBank AGU90035.1; SEQ ID NO:5); mumps virus F (GenBank BAA94388.1; SEQ ID NO:6); and PIV5 (also known as mumps virus 5) F (GenBank AAC95515.1; SEQ ID NO:7).

[0064] II. Stabilization of PIV F Protein in a Prefusion Conformation

[0065] The three PIV F protomers oligomerize in the mature F protein, which adopts a metastable prefusion conformation that triggers a conformational change to a postfusion conformation upon contact with the target cell membrane. This conformational change exposes a hydrophobic sequence, called the fusion peptide, located at the N-terminus of the F1 extracellular domain and associates with the host cell membrane, promoting fusion of the viral or infected cell membrane with the target cell membrane.

[0066] Provided herein are engineered PIV3 F ectodomain trimers comprising a protomer containing one or more amino acid substitutions that stabilize the F ectodomain trimer in a prefusion conformation.

[0067] As used herein, "prefusion conformation" refers to a structural conformation adopted by a polypeptide that differs from the postfusion conformation of PIV F in at least molecular size or three-dimensional coordinates. The prefusion conformation refers to the structural conformation adopted by PIV F before the fusion event is triggered, resulting in the transition of F to the postfusion conformation. Isolating PIV F in a stable prefusion conformation can help provide information and guidance for the development of improved vaccines and immunogenic compositions to address the important public health issue of PIV infection. The prefusion conformation can be a conformation that can bind to a prefusion-specific antibody.

[0068] A PIV F ectodomain trimer that is "stabilized in a prefusion conformation" comprises one or more amino acid substitutions, deletions, or insertions compared to the corresponding native PIV F sequence, which increases the retention of the prefusion conformation compared to the PIV F ectodomain trimer formed by the corresponding native hPIV F sequence. "Stabilization" of the prefusion conformation can be, for example, energy stabilization (e.g., reducing the energy of the prefusion conformation relative to the postfusion open conformation) and / or kinetic stabilization (e.g., reducing the rate of transition from the prefusion conformation to the postfusion conformation). In addition, the stability of the PIV F ectodomain trimer in the prefusion conformation can include an increase in resistance to denaturation compared to the corresponding native PIV F sequence. Methods for determining whether an hPIVF ectodomain trimer is in a prefusion conformation are provided herein and include, but are not limited to, negative stain electron microscopy and antibody binding assays using prefusion conformation-specific antibodies (such as, in the case of hPIV3, PIA3 or PIA174 antibodies).

[0069] The present disclosure provides engineered proteins comprising amino acid substitutions relative to the amino acid sequence of the corresponding native hPIV3 F protein (e.g., SEQ ID NO: 1 or 2). Amino acid mutations include amino acid substitutions, deletions, or additions relative to the native hPIV3 F protein. Thus, the engineered protein is a mutant of the native hPIV3 F protein.

[0070] The engineered PIV F extracellular domain trimer may be derived from a human PIV strain other than hPIV3, such as hPIV1, hPIV2, or hPIV4. Based on the high sequence identity between the hPIV3F sequence and other hPIV F sequences, residues from other hPIV F sequences corresponding to the hPIV3 F sequence can be readily obtained. Any amino acid substitution (or combination of substitutions) described herein for stabilizing hPIV3 F in its prefusion conformation can be introduced into another hPIV F sequence to achieve prefusion stabilization.

[0071] The engineered PIV F extracellular domain trimer can be derived from a non-human PIV strain, such as a bovine or ovine PIV strain. Based on the high sequence identity between the human PIV F sequence and the non-human PIV F gene sequence, residues of the non-human PIV F sequence corresponding to the hPIV F sequence can be easily obtained. Any amino acid substitution (or combination of substitutions) described herein for stabilizing hPIV3 F in its pre-fusion conformation can be introduced into a non-human PIV3 F sequence (e.g., GENBANK: AHZ90086.1 or AIW42876.1) to achieve pre-fusion stabilization.

[0072] The engineered hPIV3 F extracellular domain trimer comprises protomers that are "single-chain" proteins, in which the F2 polypeptide and F1 extracellular domain of each protomer are directly linked or connected by a peptide linker to form a continuous polypeptide chain. Some examples of natural hPIV3 F proteins (such as GENBANK: AGW51052.1) do not contain a consensus furin cleavage site between the F1 and F2 proteins; hPIV3 F immunogens based on such natural hPIV3 F proteins generally do not need to be modified to produce single-chain F proteins. However, other natural hPIV3 F proteins (such as SWISS-PROT: P06828.2) do contain a consensus furin cleavage site between the F1 and F2 proteins; hPIV3F immunogens based on such natural hPIV3F proteins can be modified to produce single-chain F proteins. Exemplary modifications include amino acid substitutions to remove the consensus furin cleavage site, such as K108E substitutions.

[0073] The protomer of the engineered PIV F extracellular domain trimer includes PIV F positions 19-481 and may include any of the following: an amino acid substitution (such as K108E) to remove the consensus furin cleavage site between F2 and F1 (if the consensus site is present in the native sequence), K or R at position 87, T or S at position 95, K or R at position 141, V or I at position 165, L or Q at position 168, K or R at position 295, T or V at position 267, T or K at position 369, and D or N at position 441.

[0074] The engineered PIV F ectodomain trimer can be a soluble protein complex, for example, used as a recombinant subunit vaccine. In several such embodiments, the protomer of the engineered PIV F ectodomain trimer can each comprise a C-terminal connection with a trimerization domain such as a GCN4 trimerization domain. The trimerization domain promotes trimerization and stability of the membrane proximal end of the engineered PIV F ectodomain trimer. For example, the C-terminal residue (such as the residue in the stem region of the trimer) of the protomer of the engineered PIV F ectodomain trimer can be directly connected to the trimerization domain, or can be indirectly connected to the trimerization domain by a peptide linker. Exemplary linkers include glycine and glycine-serine linkers. Non-limiting examples of exogenous multimerization domains that promote stable trimers of soluble recombinant proteins include: a GCN4 leucine zipper, a trimerization motif from pulmonary surfactant protein (Hoppe et al. 1994 FEBS Lett 344: 191-195), collagen (McAlinden et al. 2003 J Biol Chem 278: 42200-42207), any of which can be linked to the C-terminus of the engineered PIV F ectodomain protomer to promote trimerization, as long as the recombinant PIV F ectodomain trimer retains the pre-fusion conformation. In some examples, the protomer of the engineered PIV F ectodomain trimer can be linked to a GCN4 trimerization domain, for example, each protomer in the trimer can include a C-terminal connection to the GCN4 trimerization domain, such as a connection to any one of positions 475-485 of hPIV3 F (such as position 481 of hPIV3 F).

[0075] The engineered PIV F ectodomain trimer can be a membrane-anchored protein complex, for example, for attenuated viruses or virus-like particle vaccines. Membrane anchoring can be achieved, for example, by connecting the protomer of the engineered PIV F ectodomain trimer to the C-terminus of a transmembrane domain and an optional cytoplasmic tail (such as a PIV F transmembrane domain and a cytoplasmic tail). One or more peptide linkers (such as a glycine-serine linker, for example a 10 amino acid glycine-serine peptide linker) can be used to connect the protomer of the engineered PIV F ectodomain trimer to the transmembrane domain. Non-limiting examples of transmembrane domains for disclosed embodiments include hPIV3 F transmembrane domains.

[0076] The engineered proteins may have certain beneficial properties, such as immunogenicity. The engineered proteins may have enhanced immunogenicity or improved pre-fusion conformational stability compared to the corresponding native hPIV3 F protein. Stability refers to the extent to which the transition of hPIV3 F from the pre-fusion conformation to the post-fusion conformation is hindered or prevented. The engineered protein may exhibit one or more introduced mutations as described herein, which may also result in increased stability of the pre-fusion conformation. Amino acid mutations introduced into the hPIV3F protein include amino acid substitutions, deletions and / or additions. The mutations in the amino acid sequence of the engineered protein may be amino acid substitutions, insertions and / or deletions relative to the native hPIV3-F extracellular domain.

[0077] Several ways to stabilize the conformation of the engineered protein compared to the native hPIV3 F protein include, but are not limited to, amino acid substitutions that introduce disulfide bonds (intra- and inter-protomers), modify salt bridges, introduce electrostatic interactions, introduce hydrogen bonds, introduce proline, fill cavities, change the packing of residues, and combinations thereof.

[0078] The engineered protein can be isolated, i.e., separated from the hPIV3 F protein in the post-fusion conformation. Thus, the engineered protein can be, for example, at least 80% separated, at least 90% separated, at least 95% separated, at least 98% separated, at least 99% separated, or at least 99.9% separated from the hPIV3 F polypeptide in the post-fusion conformation. The engineered protein can specifically bind to an hPIV3 F pre-fusion-specific antibody.

[0079] It will be understood that a homogeneous population of engineered proteins of a particular conformation may include changes that do not alter the conformational state of the engineered protein (such as changes in polypeptide modifications, e.g., glycosylation state). The population of engineered proteins may remain homogeneous over time. For example, when the engineered protein is dissolved in an aqueous solution, a population of proteins may be formed that is stable in a pre-fusion conformation for at least 12 hours, at least 24 hours, at least 48 hours, at least one week, at least two weeks, or longer. One of ordinary skill in the art will appreciate that the engineered proteins provided herein can be used to elicit an immune response to hPIV3 in a mammal.

[0080] Compared to native PIV F protein, engineered proteins can include introduced cysteine ​​substitutions. Engineered proteins can include any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cysteine ​​substitutions. Without being bound by theory or mechanism, the cysteine ​​substitutions described herein are believed to promote the stability of the polypeptide in a non-PIV F post-fusion conformation. The introduced cysteine ​​substitutions can be introduced by protein engineering, for example, by including one or more substituted cysteine ​​residues that form disulfide bonds. The amino acid position of the cysteine ​​may be close enough to form a disulfide bond in the PIV F protein conformation before fusion rather than after fusion.

[0081] The engineered protein may include a recombinant PIV F protein that is stabilized in a prefusion conformation by a disulfide bond between cysteines introduced into a pair of amino acid positions that are close to each other in the prefusion conformation but further apart in the postfusion conformation. The cysteine ​​pair may be present simultaneously in a single protomer, thereby forming an intraprotomer disulfide bond, or the cysteine ​​pair may be present in different protomers, thereby forming an interprotomer disulfide bond.

[0082] Compared to the native PIV F protein, exemplary cysteine ​​substitutions include any of the disulfide bond substitutions in Table 1, whose numbering is based on the numbering of SEQ ID NO: 1. The engineered protein may include a combination of two or more disulfide bonds between pairs of cysteine ​​residues listed in Table 1.

[0083] The engineered protein may include a combination of two or more different types of mutations selected from engineered disulfide bond mutations, cavity filling mutations, and proline mutations. The engineered protein may include at least one disulfide bond mutation and at least one proline mutation. The engineered protein may include at least one cysteine ​​substitution and at least one cavity filling substitution. The engineered protein may include at least one cysteine ​​substitution and at least one charge reducing substitution. The engineered protein may include at least one mutation selected from any one or group of mutations in Tables 1 or 2.

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110] III. Protein Preparation

[0111] Proteins as described herein can be prepared by conventional methods known in the art, such as by expressing in a recombinant host system using a suitable vector. Suitable recombinant host cells include, for example, insect cells, mammalian cells, avian cells, bacteria and yeast cells. The example of suitable insect cells includes, for example, Sf9 cells, Sf21 cells, Tn5 cells, Schneider S2 cells and HIGH FIVE cells (clonal isolates derived from parent Trichoplusia ni BTI-TN-5B1-4 cell lines). The example of suitable mammalian cells includes Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK293 or Expi 293 cells, usually transformed by sheared adenovirus type 5 DNA), NIH-3T3 cells, 293-T cells, Vero cells and HeLa cells. Suitable avian cells include, for example, chicken embryonic stem cells (e.g., Cells), chicken embryonic fibroblasts, chicken embryonic germ cells, quail fibroblasts (e.g., ELL-O), and duck cells. Suitable insect cell expression systems, such as baculovirus vector systems, are known to those skilled in the art. Materials and methods for baculovirus / insect cell expression systems are commercially available in kit form. Avian cell expression systems are also known to those skilled in the art. Similarly, bacterial and mammalian cell expression systems are also known to those skilled in the art.

[0112] A variety of suitable vectors for expressing recombinant proteins in insect or mammalian cells are known and routinely used in the art. Suitable vectors may contain a variety of elements, including but not limited to one or more of the following: an origin of replication; a selection marker gene; one or more expression control elements, such as transcription control elements (e.g., promoters, enhancers, terminators) and / or one or more translation signals; and a signal sequence or leader sequence for targeting to the secretory pathway in a selected host cell (e.g., mammalian origin or from a heterologous mammal or non-mammalian species). For example, in order to express in insect cells, a suitable baculovirus expression vector such as PFASTBAC is used to produce recombinant baculovirus particles. Baculovirus particles are amplified and used to infect insect cells to express recombinant proteins. For expression in mammalian cells, a vector that drives the expression of the construct in desired mammalian host cells (e.g., Chinese hamster ovary cells) is used.

[0113] Any suitable method can be used to purify the protein. For example, methods for purifying proteins by immunoaffinity chromatography are known in the art. Suitable methods for purifying the desired protein include precipitation and various types of chromatography, such as hydrophobic interaction chromatography, ion exchange chromatography, affinity chromatography, chelate chromatography, and size exclusion chromatography, which are also known in the art. Two or more of these or other suitable methods can be used to create a suitable purification scheme. If desired, the protein can include a "tag" that aids in purification, such as an epitope tag or a histidine tag. Such tagged proteins can be purified from conditioned medium by, for example, chelate chromatography or affinity chromatography.

[0114] IV. Protein-Encoding Nucleic Acids

[0115] Also provided are nucleic acid molecules encoding the proteins described herein. These nucleic acid molecules include DNA, cDNA, and RNA sequences. Nucleic acid molecules encoding only the extracellular domain of a protein are also contemplated. The nucleic acid molecules can be incorporated into a vector, such as an expression vector.

[0116] The nucleic acid can be a self-replicating RNA molecule. The nucleic acid can include a modified RNA molecule. Compositions comprising the nucleic acids described herein are also provided.

[0117] V. Preparations and Methods of Use

[0118] Provided herein are methods for inducing an immune response against PIV in a mammal, the methods comprising administering to the mammal an immune composition in an amount effective to induce an immune response, wherein the composition comprises an engineered PIV F prefusion protein or a polynucleotide encoding an engineered PIV F prefusion protein. The induced immune response may be a protective immune response, i.e., the response reduces the risk or severity or clinical consequences of PIV infection. The immune response may comprise a humoral immune response, a cell-mediated immune response, or both. The immune response may comprise a T cell response or a B cell response. The cell-mediated immune response may comprise a helper T cell (Th) response, a CD8+ cytotoxic T cell (CTL) response, or both. The humoral immune response may comprise antibody presenting B cells, and the antibody may be a neutralizing antibody against PIV. Neutralizing antibodies block viral infection of cells. The immune response may reduce or prevent cellular infection. The neutralizing antibody response may be complement dependent or complement independent. The neutralizing antibody response may be complement independent. The neutralizing antibody response can be cross-neutralizing; that is, antibodies raised against the administered composition neutralize a related PIV virus of a different strain than the strain used in the composition.

[0119] The method may involve a single administration of the composition. The method may further comprise administering a booster dose of the composition to the subject.

[0120] The desired response is to inhibit or reduce or prevent PIV infection. The desired response is to reduce PIV virus shedding. The method may reduce PIV virus shedding in saliva. PIV virus shedding in a mammal is reduced compared to virus shedding in a mammal not administered the engineered PIVF protein. The term "viral shedding" is used herein in accordance with its ordinary meaning in medicine and virology and refers to the production and release of virus from infected cells. Viruses may be released from mammalian cells. Viruses may be released from the body of an infected mammal into the environment. Viruses may be released from cells in the body of a mammal. The ideal response is to reduce PIV virus titer. The method may reduce PIV nucleic acid in serum.

[0121] In order for the method to be effective, it is not necessary to completely eliminate or reduce or prevent PIV infection, viral shedding or viral titer. For example, administration of an effective amount of an agent can reduce PIV infection (e.g., as measured by cell infection or by the number or percentage of subjects infected with PIV), viral shedding or viral titer by a desired amount, such as by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or even at least 100% (eliminating or preventing detectable PIV infection, viral shedding or viral titer) compared to a suitable control.

[0122] The engineered proteins described herein can be directly delivered as components of immunogenic compositions or vaccines. Alternatively, nucleic acids encoding proteins described herein can be administered to produce proteins or immunogenic fragments in vivo. Protein preparations, recombinant nucleic acids (e.g., DNA, RNA, mRNA, self-replicating RNA or any variants thereof) and / or viral vectors (e.g., live, single-round, non-replicating assembled virions or other virus-like particles or alphavirus VRPs) containing sequences encoding engineered proteins provided herein may be included in immunogenic compositions or vaccines. Such compositions can produce proteins described herein after translating a codon-optimized open reading frame. The composition may include at least one RNA polynucleotide encoding at least one PIV F antigen polypeptide or its immunogenic fragment and at least one 5' end cap. The 5' end cap can be 7mG(5')ppp(5')NImpNp.

[0123] In the case where a nucleic acid molecule encoding an engineered PIV F protein is used in a pharmaceutical composition, the nucleic acid molecule may comprise or consist of deoxyribonucleotides and / or ribonucleotides or analogs thereof covalently linked together. Nucleic acid molecules as described herein typically contain phosphodiester bonds, but in some cases may include nucleic acid analogs having at least one different linkage, for example, phosphoramidate, phosphorothioate, phosphorodithioate or O-methylphosphoramidite linkages and peptide nucleic acid backbones and linkages. A mixture of naturally occurring polynucleotides and analogs can be prepared; alternatively, a mixture of different polynucleotide analogs, as well as a mixture of naturally occurring polynucleotides and analogs can be prepared. The nucleic acid molecule may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, the nucleotide structure may be modified before or after polymer assembly. The nucleotide sequence may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. The term also includes both double-stranded molecules and single-stranded molecules. Unless otherwise specified or required, the term polynucleotide encompasses each of a double-stranded form and two complementary single-stranded forms known or predicted to constitute the double-stranded form. Nucleic acid molecules are composed of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), and when the polynucleotide is RNA, thymine is uracil (U). Therefore, the term "nucleic acid sequence" is an alphabetical representation of a nucleic acid molecule. Unless otherwise specified or required, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced by mixed bases and / or deoxyinosine residues.

[0124] At least one polynucleotide may have at least one chemical modification. At least one polynucleotide may also include a second chemical modification. The polynucleotide may be RNA. At least one polynucleotide having at least one chemical modification may have a 5′. The at least one chemical modification may be selected from pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thiol-1-1-methyl-1-deaza-pseudouridine, 2-thiol-1-methyl-pseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxy-pseudouridine, 4-thiol-1-methyl-pseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-0-methyluridine. At least 80% (e.g., 85%, 90%, 95%, 98%, 99%, 100%) of the uracils in the open reading frame may have a chemical modification, optionally wherein the composition is formulated in a lipid nanoparticle. All uracils in the open reading frame may have a chemical modification. The chemical modification may occur at the 5 position of the uracil. The chemical modification may be N1-methyl pseudouridine.

[0125] Nucleic acid of the present disclosure may comprise one or more modified nucleosides, the modified nucleosides comprising modified sugar moieties. Relative to oligonucleotides comprising only nucleosides containing naturally occurring sugar moieties, such compounds comprising one or more sugar-modified nucleosides may have desirable characteristics, such as enhanced nuclease stability or increased binding affinity to the target nucleic acid. In some embodiments, the modified sugar moieties are substituted sugar moieties. In some embodiments, the modified sugar moieties are sugar surrogates. Such sugar surrogates may comprise one or more substitutions corresponding to those substituted sugar moieties.

[0126] In some embodiments, the modified sugar moiety is a substituted sugar moiety comprising one or more non-bridging sugar substituents, including but not limited to substituents at the 2' position and / or the 5' position. Examples of sugar substituents suitable for the 2'-position include, but are not limited to, 2'-F, 2'-OCH3 ("OMe" or "O-methyl"), and 2'-O(CH2)2OCH3 ("MOE"). In certain embodiments, the sugar substituent at the 2' position is selected from allyl, amino, azido, thio, O-allyl, O--C1-C10 alkyl, O--C1-C10 substituted alkyl; OCF3, O(CH2)2SCH3, O(CH2)2--O--N(Rm)(Rn), and O--CH2--C(=O)--N(Rm)(Rn), wherein each Rm and Rn is independently H or substituted or unsubstituted C1-C10 alkyl. Examples of sugar substituents at the 5'-position include, but are not limited to, 5'-methyl (R or S); 5'-vinyl and 5'-methoxy. In some embodiments, the substituted sugar comprises more than one non-bridging sugar substituent, e.g., TF-5'-methyl sugar moiety (for additional 5',2'-disubstituted sugar moieties and nucleosides, see, e.g., PCT International Application No. WO 2008 / 101157).

[0127] Nucleosides comprising 2'-substituted sugar moieties are referred to as 2'-substituted nucleosides. In some embodiments, 2'-substituted nucleosides comprise 2'-substituent groups selected from the group consisting of halo, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, O, S or N(Rm)-alkyl; O, S or N(Rm)-alkenyl; O, S or N(Rm)-alkynyl; O-alkylene-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O(CH2)2--O--N(Rm)(Rn) or O--CH2--C(=O)--N(Rm)(Rn), wherein each Rm and Rn are independently H, an amino protecting group or a substituted or unsubstituted C1-C10 alkyl. These 2'-substituents may be further substituted by one or more substituents independently selected from the group consisting of hydroxy, amino, alkoxy, carboxyl, benzyl, phenyl, nitro (NO2), mercapto, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl, and alkynyl.

[0128] In some embodiments, the 2'-substituted nucleoside comprises a 2'-substituent group selected from the group consisting of F, NH2, N3, OCF3, O--CH3, O(CH2)3NH2, CH2-CH=CH2, O--CH2-CH=CH2, OCH2CH2OCH3, O(CH2)2SCH3, O--(CH2)2--O--N(Rm)(Rn), O(CH2)2O(CH2)2N(CH3)2 and N-substituted acetamide (O--CH2--C(=O)--N(Rm)(Rn), wherein each Rm and Rn is independently H, an amino protecting group, or a substituted or unsubstituted C1-C10 alkyl group.

[0129] In some embodiments, the 2'-substituted nucleoside comprises a sugar moiety comprising a 2'-substituent group selected from the group consisting of F, OCF3, O--CH3, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2--O--N(CH3)2, --O(CH2)2O(CH2)2N(CH3)2, and O--CH2--C(=O)--N(H)CH3.

[0130] In some embodiments, the 2'-substituted nucleoside comprises a sugar moiety comprising a 2'-substituent group selected from the group consisting of F, O--CH3, and OCH2CH2OCH3.

[0131] In some embodiments, the nucleosides of the present disclosure comprise one or more unmodified nucleobases.In certain embodiments, the nucleosides of the present disclosure comprise one or more modified nucleobases.

[0132] In some embodiments, the modified nucleobase is selected from the group consisting of universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine; 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (CH3) uracil and cytosine, and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine, and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo, specifically 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases, hydrophobic bases, mixed bases, size-expanded bases, and fluorinated bases, as defined herein. Additional modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine ([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-13][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced by other heterocycles such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone. Other nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, edited by Kroschwitz, JI, John Wiley & Sons, 1990, 858-859; those disclosed by Englisch et al., 1991; and those disclosed by Sanghvi, YS, 1993.

[0133] Representative U.S. patents that teach the preparation of some of the above-mentioned modified nucleobases and other modified nucleobases include, but are not limited to, U.S. Patents 3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,50 5,763,588; 5,830,653 and 6,005,096, each of which is incorporated herein by reference in its entirety.

[0134] Additional modifications can also be made at other positions on the oligonucleotide, particularly at the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of the 5' terminal nucleotide. For example, an additional modification of the ligand-conjugated oligonucleotide of the present disclosure involves chemically connecting one or more additional non-ligand moieties or conjugates to the oligonucleotide, which enhances the activity, cellular distribution or cellular uptake of the oligonucleotide. Such moieties include, but are not limited to, lipid moieties, such as cholesterol moieties (Letsinger et al., 1989), cholic acid (Manoharan et al., 1994), thioethers, such as hexyl-5-tritylthiol (Manoharan et al., 1992; Manoharan et al., 1993), thiocholesterol (Oberhauser et al., 1992), fatty chains, such as dodecanediol or undecyl residues (Saison-Behmoaras et al., 1991; Kabanov et al., 1990; Sv Inarchuk et al., 1993), phospholipids, such as hexadecylrac glycerol or 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonic acid triethylammonium (Manoharan et al., 1995; Shea et al., 1990), polyamines or polyethylene glycol chains (Manoharan et al., 1995) or adamantane acetic acid (Manoharan et al., 1995), palmityl moieties (Mishra et al., 1995) or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties (Crooke et al., 1996). In some aspects, the nucleic acid molecule encoding the engineered PIV F protein is a modified RNA, such as, for example, a modified mRNA. Modified (m)RNA contemplates certain chemical modifications that confer increased stability and low immunogenicity to mRNA, thereby promoting expression of therapeutically important proteins. For example, N1-methyl-pseudouridine (N1mΨ) outperforms several other nucleoside modifications and combinations thereof in terms of translational capacity. In some embodiments, the (m)RNA molecules used herein may replace uracil with a pseudouracil, such as a 1-methyl-3'-pseudouridylyl base. In some embodiments, some uracils are replaced, but in other embodiments, all uracils are replaced. The (m)RNA may comprise a 5' cap, a 5' UTR element, an optionally codon-optimized open reading frame, a 3' UTR element, and a poly(A) sequence and / or a polyadenylation signal.

[0135] Nucleic acid molecules, whether natural or modified, can be delivered as naked nucleic acid molecules or in a delivery vehicle such as a lipid nanoparticle. The lipid nanoparticle can comprise one or more nucleic acids in a weight ratio of about 5:1 to about 1:100 to the lipid nanoparticle. In some embodiments, the weight ratio of nucleic acid to lipid nanoparticle is about 5:1, 2.5:1, 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100, or any value derivable therein.

[0136] In some embodiments, the lipid nanoparticles used herein may contain one, two, three, four, five, six, seven, eight, nine, or ten lipids. These lipids may include triglycerides, phospholipids, steroids or sterols, pegylated lipids, or groups having ionizable groups such as alkylamines and one or more hydrophobic groups such as C6 or larger alkyl groups.

[0137] In some aspects of the present disclosure, lipid nanoparticles are mixed with one or more steroids or steroid derivatives. In some embodiments, the steroid or steroid derivative includes any steroid or steroid derivative. As used herein, in some embodiments, the term "steroid" is a class of compounds having a tetracyclic 17-carbon ring structure, which may further contain one or more substituents, including alkyl, alkoxy, hydroxy, oxo, acyl, or double bonds between two or more carbon atoms.

[0138] In some aspects of the present disclosure, lipid nanoparticles are mixed with one or more pegylated lipids (or PEG lipids). In some embodiments, the present disclosure includes the use of any lipid to which a PEG group has been attached. In some embodiments, the PEG lipid is a diglyceride, which also includes a PEG chain attached to a glycerol group. In other embodiments, the PEG lipid is a compound containing one or more C6-C24 long-chain alkyl or alkenyl groups or C6-C24 fatty acid groups, which are attached to a linker group with a PEG chain. Some non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG ceramide conjugated, PEG-modified dialkylamines and PEG-modified 1,2-diacyloxypropane-3-amines, PEG-modified diacylglycerols and dialkylglycerols. In some embodiments, PEG-modified distearoylphosphatidylethanolamine or PEG-modified dimyristoyl-sn-glycerol. In some embodiments, PEG is modified by measuring the molecular weight of the PEG component of the lipid. In some embodiments, PEG is modified to have a molecular weight of about 100 to about 15,000. In some embodiments, the molecular weight is from about 200 to about 500, from about 400 to about 5,000, from about 500 to about 3,000, or from about 1,200 to about 3,000. The molecular weight of the PEG modification is from about 100, 200, 400, 500, 600, 800, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,500 to about 15,000. Some non-limiting examples of lipids useful in the present disclosure are taught in US Patent 5,820,873, WO 2010 / 141069, or US Patent 8,450,298, which are incorporated herein by reference.

[0139] In some aspects of the present disclosure, lipid nanoparticles are mixed with one or more phospholipids. In some embodiments, any lipid further comprising a phosphate group. In some embodiments, a phospholipid is a structure containing one or two long-chain C6-C24 alkyl or alkenyl groups, glycerol or sphingosine, one or two phosphate groups and an optional small organic molecule. In some embodiments, the small organic molecule is an alkoxy group substituted with an amino acid, a sugar or an amino group, such as choline or ethanolamine. In some embodiments, the phospholipid is a phosphatidylcholine. In some embodiments, the phospholipid is a distearoylphosphatidylcholine or a dioleoylphosphatidylethanolamine. In some embodiments, other zwitterionic lipids are used, wherein the zwitterionic lipid definition has lipids and lipid-like molecules that have both a positive charge and a negative charge.

[0140] In some aspects of the present disclosure, there is provided a lipid nanoparticle comprising a compound containing a lipophilic and cationic component, wherein the cationic component is ionizable. In some embodiments, the cationic ionizable lipid contains one or more protonated at physiological pH but can be deprotonated and uncharged groups at a pH higher than 8,9,10,11 or 12. The ionizable cationic group can contain one or more protonated amines that can form cationic groups at physiological pH. The cationic ionizable lipid compound can also further include one or more lipid components, such as two or more fatty acids with C6-C24 alkyl or alkenyl carbon groups. These lipid groups can be attached by ester linkage or can be further added to the sulphur atom by Michael addition (Michael addition). In some embodiments, these compounds can be dendrimers (dendrimer), dendrimers (dendron), polymers or combinations thereof.

[0141] In some aspects of the present disclosure, there is provided a composition comprising a compound containing lipophilic and cationic components, wherein the cationic component is ionizable. In some embodiments, ionizable cationic lipids refer to lipids and lipid-like molecules with nitrogen-atoms that can obtain an electric charge (pKa). These lipids can be referred to as cationic lipids in the literature. These molecules with amino groups generally have 2 to 6 hydrophobic chains, typically alkyl or alkenyl such as C6-C24 alkyl or alkenyl, but can have at least 1 or more than 6 tails (tail).

[0142] In some embodiments, the amount of lipid nanoparticles encapsulating nucleic acid molecules in the pharmaceutical composition is about 0.1% weight / weight to about 50% weight / weight, about 0.25% weight / weight to about 25% weight / weight, about 0.5% weight / weight to about 20% weight / weight, about 1% weight / weight to about 15% weight / weight, about 2% weight / weight to about 10% weight / weight, about 2% weight / weight to about 5% weight / weight, or about 6% weight / weight to about 10% weight / weight. In some embodiments, the amount of lipid nanoparticles encapsulating nucleic acid molecules in the pharmaceutical composition is about 0.1% w / w, 0.25% w / w, 0.5% w / w, 1% w / w, 2.5% w / w, 5% w / w, 7.5% w / w, 10% w / w, 15% w / w, 20% w / w, 25% w / w, 30% w / w, 35% w / w, 40% w / w, 45% w / w, 50% w / w, 55% w / w, 60% w / w, 65% w / w, 70% w / w, 75% w / w, 80% w / w, 85% w / w, 90% w / w to about 95% w / w, or any range derivable therein.

[0143] In some aspects, the present disclosure includes one or more sugars formulated into pharmaceutical compositions. In some embodiments, the sugar used herein is a saccharide. These saccharides can be used to serve as freeze-drying protectants that prevent the pharmaceutical composition from being unstable during the drying process. These water-soluble excipients include carbohydrates or saccharides such as disaccharides such as sucrose, trehalose or lactose, trisaccharides such as raffinose comprising fructose, glucose, galactose, polysaccharides such as starch or cellulose, or sugar alcohols such as xylitol, sorbitol or mannitol. In some embodiments, these excipients are solid at room temperature. Some non-limiting examples of sugar alcohols include erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotritol, maltotetraitol, or a polyglycitol.

[0144] In some embodiments, the amount of sugar in the pharmaceutical composition is about 25% weight / weight to about 98% weight / weight, 40% weight / weight to about 95% weight / weight, 50% weight / weight to about 90% weight / weight, 50% weight / weight to about 70% weight / weight, or about 80% weight / weight to about 90% weight / weight. In some embodiments, the amount of sugar in the pharmaceutical composition is about 10% weight / weight, 15% weight / weight, 20% weight / weight, 25% weight / weight, 30% weight / weight, 35% weight / weight, 40% weight / weight, 45% weight / weight, 50% weight / weight, 52.5% weight / weight, 55% weight / weight, 57.5% weight / weight, 60% weight / weight, 62.5% weight / weight, 65% weight / weight, 67.5% weight / weight, 70% weight / weight, 75% weight / weight, 80% weight / weight, 82.5% weight / weight, 85% weight / weight, 87.5% weight / weight, 90% weight / weight to about 95% weight / weight, or any range derivable therein.

[0145] In some embodiments, the pharmaceutically acceptable polymer is a copolymer. The pharmaceutically acceptable polymer may further comprise one, two, three, four, five, or six separate subunits of different types of polymer subunits. These polymer subunits may include polyoxypropylene, polyoxyethylene, or similar subunits. In particular, the pharmaceutically acceptable polymer may comprise at least one hydrophobic subunit and at least one hydrophilic subunit. In particular, the copolymer may have a hydrophilic subunit on each side of the hydrophobic unit. The copolymer may have a hydrophilic subunit polyoxyethylene and a hydrophobic subunit polyoxypropylene.

[0146] In some embodiments, the expression cassette is used to express the PIV F protein for subsequent purification and delivery to cells / subjects, or directly for use in viral-based delivery methods. Provided herein are expression vectors containing one or more nucleic acids encoding the PIV F protein.

[0147] Expression requires the provision of appropriate signals within the vector and the inclusion of various regulatory elements, such as enhancers / promoters from both viral and mammalian sources, that drive expression of the engineered hMPV F protein in cells. Throughout this application, the term "expression cassette" is intended to include any type of genetic construct containing a nucleic acid encoding a gene product, wherein part or all of the nucleic acid coding sequence is capable of being transcribed and translated, i.e., under the control of a promoter. A "promoter" refers to a DNA sequence recognized by the cell's synthetic machinery or introduced synthetic machinery to initiate specific transcription of a gene. The phrase "under transcriptional control" means that the promoter is in the correct position and orientation relative to the nucleic acid to control RNA polymerase initiation and gene expression. An "expression vector" is intended to include an expression cassette contained in a replicable genetic construct, thus including one or more of an origin of replication, a transcription termination signal, a polyadenylation region, a selectable marker, and a multipurpose cloning site.

[0148] The term promoter will be used herein to refer to a group of transcriptional control modules that are clustered around the start site of RNA polymerase II. Much of the thinking about how promoters are organized stems from analyses of several viral promoters, including those of the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, supplemented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA and containing one or more recognition sites for transcriptional activators or repressors.

[0149] The function of at least one module in each promoter is to locate the start site for RNA synthesis. The best-known example is the TATA box, but in some promoters that lack a TATA box, such as the promoter of the mammalian terminal deoxynucleotidyl transferase gene and the promoter of the SV40 late gene, discrete elements overlapping the start site themselves help determine the start position.

[0150] Additional promoter elements regulate the frequency of transcription initiation. Typically, they are located in the region 30-110 bp upstream of the start site, although recently many promoters have been found to also contain functional elements downstream of the start site. The spacing between promoter elements is usually flexible, so when elements are inverted or moved relative to each other, promoter function is retained. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it seems that individual elements can work collaboratively or independently to activate transcription.

[0151] In certain embodiments, viral promoters such as human cytomegalovirus (CMV) immediate early gene promoter, SV40 early promoter, Rous sarcoma virus long terminal repeat, rat insulin promoter and glyceraldehyde-3-phosphate dehydrogenase can be used to obtain high-level expression of the coding sequence of interest. It is also envisioned that other viral or mammalian cell or bacterial phage promoters well known in the art can be used to achieve expression of the coding sequence of interest, provided that the expression level is sufficient for a given purpose. By adopting a promoter with known properties, the expression level and pattern of the protein of interest after transfection or transformation can be optimized. In addition, the selection of a promoter that is regulated in response to a specific physiological signal can allow for inducible expression of the gene product.

[0152] Enhancers are genetic elements that increase transcription from a promoter located at a distant site on the same DNA molecule. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. The fundamental difference between enhancers and promoters is operational. The entire enhancer region must be able to stimulate transcription from a distance; this is not necessarily true for the promoter region or its constituent elements. On the other hand, promoters must have one or more elements that direct the initiation of RNA synthesis at a specific site and in a particular direction, while enhancers lack these specificities. Promoters and enhancers are often overlapping and adjacent and appear to generally have a very similar modular organization.

[0153] The following is a list of promoters / enhancers and inducible promoters / enhancers that can be used in combination with nucleic acids encoding a gene of interest in an expression construct. In addition, any promoter / enhancer combination (according to the eukaryotic promoter database EPDB) can also be used to drive the expression of a gene. If appropriate bacterial polymerase is provided, then, whether as a part for delivering the complex or as another genetic expression construct, eukaryotic cells can support the cytoplasm transcription caused by some bacterial promoters.

[0154] The promoter and / or enhancer can be, for example, an immunoglobulin light chain, an immunoglobulin heavy chain, a T cell receptor, HLA DQ alpha and / or DQ beta, beta-interferon, interleukin-2, interleukin-2 receptor, MHC class II 5, MHC class II HLA-Dra, beta-actin, muscle creatine kinase (MCK), prealbumin (transthyretin), elastase I, metallothionein (MTII), collagenase, albumin, alpha-fetoprotein, t-globin, beta-globin, c-fos, c-HA-ras, insulin, neural cell adhesion molecule (NCAM), alpha 1-antitrypsin, H2B (TH2B) histone, mouse and / or type I collagen, glucose-regulated proteins (GRP94 and GRP78), rat growth hormone, human serum amyloid A (SAA), troponin I (TN), I), platelet-derived growth factor (PDGF), SV40, polyomavirus, retrovirus, papillomavirus, hepatitis B virus, human immunodeficiency virus, cytomegalovirus (CMV), and gibbon ape leukemia virus.

[0155] In the case of using a cDNA insert, one would typically desire to include a polyadenylation signal to achieve correct polyadenylation of the gene transcript. Any polyadenylation sequence can be used, such as human growth hormone and SV40 polyadenylation signals. Also contemplated as elements of the expression cassette are terminators. These elements can be used to enhance message levels and minimize read-through from the cassette into other sequences.

[0156] There are many ways to introduce expression vectors into cells. In certain embodiments, the expression construct comprises a virus or an engineered construct derived from a viral genome. The ability of certain viruses to enter cells via receptor-mediated endocytosis, integrate into the host cell genome, and stably and efficiently express viral genes makes them attractive candidates for transferring foreign genes into mammalian cells. These viruses have a relatively low capacity for foreign DNA sequences and have a restricted host spectrum. In addition, their carcinogenic potential and cytopathic effects in permissive cells have caused safety issues. They can only accommodate up to 8kB of foreign genetic material, but can be easily introduced into a variety of cell lines and laboratory animals.

[0157] A method for in vivo delivery involves the use of an adenoviral expression vector. "Adenoviral expression vector" is intended to include constructs containing adenoviral sequences sufficient to (a) support the packaging of the construct and (b) express the engineered PIV F protein cloned therein. In this case, expression does not require a synthetic gene product.

[0158] The expression vector comprises adenovirus in a genetically engineered form. Understanding the genetic organization of adenovirus (a 36kB linear double-stranded DNA virus) allows the replacement of large pieces of adenoviral DNA with up to 7kB of foreign sequences. In contrast to retroviruses, adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can be replicated in an episomal manner without potential genotoxicity. In addition, adenovirus structure is stable, and no genome rearrangement has been detected after extensive amplification. Adenovirus can infect almost all epithelial cells, regardless of their cell cycle stage. So far, adenoviral infection seems to be associated only with mild diseases, such as acute respiratory diseases in humans.

[0159] Adenovirus is particularly well-suited for use as a gene transfer vector due to its medium-sized genome, ease of manipulation, high titer, broad target cell range, and high infectivity. The viral genome contains 100-200 base pair inverted repeats (ITRs) at both ends, which are cis-elements essential for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain distinct transcription units, which are divided according to the onset of viral DNA replication. The E1 region (E1A and E1B) encodes proteins responsible for regulating transcription of the viral genome and some cellular genes. Expression of the E2 region (E2A and E2B) leads to the synthesis of proteins required for viral DNA replication. These proteins are involved in DNA replication, late gene expression, and host cell shutdown. The products of the late genes, including most of the viral capsid proteins, are expressed only after significant processing of a single primary transcript emanating from the major late promoter (MLP). The MLP (located at 16.8 mu) is particularly efficient late in infection, and all mRNAs emanating from this promoter possess a 5'-triplet leader (TPL) sequence, making them preferred mRNAs for translation. In one system, recombinant adenovirus is generated by homologous recombination between a shuttle vector and a proviral vector. Because recombination between the two proviral vectors can occur, wild-type adenovirus can be generated from this process. Therefore, it is crucial to isolate individual viral clones from individual plaques and examine their genomic structure.

[0160] The production and propagation of current replication-deficient adenoviral vectors rely on a unique helper cell line, designated 293, which is derived from human embryonic kidney cells transformed with Ad5 DNA fragments and constitutively expresses the E1 protein. Because the E3 region is dispensable in the adenoviral genome, current adenoviral vectors, with the help of 293 cells, carry foreign DNA in either the E1, D3, or both regions. In nature, adenovirus can package approximately 105% of the wild-type genome, providing capacity for approximately 2 kb of additional DNA. Combined with the approximately 5.5 kb of DNA that can be replaced in the E1 and E3 regions, the maximum capacity of current adenoviral vectors is less than 7.5 kb, or approximately 15% of the total vector length. Over 80% of the adenoviral viral genome is retained in the vector backbone and is the source of vector-borne cytotoxicity. Furthermore, the replication defect of E1-deleted viruses is incomplete.

[0161] Helper cell lines can be derived from human cells, such as human embryonic kidney cells, muscle cells, hematopoietic cells, or other human embryonic mesenchymal or epithelial cells. Alternatively, helper cells can be derived from cells of other mammalian species that are permissive for human adenoviruses. Such cells include, for example, Vero cells or other monkey embryonic mesenchymal or epithelial cells. As described above, a preferred helper cell line is 293.

[0162] The adenoviruses of the present disclosure are replication-defective, or at least conditionally replication-defective. Adenoviruses can be of any of the 42 different known serotypes or subgroups A to F. Adenovirus type 5 of subgroup C is an exemplary starting material that can be used to obtain conditionally replication-defective adenoviral vectors for use in the present disclosure.

[0163] Other viral vectors can be used as expression constructs in the present disclosure. Vectors derived from viruses such as vaccinia virus, adeno-associated virus (AAV) and herpes virus can be used. They provide several attractive features for various mammalian cells.

[0164] In an embodiment, a specific embodiment, the vector is an AAV vector. AAV is a small virus that infects humans and some other primate species. AAV is not yet known to cause disease. The virus causes a very mild immune response, further supporting its apparent lack of pathogenicity. In many cases, AAV vectors are integrated into the host cell genome, which may be important for certain applications, but may also have adverse consequences. Gene therapy vectors using AAV can infect both dividing cells and resting cells and continue to exist in an extrachromosomal state without integrating into the host cell genome, although some viral-carried genes do integrate into the host genome in natural viruses. These characteristics make AAV a very attractive candidate for creating viral vectors for gene therapy and for creating isogenic human disease models. Recent human clinical trials using AAV for retinal gene therapy have shown promise. AAV belongs to the genus Dependinovirus, which in turn belongs to the family Parvoviridae. The virus is a small (20nm), replication-defective, non-enveloped virus.

[0165] Wild-type AAV has garnered significant interest among gene therapy researchers due to a number of characteristics. Chief among these is the virus's apparent lack of pathogenicity. It can also infect non-dividing cells and stably integrate into the host cell genome at a specific site on human chromosome 19 (designated AAVS1). This characteristic makes it more predictable than retroviruses, which carry the risk of random insertions and mutagenesis, sometimes leading to the development of cancer. The AAV genome most frequently integrates at the aforementioned site, while random incorporation into the genome is negligible. However, the development of AAV as a gene therapy vector eliminated this integration ability by removing the rep and cap sequences from the vector DNA. The desired gene, along with the promoter driving gene transcription, is inserted between inverted terminal repeats (ITRs). This facilitates the formation of concatemers in the cell nucleus after the single-stranded vector DNA is converted to double-stranded DNA by the host cell DNA polymerase complex. AAV-based gene therapy vectors form episomal concatemers in the host cell nucleus. In non-dividing cells, these concatemers remain intact throughout the host cell's lifespan. In dividing cells, AAV DNA is lost through cell division because the episomal DNA is not replicated along with the host cell DNA. Random integration of AAV DNA into the host genome is detectable but occurs at a very low frequency. AAVs also exhibit very low immunogenicity, which appears to be limited to the production of neutralizing antibodies, while they do not induce a well-defined cytotoxic response. This feature, along with the ability to infect quiescent cells, suggests that they are superior to adenoviruses as vectors for human gene therapy.

[0166] The AAV genome consists of single-stranded deoxyribonucleic acid (ssDNA), which can be positive or negative sense and is approximately 4.7 kilobases long. The genome contains inverted terminal repeats (ITRs) at each end of the DNA strand and two open reading frames (ORFs): rep and cap. The former consists of four overlapping genes encoding the Rep protein, which is essential for the AAV life cycle, while the cap contains overlapping capsid protein nucleotide sequences: VP1, VP2, and VP3, which interact to form the icosahedral capsid.

[0167] Inverted terminal repeat (ITR) sequences each comprise 145 bases. They are so named because of their symmetry, which has been shown to be required for the effective proliferation of the AAV genome. The feature of these sequences that gives them this characteristic is the ability to form hairpins, which contributes to so-called self-priming, which allows the synthesis of the second DNA chain independent of the primase. ITR has also been shown to be required for the integration of AAV DNA into the host cell genome (human chromosome 19) and for the rescue thereof, as well as for the combination of the generation of the effective encapsidation of AAV DNA and the deoxyribonuclease-resistant AAV particles that are fully assembled.

[0168] With regard to gene therapy, the ITR appears to be the only sequence required in cis alongside the therapeutic gene: structural (cap) and packaging (rep) proteins can be delivered in trans. With this assumption, numerous methods have been established for the efficient production of recombinant AAV (rAAV) vectors containing reporter or therapeutic genes. However, it has also been shown that the ITR is not the only element required in cis for efficient replication and encapsidation. Several research groups have identified a sequence within the coding sequence of the rep gene, termed the cis-acting Rep-dependent element (CARE). When present in cis, the CARE has been shown to enhance replication and encapsidation.

[0169] Each immunogenic composition discussed herein can be used alone or in combination with one or more other antigens, the latter being derived from the same viral pathogen or from another pathogenic source or sources. These compositions can be used for prophylactic (preventing infection) or therapeutic (treating disease following infection) purposes.

[0170] The term "pharmaceutically acceptable" may mean approved by federal regulatory agencies or state governments or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeia for use in animals and more particularly in humans. The term "carrier" refers to a diluent, excipient, or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water, and can preferably include an adjuvant. When the pharmaceutical composition is administered by injection, such as intramuscular injection, water is a specific carrier. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc.

[0171] The immunogenic composition may comprise a diluent such as water, saline, glycerol, ethanol, or the like. In addition, auxiliary substances such as wetting agents or emulsifiers, pH buffering substances, or the like may be present in such carriers. The immunogenic composition may contain one or more salts. The salt may be an inorganic potassium or sodium salt, such as potassium chloride, sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, or sodium dihydrogen phosphate. The immunogenic composition may comprise one or more phosphates to produce a phosphate buffered solution. The phosphate buffered solution may comprise each of the phosphates to buffer the solution to a pH of about 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0, or any range derivable therein.

[0172] The immunogenic composition may include an adjuvant. Exemplary adjuvants that enhance the effectiveness of the composition include: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, and the like; (2) oil-in-water emulsion formulations (with or without other specific adjuvants, such as murein peptides (see below) or bacterial cell wall components), such as, for example, (a) MF59 (PCT Publication No. WO 90 / 14837), containing 5% squalene, 0.5% TWEEN 80, and 0.5% Span 85, formulated into submicron particles using a microfluidizer, (b) SAF, containing 10% squalane, 0.4% Tween 80, 5% Pluronic blocked polymer L121, and thr-MDP, prepared into a submicron emulsion by microfluidization or vortexing to produce an emulsion of larger particle size, and (c) RIBI TM Adjuvant system (RAS) (RibiImmunochem, Hamilton, Mont.) containing 2% squalene, 0.2% Tween 80 and one or more bacterial cell wall components selected from the group consisting of monophosphoryl lipid A (MPL), trehalose dimycolate (TDM) and cell wall skeleton (CWS), preferably MPL + CWS (DETOXTM ); (3) saponin adjuvants, such as QS-21, STIMULON, which can be used TM (Cambridge Bioscience, Worcester, MA) or particles produced therefrom, such as ISCOM (immunostimulating complex); (4) complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA); (5) cytokines, such as interleukins (IL-1, IL-2, etc.), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; (6) Toll-like receptor agonists; and (7) other substances that act as adjuvants to enhance the effectiveness of the composition. The composition may not contain an adjuvant. The composition may also contain lipid nanoparticles. The composition may be formulated as nanoparticles. The composition may also contain cationic or polycationic compounds, including protamine or other cationic peptides or proteins, such as poly-L-lysine (PLL).

[0173] Typically, the ingredients of the compositions of the present disclosure are provided individually or mixed together in a unit dose form, for example as a dry lyophilized powder or anhydrous concentrate, in a sealed container such as an ampoule or sachet indicating the amount of active agent. When the composition is administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule containing sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0174] The compositions described herein may include an immunologically effective amount of polypeptides or polynucleotides, as well as any other components described above, as needed. "Immunologically effective amount" means that an immune response can be effectively elicited by administering the amount to an individual as a portion of a single dose or a series of doses. The immune response elicited may be sufficient for, for example, treating and / or preventing and / or reducing the incidence of illness, infection, or disease. This amount varies depending on the health and physical condition of the individual to be treated, the taxonomic group of the individual to be treated (e.g., non-human primates, primates, etc.), the ability of the individual immune system to synthesize antibodies, the degree of protection required, the formulation of the vaccine, the assessment of the medical condition by the treating physician, and other relevant factors. The expected amount will fall within a relatively wide range, and the amount can be determined by routine experiments.

[0175] Any suitable route of administration can be used. For example, the engineered proteins of the present disclosure or nucleic acids encoding the engineered proteins can be formulated for parenteral administration, such as for injection via intradermal, intravenous, intraarterial, intramuscular, subcutaneous, intratumoral, or even intraperitoneal routes. Particularly preferred routes of administration include intramuscular, intradermal, and subcutaneous injection. Alternatively, the formulation can be administered directly to the mucosa via a topical route, such as by nasal drops, inhalation, or by a nebulizer.

[0176] Compositions can be administered according to any suitable schedule. Dosage therapy can be a single dose regimen or a multiple dose regimen. Multiple doses can be used for primary immunization regimens and / or booster immunization regimens. In multiple dose regimens, various dosages can be given by the same or different approaches. Multiple doses are typically administered at intervals of at least 1 week (e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.). Immunogenic compositions can be administered in combination with other immunomodulators.

[0177] The compositions disclosed herein can be used to treat both children and adults. Thus, the human subject can be less than 1 year old, 1-5 years old, 5-16 years old, 16-55 years old, 55-65 years old, or at least 65 years old.

[0178] VI. Antibodies and Diagnostic Uses

[0179] The above polypeptides can be used to produce polyclonal and monoclonal antibodies. If polyclonal antibodies are needed, mammals (e.g., mice, rabbits, goats, guinea pigs, horses, etc.) selected by immunogenic polypeptide immunity with PIV F prefusion epitopes are used. Serum from immune animals is collected and processed according to known procedures. If the serum containing polyclonal antibodies against PIV F prefusion epitopes contains antibodies against other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. The technology for producing and processing polyclonal antisera is known in the art.

[0180] Monoclonal antibodies directed against the PIV F prefusion epitope can also be readily prepared by one skilled in the art. General methods for producing monoclonal antibodies using hybridomas are known. Immortalized antibody-producing cell lines can be created by cell fusion or by other techniques, such as direct transformation of B lymphocytes with oncogenic DNA or transfection with Epstein-Barr virus. Panels of monoclonal antibodies generated against the PIV F prefusion epitope can be screened for various properties; i.e., isotype, epitope affinity, etc.

[0181] Monoclonal and polyclonal antibodies against the PIV F prefusion epitope are particularly useful in diagnosis, while neutralizing antibodies are useful in passive immunotherapy. Monoclonal antibodies, in particular, can be used to generate anti-idiotypic antibodies.

[0182] Polypeptides that immunoreact with serum containing PIV F antibodies and antibodies raised against these polypeptides can be used in immunoassays to detect the presence of PIV F antibodies or the presence of virus in biological samples (including, for example, blood or serum samples). There is a wide variety of immunoassay designs, many of which are known in the art. For example, an immunoassay can utilize a polypeptide having a sequence as shown in any one of SEQ ID NOs: 1-7.

[0183] Alternatively, the immunoassay may use a combination of viral antigens derived from the polypeptides described herein. For example, monoclonal antibodies to at least one polypeptide described herein, a combination of monoclonal antibodies to polypeptides described herein, monoclonal antibodies to different viral antigens, polyclonal antibodies to polypeptides described herein, or polyclonal antibodies to different viral antigens may be used. For example, the protocol may be based on a competitive or direct reaction, or may be a sandwich type assay. For example, the protocol may also use a solid support, or may be achieved by immunoprecipitation. Most assays involve the use of labeled antibodies or polypeptides; the label may be, for example, fluorescent, chemiluminescent, radioactive, or a dye molecule. Assays that amplify probe signals are also known; examples are assays utilizing biotin and avidin, and enzyme-labeled and mediated immunoassays, such as ELISA assays.

[0184] Kits suitable for immunodiagnostics and containing appropriately labeled reagents are constructed by packaging appropriate materials (including engineered PIV F proteins containing PIV F prefusion epitopes or antibodies against the epitopes) in a suitable container along with the remaining reagents and materials required to perform the assay and a set of appropriate assay instructions.

[0185] The polynucleotide probes can also be packaged into diagnostic kits. The diagnostic kits include the probe DNA, which can be labeled; alternatively, the probe DNA can be unlabeled, and the labeled component can be included in the kit. The kits can also contain other appropriately packaged reagents and materials required for a specific hybridization protocol, such as standards, and instructions for performing the test.

[0186] VII. Immunoassay Methods

[0187] The present disclosure relates to immunoassays for binding, purification, removal, quantification, and otherwise general detection of PIV F protein. While such methods can be used in a conventional sense, another application would be quality control and monitoring of vaccine stocks, where antibodies according to the present disclosure can be used to assess the amount or integrity (i.e., long-term stability) of the antigen. Alternatively, the methods can be used to screen various antibodies for appropriate / desired reactivity characteristics.

[0188] Some immunoassays include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluorescent immunoassay, chemiluminescent assay, bioluminescent assay and Western blot. In particular, competitive assays for detecting and quantifying PIV F protein are also provided. The steps of various useful immunoassays have been described in the scientific literature, such as, for example, Doolittle and Ben-Zeev (1999), Gulbis and Galand (1993), De Jager et al. (1993) and Nakamura et al. (1987). In general, the immunobinding method includes obtaining a sample suspected of containing PIV F protein, and contacting the sample with a first antibody according to the present disclosure (as the case may be) under conditions that effectively allow the formation of an immune complex.

[0189] These methods include methods for detecting or purifying PIV F protein from a sample. The antibody will preferably be attached to a solid support such as a column matrix, and a sample suspected of containing PIV F protein will be applied to the immobilized antibody. Unwanted components will be washed off the column, leaving cells expressing PIV F protein in an immunocomplex with the immobilized antibody, and the cells expressing PIV F protein will then be collected by removing the organism or antigen from the column.

[0190] Immunobinding methods also include methods for detecting and quantifying the amount of PIV F protein or related components in a sample and detecting and quantifying any immune complexes formed during the binding process. Here, one obtains a sample suspected of containing PIV F protein and contacts the sample with antibodies that bind to PIV F protein or its components, and then detects and quantifies the amount of immune complexes formed under specific conditions. In terms of antigen detection, the biological sample analyzed can be any sample suspected of containing PIV F protein, such as a tissue section or specimen, a homogenized tissue extract, a biological fluid (e.g., a nasal swab), including blood and serum, or secretions, such as feces or urine.

[0191] Contacting the selected biological sample with the antibody under effective conditions and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) is generally about the time it takes to simply add the antibody composition to the sample and incubate the mixture long enough for the antibody to form an immune complex with the hMPV F protein (i.e., to bind to the hMPV F protein-binding antibody). After this time, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot, or Western blot, is typically washed to remove any non-specifically bound antibody species, allowing only those antibodies that are specifically bound within the primary immune complex to be detected.

[0192] In general, the detection of immune complex formation is well known in the art and can be achieved by applying a variety of methods. These methods are generally based on the detection of marks or markers, such as any of those radioactive, fluorescent, biological and enzyme labels. Patents related to the use of such marks include U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241. Of course, as known in the art, by using secondary binding partners such as secondary antibodies and / or biotin / avidin ligand binding arrangements, people may find additional advantages.

[0193] The antibody used for detection can itself be linked to a detectable label, wherein one will then simply detect this label, thereby allowing the amount of the primary immune complex in the composition to be determined. Alternatively, the primary antibody bound to the primary immune complex can be detected by a secondary binding ligand that has binding affinity for the antibody. In these cases, the secondary binding ligand can be linked to a detectable label. The secondary binding ligand itself is typically an antibody and therefore can be referred to as a "secondary" antibody. The primary immune complex is contacted with a labeled secondary binding ligand or antibody under effective conditions and for a period of time sufficient to allow the formation of a secondary immune complex. The secondary immune complex is then typically washed to remove any non-specifically bound labeled secondary antibody or ligand, and the remaining label in the secondary immune complex is then detected.

[0194] Other methods include detecting primary immune complexes by a two-step approach. Secondary binding ligands, such as antibodies with binding affinity to the antibody, are used to form secondary immune complexes, as described above. After washing, the secondary immune complexes are again contacted with a tertiary binding ligand or antibody with binding affinity to the secondary antibody under effective conditions and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes). The tertiary ligand or antibody is connected to a detectable label, thereby allowing detection of the tertiary immune complexes thus formed. If desired, this system can provide signal amplification.

[0195] An immunoassay method uses two different antibodies. The first biotinylated antibody is used to detect the target antigen, and the second antibody is then used to detect the biotin attached to the complex biotin. In the method, the sample to be tested is first incubated in a solution containing the first step antibody. If the target antigen is present, some of the antibodies will bind to the antigen to form a biotinylated antibody / antigen complex. The antibody / antigen complex is then amplified by incubating in a continuous solution of streptavidin (or avidin), biotinylated DNA and / or complementary biotinylated DNA, wherein each step adds additional biotin sites to the antibody / antigen complex. The amplification step is repeated until the appropriate amplification level is reached, at which point the sample is incubated in a solution containing the second step antibody against biotin. This second step antibody is labeled, for example, with an enzyme that can be used to detect the presence of the antibody / antigen complex by tissue enzymology using a chromogenic substrate. Through appropriate amplification, a macroscopically visible conjugate can be produced.

[0196] Another known immunoassay utilizes immune PCR (polymerase chain reaction) method.The PCR method is similar to the Cantor method until hatched with biotinylated DNA, but, rather than using multiple rounds of streptavidin and biotinylated DNA hatching, the DNA / biotin / streptavidin / antibody complex is washed away with low pH or high salt buffer that releases the antibody.Then the gained washing solution is used to carry out the PCR reaction with suitable primers and appropriate controls.At least in theory, the huge amplification capacity and specificity of PCR can be used to detect single antigen molecules.

[0197] A.ELISA

[0198] In its simplest and most direct sense, immunoassay is a binding assay. Some preferred immunoassays are various types of enzyme-linked immunosorbent assays (ELISA) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily understood that detection is not limited to such techniques, and Western blots, dot blots, FACS analyses, etc. may also be used.

[0199] In an exemplary ELISA, the antibodies of the present disclosure are immobilized on a selected surface that exhibits protein affinity, such as a well in a polystyrene microtiter plate. A test composition suspected of containing PIV F protein is then added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen can be detected. Detection can be achieved by adding another anti-PIV F protein antibody linked to a detectable label. This type of ELISA is a simple "sandwich ELISA." Detection can also be achieved by adding a secondary anti-PIV F protein antibody, followed by a tertiary antibody that has binding affinity for the secondary antibody, wherein the tertiary antibody is linked to a detectable label.

[0200] In another exemplary ELISA, a sample suspected of containing PIV F protein (e.g., potentially infected cells) is immobilized on a well surface and then contacted with an anti-PIV F protein antibody of the present disclosure. After binding and washing to remove non-specifically bound immune complexes, bound anti-PIV F protein antibodies are detected. When the primary anti-PIV F protein antibody is linked to a detectable label, the immune complex can be directly detected. Similarly, immune complexes can be detected using a secondary antibody that has binding affinity for the primary anti-PIV F protein antibody, wherein the secondary antibody is linked to a detectable label.

[0201] Regardless of the format used, ELISAs share certain common features, such as coating, incubation and binding, washing to remove non-specifically bound substances, and detection of bound immune complexes. These are described below.

[0202] When coating a plate with an antigen or antibody, people typically incubate the wells of the plate with a solution of the antigen or antibody overnight or for a specified number of hours. The wells of the plate will then be washed to remove any incompletely adsorbed material. Any remaining available surface of the wells is then "coated" with a nonspecific protein that is antigenically neutral with respect to the test antiserum. These include bovine serum albumin (BSA), casein, or a powdered milk solution. The coating allows for the blocking of nonspecific adsorption sites on the fixed surface, thereby reducing the background caused by the nonspecific binding of the antiserum to the surface.

[0203] In ELISA, it may be more customary to use secondary or tertiary detection methods rather than direct procedures. Thus, after the protein or antibody binds to the wells, is coated with a non-reactive material to reduce background, and washed to remove unbound material, the fixed surface is contacted with the biological sample to be tested under conditions effective to allow the formation of immune complexes (antigen / antibody). Detection of the immune complex then requires a labeled secondary binding ligand or antibody, as well as a secondary binding ligand or antibody that binds to the labeled tertiary antibody or tertiary binding ligand.

[0204] "Under conditions effective to allow immune complex (antigen / antibody) formation" means that the conditions preferably include diluting the antigen and / or antibody with a solution such as BSA, bovine gamma globulin (BGG) or phosphate buffered saline (PBS) / Tween. These added agents also help to reduce nonspecific background.

[0205] "Suitable" conditions also mean incubation at a temperature or for a period of time sufficient to allow effective binding. The incubation step is typically about 1 to 2 to 4 hours, preferably approximately 25°C to 27°C, or can be overnight at about 4°C.

[0206] After all incubation steps in ELISA, the contacted surfaces are washed to remove uncomplexed material. Preferred washing procedures include washing with solutions such as PBS / Tween or borate buffer. After the formation of specific immune complexes between the test sample and the initially bound material and subsequent washings, even the appearance of trace immune complexes can be determined.

[0207] In order to provide detection means, secondary or tertiary antibodies will have relevant labels to allow detection. Preferably, this will be an enzyme that will produce a color development after hatching with a suitable chromogenic substrate. Therefore, for example, people will wish to contact one-level and secondary immune complexes with urease, glucose oxidase, alkaline phosphatase or catalase-conjugated antibodies for a period of time or to hatch both together for a period of time under the conditions of development that is conducive to further immune complex formation (for example, hatching 2 hours at room temperature in a solution containing PBS such as PBS-Tween).

[0208] After incubation with the labeled antibody and subsequent washing to remove unbound material, the amount of label is quantified, for example by incubation with a chromogenic substrate such as urea, or bromocresol purple, or 2,2'-azino-di-(3-ethyl-benzothiazoline-6-sulfonic acid (ABTS), or H2O2 in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the extent of color produced, for example using a visible spectrum spectrophotometer.

[0209] B. Western Blot

[0210] Western blotting (or immunoblotting) is an analytical technique used to detect specific proteins in a given tissue homogenate or extract sample. It uses gel electrophoresis to separate native or denatured proteins based on polypeptide length (denaturing conditions) or protein 3-D structure (native / non-denaturing conditions). The proteins are then transferred to a membrane (usually nitrocellulose or PVDF), where they are probed (detected) using antibodies specific for the target protein.

[0211] Samples can be taken from whole tissues or cell cultures. In most cases, solid tissues are first mechanically broken down using a stirrer (for larger sample volumes), a homogenizer (for smaller volumes), or by ultrasonic treatment. Cells can also be opened by one of the above-mentioned mechanical methods. Various detergents, salts, and buffers can be used to promote cell lysis and solubilize proteins. Protease and phosphatase inhibitors are usually added to prevent the sample from being digested by its own enzymes. Tissue preparation is usually carried out at low temperatures to avoid protein denaturation.

[0212] Gel electrophoresis is used to separate the proteins of a sample. Proteins can be separated by isoelectric point (pI), molecular weight, charge, or a combination of these factors. The nature of the separation depends on the sample preparation and the properties of the gel. This is a very useful way to identify proteins. Two-dimensional (2-D) gels can also be used to disperse proteins from a single sample into two dimensions. Proteins are separated in the first dimension according to their isoelectric point (the pH at which they have a neutral net charge) and in the second dimension according to their molecular weight.

[0213] To make proteins available for antibody detection, they are moved from the gel to a membrane made of nitrocellulose or polyvinylidene fluoride (PVDF). The membrane is placed on top of the gel, and a stack of filter paper is placed on top. The entire stack is placed in a buffer solution, which causes the paper to move upward through capillary action, bringing the proteins with it. Another method of transferring proteins is called electroblotting, which uses an electric current to pull the proteins from the gel to a PVDF or nitrocellulose membrane. The proteins move from the gel to the membrane while maintaining their organization within the gel. As a result of this blotting process, the proteins are exposed to a thin surface layer for detection (see below). These two membranes are chosen because they have non-specific protein binding properties (i.e., they bind equally to all proteins). Protein binding is based on hydrophobic interactions and charged interactions between the membrane and the protein. Nitrocellulose membranes are cheaper than PVDF, but are much more fragile and do not withstand repeated probing well. The uniformity and overall effectiveness of protein transfer from the gel to the membrane can be checked by staining the membrane with Coomassie Brilliant Blue or Ponceau Red dyes. Once transferred, the protein is detected using either a labeled primary antibody or an unlabeled primary antibody followed by indirect detection using labeled protein A or a labeled secondary antibody that binds to the Fc region of the primary antibody.

[0214] C. Immunohistochemistry

[0215] The antibodies disclosed herein can also be used in conjunction with fresh frozen and / or formalin-fixed, paraffin-embedded tissue blocks prepared for studies by immunohistochemistry (IHC). Methods for preparing tissue blocks from these microparticle specimens have been successfully used in previous IHC studies of various prognostic factors and are well known to those skilled in the art (Brown et al., 1990; Abbondanzo et al., 1990; Allred et al., 1990).

[0216] Briefly, cryosections can be prepared by the following steps: rehydrating 50 ng of frozen "pulverized" tissue in phosphate-buffered saline (PBS) at room temperature in a small plastic capsule; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule again and / or pelleting by centrifugation; snap freezing in isopentane at -70°C; cutting the plastic capsule and / or removing the frozen tissue cylinder; securing the tissue cylinder to a cryostat chuck; and / or cutting 25-50 serial sections from the capsule. Alternatively, the entire frozen tissue sample can be used for serial sectioning.

[0217] Permanent sections can be prepared by a similar method involving the following steps: rehydrating 50 mg of sample in a plastic microcentrifuge tube; pelleting; resuspending and fixing in 10% formalin for 4 hours; washing / pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue / agar block from the tube; infiltrating and / or embedding the block in paraffin; and / or cutting up to 50 serial permanent sections. Likewise, whole tissue samples can be substituted.

[0218] D. Immunoassay Kit

[0219] In yet other embodiments, the present disclosure relates to immunoassay kits for use with the above-described immunoassay methods. Since antibodies can be used to detect PIV F protein, the antibodies can be included in the kit. Thus, the immunoassay kit will include a primary antibody that binds to PIV F protein and, optionally, an immunoassay reagent in a suitable container.

[0220] In certain embodiments, the antibodies can be pre-bound to a solid support such as a column matrix and / or the wells of a microtiter plate. The immunodetection reagents of the kit can take any of a variety of forms, including those with detectable labels associated or linked to a given antibody. Detectable labels associated with or attached to a secondary binding ligand are also contemplated. Exemplary secondary ligands are those that have binding affinity for the primary antibody.

[0221] Other suitable immunodetection reagents for use in the present kits include a two-component reagent comprising a secondary antibody having binding affinity for the primary antibody, together with a tertiary antibody having binding affinity for the secondary antibody, wherein the tertiary antibody is linked to a detectable label. As described above, many exemplary labels are known in the art and all such labels can be used in conjunction with the present disclosure.

[0222] The kit may further include a suitable aliquot of a PIV F protein composition, whether labeled or unlabeled, such as may be used to prepare a standard curve for a detection assay. The kit may contain an antibody-marker conjugate, either in fully conjugated form, in the form of an intermediate, or as a separate part to be conjugated by the user of the kit. The components of the kit may be packaged in an aqueous medium or in a lyophilized form.

[0223] The container means of the test kit typically includes at least one vial, test tube, flask, bottle, syringe or other container means that can be placed or preferably suitably aliquoted into the antibody. The test kit of the present disclosure will also typically include a device for hermetically sealing and containing the antibody, antigen and any other reagent containers for commercial sale. Such containers may include injection-molded or blow-molded plastic containers in which the desired vials are retained.

[0224] E. Flow cytometry and FACS

[0225] The antibodies disclosed herein can also be used in flow cytometry or FACS. Flow cytometry is a laser- or impedance-based technique used in many detection assays, including cell counting, cell sorting, biomarker detection, and protein engineering. The technique suspends cells in a fluid stream and passes them through an electronic detection device, allowing for multi-parameter analysis of the physical and chemical characteristics of up to thousands of particles per second. Flow cytometry is commonly used for the diagnosis of conditions, particularly blood cancers, but has many other applications in basic research, clinical practice, and clinical trials.

[0226] Fluorescence-activated cell sorting (FACS) is a special type of cytometry. It provides a method for sorting a heterogeneous mixture of biological cells, one cell at a time, into two or more containers based on each cell's specific light scattering and fluorescence characteristics. Generally speaking, the technique involves entraining a cell suspension in the center of a narrow, rapidly flowing liquid stream. The flow is arranged so that cells are widely separated relative to their diameters. A vibration mechanism causes the cell stream to break up into individual droplets. Just before the cell stream breaks up into droplets, the cell stream passes through a fluorescence measurement station, where the fluorescence of each cell is measured. A charged ring is placed at the point where the cell stream breaks up into droplets. An electrical charge is placed on the ring immediately before the fluorescence intensity is measured, and an opposite charge is captured on the droplets as they break off the stream. The charged droplets then fall through an electrostatic deflection system, which diverts them into containers based on their charge.

[0227] In certain embodiments, for use in flow cytometry or FACS, antibodies of the disclosure are labeled with a fluorophore and then allowed to bind to cells of interest, which are analyzed in a flow cytometer or sorted by a FACS machine.

[0228] VIII. Definitions

[0229] It should be understood that the above overview and the following detailed description are merely exemplary and explanatory and do not limit the claimed invention. In this application, unless otherwise specifically stated, the use of the singular includes the plural. In this application, unless otherwise specified, the use of "or" means "and / or". In addition, the use of the term "including" and other forms such as "includes" and "included" is not restrictive. In addition, unless otherwise specifically stated, terms such as "element" or "component" cover both elements and components comprising one unit and elements and components comprising more than one subunit. In addition, the use of the term "portion" may include a portion or the entire portion of a portion.

[0230] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. As used in the specification herein, "a" may mean one or more than one. As used herein in the claims, when used in conjunction with the word "comprising," the word "a" may mean one or more than one.

[0231] As used herein, the term "about" is intended to encompass variations of up to ±10% from the specified value when referring to measurable values ​​such as amounts, time intervals, etc. Unless otherwise indicated, all numerals used in the specification and claims representing the quantity of ingredients, characteristics such as molecular weight, reaction conditions, etc. should be understood to be modified by the term "about" in all cases. Therefore, unless otherwise indicated, the numerical parameters set forth in the following specification and the appended claims are approximate values, which may vary depending on the desired properties sought to be obtained by the disclosed subject matter. At a minimum, and without attempting to limit the application of the principle of equivalent scope to the scope of the claims, each numerical parameter should be interpreted at least according to the numerical value of the reported significant digits and by applying ordinary rounding techniques. Although the numerical ranges and parameters setting forth the broad scope of the present invention are approximate values, the numerical values ​​set forth in the specific embodiments are reported as accurately as possible. However, any numerical value inherently contains certain errors caused by the standard deviation found in its respective test measurements.

[0232] As used herein, "substantially free" with respect to a specified component is used herein to mean that any of the specified components are not purposefully formulated into the composition and / or are present only as contaminants or in trace amounts. The total amount of the specified component resulting from any unintentional contamination of the composition is thus well below 0.05%, preferably below 0.01%. Most preferred are compositions in which no amount of the specified component can be detected using standard analytical methods.

[0233] The term "antibody" refers to a complete immunoglobulin or fragment thereof of any isotype that can compete with a complete antibody for specific binding to a target antigen, and includes, for example, chimeric antibodies, humanized antibodies, fully human antibodies, and bispecific antibodies. An "antibody" is an antigen-binding protein. A complete antibody will typically comprise at least two full-length heavy chains and two full-length light chains, but may comprise fewer chains in some cases, such as antibodies naturally present in camelids that may comprise only heavy chains. An antibody may be derived from only a single source or may be "chimeric," i.e., different parts of the antibody may be derived from two different antibodies, as further described below. Antigen-binding proteins, antibodies, or binding fragments may be produced in hybridomas by recombinant DNA technology or by enzymatic or chemical cleavage of a complete antibody. Unless otherwise indicated, the term "antibody" includes, in addition to antibodies comprising two full-length heavy chains and two full-length light chains, derivatives, variants, fragments, and mutant proteins thereof, examples of which are described below. In addition, unless expressly excluded, antibodies include monoclonal antibodies, bispecific antibodies, miniantibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), and fragments thereof, respectively. In some embodiments, the term also encompasses peptibodies.

[0234] Naturally occurring antibody structural units typically comprise tetramers. Each such tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one full-length "light" chain (in certain embodiments, about 25 kDa) and one full-length "heavy" chain (in certain embodiments, about 50-70 kDa). The amino-terminal portion of each chain typically includes a variable region of about 100 to 110 or more amino acids, which is typically responsible for antigen recognition. The carboxyl-terminal portion of each chain typically defines a constant region that may be responsible for effector function. Human light chains are typically divided into kappa and lambda light chains. Heavy chains are typically classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4. Subclasses of IgM include but are not limited to IgM1 and IgM2. IgA is similarly subdivided into subclasses, including but not limited to IgA1 and IgA2. In full-length light and heavy chains, the variable and constant regions are typically joined by a "J" region of about 12 or more amino acids, while the heavy chain also includes a "D" region of about 10 or more amino acids. See, e.g., Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)) (incorporated by reference in its entirety for all purposes). The variable regions of each light chain / heavy chain pair typically form an antigen-binding site.

[0235] The term "variable region" or "variable domain" refers to a portion of the light and / or heavy chains of an antibody, typically including approximately the amino-terminal 120 to 130 amino acids in the heavy chain and approximately 100 to 110 amino-terminal amino acids in the light chain. In certain embodiments, the variable regions of different antibodies vary greatly in amino acid sequence, even between antibodies of the same species. The variable region of an antibody generally determines the specificity of a particular antibody for its target.

[0236] The variable region generally exhibits the same general structure of a relatively conservative framework region (FR) joined by three hypervariable regions, also referred to as complementary determining regions or CDRs. The CDRs from each pair of two chains are generally aligned by the framework region, which can achieve binding to a specific epitope. From N-terminal to C-terminal, the light chain and heavy chain variable regions generally include domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The allocation of amino acids to each domain is generally based on Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), Chothia & Lesk, J. Mol. Biol., 196: 901-917 (1987) or Chothia et al., Nature, 342: 878-883 (1989) definitions.

[0237] In certain embodiments, the antibody heavy chain binds to the antigen in the absence of the antibody light chain. In certain embodiments, the antibody light chain binds to the antigen in the absence of the antibody heavy chain. In certain embodiments, the antibody binding region binds to the antigen in the absence of the antibody light chain. In certain embodiments, the antibody binding region binds to the antigen in the absence of the antibody heavy chain. In certain embodiments, a single variable region specifically binds to the antigen in the absence of other variable regions.

[0238] The clear delineation of CDRs and the identification of residues comprising the antibody binding site can be achieved by solving the structure of the antibody and / or solving the structure of the antibody-ligand complex, which can be achieved by any of the various techniques known to those skilled in the art, such as X-ray crystallography. Various analytical methods can be used to identify or roughly estimate CDR regions. Examples of such methods include, but are not limited to, Kabat definition, Chothia definition, AbM definition, and contact definition.

[0239] The Kabat definition is a standard for numbering residues in antibodies and is commonly used to identify CDR regions. See, for example, Johnson & Wu, Nucleic Acids Res., 28:214-8 (2000). The Chothia definition is similar to the Kabat definition, but the Chothia definition takes into account the position of certain structural loop regions. See, for example, Chothia et al., J. Mol. Biol., 196:901-17 (1986); Chothia et al., Nature, 342:877-83 (1989). The AbM definition uses an integrated set of computer programs produced by the Oxford Molecular Group that model antibody structure. See, for example, Martin et al., Proc Natl Acad Sci (USA), 86:9268-9272 (1989); "AbM TM , A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd. The AbM definition models the tertiary structure of an antibody from the primary sequence using a combination of knowledge databases and ab initio computational methods, such as those described by Samudrala et al., "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," PROTEINS, Structure, Function and Genetics Suppl., 3:194-198 (1999). The contact definition is based on analysis of available complex crystal structures. See, e.g., MacCallum et al., J. Mol. Biol., 5:732-45 (1996).

[0240] By convention, the CDR regions in the heavy chain are usually referred to as H1, H2, and H3, and are numbered sequentially from the amino terminus to the carboxyl terminus. The CDR regions in the light chain are usually referred to as L1, L2, and L3, and are numbered sequentially from the amino terminus to the carboxyl terminus.

[0241] The term "light chain" includes full-length light chains and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length light chain comprises a variable region domain, VL, and a constant region domain, CL. The variable region domain of a light chain is located at the amino terminus of a polypeptide. Light chains include kappa chains and lambda chains.

[0242] The term "heavy chain" includes full-length heavy chains and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length heavy chain comprises a variable region domain, VH, and three constant region domains, CHI, CHI, and CHI. The VH domain is located at the amino terminus of the polypeptide, while the CHI domain is located at the carboxyl terminus, with CHI being closest to the carboxyl terminus of the polypeptide. The heavy chain can be of any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0243] Bispecific or bifunctional antibodies are typically artificial hybrid antibodies with two different pairs of heavy / light chains and two different binding sites. Bispecific antibodies can be produced by a variety of methods, including but not limited to fusion of hybridomas or linking of Fab' fragments. See, for example, Songsivilai et al., Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol., 148:1547-1553 (1992).

[0244] The term "antigen" refers to a substance capable of inducing an adaptive immune response. Specifically, an antigen is a substance that serves as a target for an adaptive immune response receptor. Generally, an antigen is a molecule that binds to an antigen-specific receptor but cannot induce an immune response in the body on its own. Antigens are typically proteins and polysaccharides, and a few are also lipids. As used herein, antigens also include immunogens and haptens.

[0245] The "Fc" region comprises two heavy chain fragments comprising the CH1 and CH2 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions with the CH3 domains.

[0246] The "Fv region" comprises the variable regions from the heavy and light chains, but lacks the constant regions.

[0247] An antibody that "specifically binds to a specific polypeptide or an epitope on a specific polypeptide" or "has specificity for a specific polypeptide or an epitope on a specific polypeptide" is an antibody that binds to the specific polypeptide or an epitope on a specific polypeptide and does not substantially bind to any other polypeptide or polypeptide epitope. For example, the PIV F protein-specific antibody of the present disclosure is specific for PIV F protein. The antibody that binds to PIV F protein may have a specificity of ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., ≤10 -8 M or smaller, such as 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13 The dissociation constant (Kd) of the

[0248] When used in the context of antigen binding proteins (e.g., antibodies or antigen binding fragments thereof) that compete for the same epitope, the term "competition" means competition between antigen binding proteins as determined by an assay in which the antigen binding protein (e.g., antibody or antigen binding fragment thereof) being tested prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., ligand or reference antibody) to a common antigen (e.g., PIV F protein or fragment thereof). Many types of competitive binding assays can be used to determine whether one antigen binding protein competes with another, for example: solid phase direct or indirect radioimmunoassays (RIA), solid phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619); solid phase direct label assays; solid phase direct label sandwich assays (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1992); Press); solid phase direct labeling RIA using I-125 labeling (see, e.g., Morel et al., 1988, Molec. Immunol. 25: 7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung et al., 1990, Virology 176: 546-552); and direct labeling RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32: 77-82). Typically, such assays involve the use of purified antigen bound to a solid surface or cell with either an unlabeled test antigen binding protein and a labeled reference antigen binding protein. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cell in the presence of the test antigen binding protein. Typically, the test antigen binding protein is present in excess. Antigen binding proteins identified by competition assays (competing antigen binding proteins) include antigen binding proteins that bind to the same epitope as the reference antigen binding protein and antigen binding proteins that bind to an epitope that is close enough to the epitope bound by the reference antigen binding protein to cause steric hindrance. Additional details on methods for determining competitive binding are provided in the Examples herein. Typically, when a competing antigen binding protein is present in excess, it will inhibit (e.g., reduce) the specific binding of the reference antigen binding protein to the common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more.In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0249] As used herein, the term "epitope" refers to a specific group of atoms or amino acids on an antigen to which an antibody binds. An epitope can be a linear epitope or a conformational epitope. A linear epitope is formed by a continuous sequence of amino acids from an antigen and interacts with the antibody based on its primary structure. On the other hand, a conformational epitope is composed of discontinuous portions of the antigen's amino acid sequence and interacts with the antibody based on the antigen's 3D structure. Typically, an epitope is approximately five or six amino acids in length. If two antibodies exhibit competitive binding to an antigen, they may bind to the same epitope within the antigen.

[0250] A useful measure of antibody potency in the art is the "50% neutralization titer." Another useful measure of antibody potency is any of the following: "60% neutralization titer"; "70% neutralization titer"; "80% neutralization titer"; and "90% neutralization titer." For example, to determine a 50% neutralization titer, serum from an immunized animal is diluted to assess how well the diluted serum retains the ability to block 50% of infectious virus entry into cells. For example, a titer of 700 indicates that serum retains the ability to neutralize 50% of infectious virus after being diluted 700-fold. Therefore, higher titers indicate a stronger neutralizing antibody response. The titer can be in a range with a lower limit of about 200, about 400, about 600, about 800, about 1000, about 1500, about 2000, about 2500, about 3000, about 3500, about 4000, about 4500, about 5000, about 5500, about 6000, about 6500, or about 7000. The upper limit of the range of 50%, 60%, 70%, 80% or 90% neutralization titer can be about 400, about 600, about 800, about 1000, about 1500, about 2000, about 2500, about 3000, about 3500, about 4000, about 4500, about 5000, about 5500, about 6000, about 6500, about 7000, about 8000, about 9000, about 10000 , about 11,000, about 12,000, about 13,000, about 14,000, about 15,000, about 16,000, about 17,000, about 18,000, about 19,000, about 20,000, about 21,000, about 22,000, about 23,000, about 24,000, about 25,000, about 26,000, about 27,000, about 28,000, about 29,000, or about 30,000. For example, a 50% neutralization titer can be about 3,000 to about 6,500. "About" means plus or minus 10% of the stated value.

[0251] The term "host cell" means a cell that has been transformed or is capable of being transformed with a nucleic acid sequence and expressing a gene of interest. The term includes offspring of a parental cell, whether or not the offspring is identical to the original parental cell in morphology or genetic makeup, as long as the gene of interest is present.

[0252] The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by alignment and comparison of sequences. "Percent identity" means the percentage of identical residues between amino acids or nucleotides in the compared molecules, and is calculated based on the size of the smallest of the compared molecules. For these calculations, gaps in the alignment, if any, are preferably resolved by a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM and Griffin, HG, eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.

[0253] When calculating percent identity, the compared sequences are usually aligned in a manner that gives the maximum match between the sequences. An example of a computer program that can be used to determine percent identity is the GCG program package, which includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12: 387; Genetics Computer Group, University of Wisconsin, Madison, Wis.). The computer algorithm GAP is used to compare two polypeptides or polynucleotides to determine the percent sequence identity. The sequences are aligned to obtain the best match ("match span" of their respective amino acids or nucleotides, as determined by the algorithm). Gap opening penalty (calculated as 3 × average diagonal, where "average diagonal" is the average value of the diagonal of the comparison matrix used; "diagonal" is a score or number assigned to each perfect amino acid match by a specific comparison matrix) and gap extension penalty (typically 1 / 10 times of gap opening penalty), and comparison matrices such as PAM 250 or BLOSUM 62 are used in combination with the algorithm. Standard comparison matrices (see, Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352, for the PAM 250 comparison matrix; Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA 89:10915-10919, for the BLOSUM 62 comparison matrix) can also be used by the algorithm.

[0254] Examples of parameters that can be used to determine percent identity of polypeptide or nucleotide sequences using the GAP program can be found in Needleman et al., 1970, J. Mol. Biol. 48:443-453.

[0255] Certain alignment schemes used to align two amino acid sequences may result in only a short region of the two sequences being matched, and this small aligned region may have a very high sequence identity, even though there is no significant relationship between the two full-length sequences. Therefore, if desired, the alignment method of choice (GAP program) may be adjusted to produce an alignment that spans at least 50 or other numbers of consecutive amino acids of the target polypeptide.

[0256] As used herein, the term "linked" refers to association via intramolecular interactions such as covalent bonds, metallic bonds, and / or ionic bonds or intermolecular interactions such as hydrogen bonds or non-covalent bonds.

[0257] The term "operably linked" refers to an arrangement of elements in which the components so described are configured so as to perform their usual functions. Thus, a given signal peptide operably linked to a polypeptide directs secretion of the polypeptide from a cell. In the case of a promoter, a promoter operably linked to a coding sequence will direct the expression of the coding sequence. A promoter or other control elements need not be adjacent to a coding sequence, as long as they serve to direct its expression. For example, there may be intervening untranslated but transcribed sequences between a promoter sequence and a coding sequence, and the promoter sequence may still be considered to be "operably linked" to the coding sequence.

[0258] The term "or" used in the claims is used to mean "and / or" unless explicitly stated to refer only to alternatives or the alternatives are mutually exclusive, although this disclosure supports a definition referring only to alternatives and "and / or." As used herein, "another" can mean at least a second or more.

[0259] The term "polynucleotide" or "nucleic acid" includes both single-stranded and double-stranded nucleotide polymers. The nucleotides constituting the polynucleotide can be modified forms of ribonucleotides or deoxyribonucleotides or any type of nucleotide. The modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2', 3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, selenophosphate, diselenphosphate, anilinothioate, anilinophosphate, and phosphoramidate.

[0260] The terms "polypeptide" or "protein" refer to a macromolecule having the amino acid sequence of a native protein, i.e., a protein produced by naturally occurring, non-recombinant cells; or a molecule produced by genetically engineered or recombinant cells and comprising a molecule having the amino acid sequence of a native protein, or having one or more amino acids deleted, added, and / or substituted from the native sequence. The terms also include amino acid polymers in which one or more amino acids are chemical analogs of the corresponding naturally occurring amino acids and polymers. The terms "polypeptide" and "protein" specifically encompass PIV F protein-binding proteins, antibodies, or sequences having one or more amino acids deleted, added, and / or substituted from an antigen-binding protein. The term "polypeptide fragment" refers to a polypeptide having amino-terminal deletions, carboxyl-terminal deletions, and / or internal deletions compared to the full-length native protein. Such fragments may also contain modified amino acids compared to the native protein. Fragments may be approximately 5 to 500 amino acids in length. For example, the length of the fragment can be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids. Useful polypeptide fragments include immunologically functional fragments of antibodies, including binding domains. In the case of PIV F protein binding antibodies, useful fragments include, but are not limited to, CDR regions, variable domains of the heavy and / or light chains, a portion of an antibody chain, or only its variable region including two CDRs, etc.

[0261] Pharmaceutically acceptable carriers are conventional. Remington's Pharmaceutical Sciences, EW Martin, Mack Publishing Co., Easton, PA, 15th edition (1975) describes compositions and preparations suitable for drug delivery of fusion proteins disclosed herein. Generally, the nature of the carrier will depend on the specific mode of administration adopted. For example, parenteral formulations generally comprise injectable fluids as vehicles, which include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, dextrose aqueous solutions, glycerol, etc. For solid compositions (e.g., powders, pills, tablets or capsule forms), conventional non-toxic solid carriers may include, for example, pharmaceutical grade mannitol, lactose, starch or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical composition to be administered may contain a small amount of non-toxic auxiliary substances, such as wetting agents or emulsifiers, preservatives and pH buffers, for example sodium acetate or sorbitan laurate.

[0262] As used herein, the term "subject" refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cow, goat, pig, sheep, horse, or primate). Humans include prenatal and postnatal forms. The subject can be a human. The subject can be a patient, which refers to a person presented to a medical provider for diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient." A subject may be suffering from or susceptible to a disease or condition, but may or may not exhibit symptoms of the disease or condition.

[0263] As used herein, the term "therapeutically effective amount" or "effective dose" refers to a dose or concentration of a drug that is effective for treating a disease or condition, for example, with respect to the use of the monoclonal antibodies or antigen-binding fragments thereof disclosed herein to treat viral infections.

[0264] As used herein, "treating" or "treatment" of a disorder includes preventing or alleviating the disorder, slowing the onset or progression of the disorder, reducing the risk of developing the disorder, preventing or delaying the development of symptoms associated with the disorder, reducing or ending symptoms associated with the disorder, causing complete or partial regression of the disorder, curing the disorder, or some combination thereof.

[0265] As used herein, "vector" refers to a nucleic acid molecule that is introduced into a host cell to produce a transformed host cell. A vector may include a nucleic acid sequence that allows it to replicate in the host cell, such as an origin of replication. A vector may also include one or more therapeutic genes and / or selectable marker genes and other genetic elements known in the art. A vector can transduce, transform, or infect a cell, thereby causing the cell to express nucleic acids and / or proteins that are different from those naturally present in the cell. The vector optionally includes materials that facilitate entry of the nucleic acid into the cell, such as viral particles, liposomes, protein coatings, and the like.

[0266] IX. Examples

[0267] The following examples are included to demonstrate preferred embodiments of the present invention. It will be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have found to work well in the practice of the present invention and therefore can be considered to constitute preferred modes for its practice. However, it will be understood by those skilled in the art that, in light of this disclosure, many changes may be made to the disclosed specific embodiments without departing from the spirit and scope of the present invention and still obtaining similar or similar results.

[0268] Example 1

[0269] The sequence used for the hPIV3 F extracellular domain design contained residues 19-481 of SEQ ID NO: 1. To screen for single substitution designs, an L168Q substitution was included to increase solubility and provide more consistent purification. This included the GCN4 CC tri2 trimerization domain. The construct also contained an HRV3C protease recognition site, an octa-histidine tag, and a tandem Twin-Strep tag cloned into the mammalian expression plasmid pαH. The sequence of the base construct with the L168Q substitution is provided in SEQ ID NO: 8, where position 141 is a lysine.

[0270] Using structure-based design, substitutions aimed at favoring prefusion structural stability were introduced into the base construct (PIV3F L168Q extracellular domain with a GCN4 trimerization tag, which adopts a postfusion conformation, Figure 3 ; SEQ ID NO: 8, wherein position 141 is lysine). Pairs of core-facing residues are substituted with aromatic side chains or aromatic and positively charged side chain pairs, respectively, to favor π-π or π-cation interactions. Alternatively, residues are replaced with extended or larger hydrophobic side chains to fill pre-existing internal cavities. Disulfide bonds are designed to increase overall stability or prevent the formation of post-fusion conformations. Charged or polar substitutions are intended to align with predicted The natural residues in the range establish hydrogen bonds or salt bridges.

[0271] The single substitution designs were carried forward to a combinatorial screening round. In this round, the L168Q substitution from the first round was converted back to leucine. For some combinatorial variants, the L168Q substitution was later reintroduced. In addition, the foldon trimerization motif of T4 fibritin is contained between the GCN4 CC tri2 trimerization domain and the HRV3C protease recognition site (SEQ ID NO: 11, where position 141 is lysine).

[0272] Table 1 provides exemplary single substitution designs. Table 2 provides exemplary substitution combinations.

[0273] Plasmids encoding hPIV3 F variants were transiently transfected into FreeStyle293F cells (Thermo Fisher) using polyethyleneimine, and 5 μM kifunensine was added 3 hours after transfection. The cultures were grown for 4-6 days, and the culture supernatants were separated by centrifugation and passed through a 0.22 μm filter. The protein was purified from the supernatant using StrepTactin resin (IBA), and the hPIV3 F variant was further purified by size exclusion chromatography (SEC) using a Superose 6 10 / 300 column (GE Healthcare) in a buffer consisting of 2 mM Tris pH 8.0, 200 mM NaCl, and 0.02% NaN3. For initial purification and characterization, single substitution and combination variants were purified from 40 mL of culture. The proteins were analyzed by SDS-PAGE (e.g., Figure 4 、 8 , 10, 11, 17, 18 and 21) and SEC (e.g., Figure 9 、 12 Protein purity, monodispersity, and expression level were determined by PCR (Figures 1A, 2B, and 2C). The first peak corresponds to hPIV3 F multimers after trimer fusion; the second peak corresponds to monomeric hPIV3 F trimers.

[0274] Negative stain electron microscopy (nsEM) analysis was performed on some hPIV3 F variants. Purified hPIV3 F variants were diluted to a concentration of 0.06 mg / mL in 2 mM Tris pH 8.0, 200 mM NaCl, and 0.02% NaN3. Each protein was deposited on CF-400-CU grids (Electron Microscopy Sciences) that had been plasma cleaned in a Solarus 950 plasma cleaner (Gatan) with an O2 / H2 ratio of 4:1 for 30 seconds and stained with 2% (w / v) uranyl acetate. The images were taken at a magnification of 60,000X (corresponding to 100 μg / mL) in a 2010f TEM (Japan Electron Optical Laboratory) operated at 200 kV and equipped with a OneView camera (Gatan). The grid is imaged with the calibrated pixel size of Figure 6 and 17 shown.

[0275] To confirm that the stabilizing substitutions did not lead to any unexpected conformational changes, cryo-EM structures of various designs were determined ( Figure 3, 13B, 14-16, 19 and 20). Purified hPIV3 F variants were diluted in 2 mM Tris pH 8.0, 200 mM NaCl, 0.02% NaN3 to a concentration range of 1-3 mg / mL and applied to plasma-cleaned CF-400 1.2 / 1.3 grids or UltrAuFoil 1.2 / 1.3 grids, then blotted for 3-6 seconds in a Vitrobot Mark IV (ThermoFisher) and frozen in liquid ethane. Micrographs were collected from individual grids using (i) a Titan Krios (ThermoFisher) equipped with a K3 direct electron detector (Gatan) or (ii) a Glacios (Thermofisher) equipped with a Falcon IV. Data are at the calibrated magnification of the Krios. and Falcon 4 calibrated magnification Collected below.

[0276] ***

[0277] All methods disclosed and claimed herein can be carried out and implemented in accordance with the present disclosure without undue experimentation. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the methods and steps or the order of steps described herein can be changed without departing from the concept, spirit and scope of the present invention. More specifically, it will be apparent that certain agents related to chemistry and physiology can replace the agents described herein while achieving the same or similar results. All similar substitutions and modifications clear to those skilled in the art are considered to be within the spirit, scope and concept of the present invention as defined by the appended claims.

[0278] References

[0279] The following references are specifically incorporated herein by reference, to the extent they provide exemplary procedural or other details supplementary to those set forth herein.

[0280] International Patent Publication No. WO 2018 / 081289

[0281] International Patent Publication No. WO 2022 / 207839

[0282] U.S. Patent Publication No. 2022 / 0024987

Claims

1. An engineered protein comprising a respiratory virus fusion protein (PIV F) an extracellular domain, the engineered protein comprising at least one substitution or a group of substitutions selected from the group consisting of: H27C / F437C; H27C / T439C; H27C / P440C; H27C / I443C; H27Y; V28M; V30I; N33C / K295C; G37C / S337C; S41C / P283C; Y48C / I169C; Y48C / I169C / A140Y; L49C / L278C; L49F; L49W; I50C / A171C; I50C / T277C; I50W; S52C / K173C; S52C / S275C; S52 L; L53C / S174C; P55C / V175C; P55C / Q176C; K56C / N155C; I57F; E58C / I183C; G64C / G196C; G64C / G200C; Q67C / L199C; Q67C / G200C; ;L86C / V266C; Q89C / A131C; K90Y; I93C / G116C; V94C / G116C; T95C / G11 6C; T117P; I118C / G381C; A119C / G381C; L120C / G381C; A123P; T124P; S1 25C / P374C; S125P; S125W; A126P; I128F; I128W; L134C / I267C; A137C / I267C; I144W; L147C / A171C; I151C / A171C; A157C / Q176C; A157C / D177 C; A157F; V158L; Q159C / A171C; L168Q; V170I; V170M; A171V; K173Q; V1 75L; V175P; V179L; E182F; E182W; P185C / A195C; G191P; G200E; I201F; I 201W; A202T; E209W; I213C / I226C; I213C / G230C; G219C / E333C; L228C / V264C; L228F; L228W; R236W; R236Y; S246V; L256Y; V264F; V264W; V26 6F; V266W; S275F; S275M; T277F; T277L; T277W; L278F; L278W; V280F; V 280W; R281Y; L282F; L282W; D327C / P344C; A334S; G345M; F346C / T369C;F346C / S370C; N349P; L356F; S361C / T444C; Q362C / N447C; P364C / N447C; T366C / V449C; T367R; N380C / G433C; G381C / K431C; G382C / G433C; V384I; T413C / A436C; G433F; I443W; I443Y; V449C / I 454C;V449C / D455C;V449C / I456C;V449C / S457C;A450F;L451P;D452P;I454F;D455K;I456W;S457C / V449C;S457C / I456C;K464C / V449C;S470C / K471C;K471A;K471L;W473A, and wherein the positions are relative to SEQ ID NO: 1 or 2. ; 2. The engineered protein of claim 1 , comprising at least one paired cysteine ​​substitution selected from the group consisting of: H27C / F437C; H27C / T439C; H27C / P440C; H27C / I443C; N33C / K295C; G37C / S337C; S41C / P283C; Y48C / I169C; Y48C / I169C / A140Y; L49C / L278C; I50C / A171C; I50C / T277C; S52C / K173C; S52C / S275C; L53C / S174C; P55C / V175C; P55C / Q176C; K56C / N155C; E58C / I183C; G64C / G196C; G64C / G200C; Q67C / L199C; Q67C / G200C; Y71C / L203C; L86C / V266C; Q89C / A131C; I93C / G116C; V94C / G116C; T95C / G116C; 20C / G381C; S125C / P374C; L134C / I267C; A137C / I267C; L147C / A171C; I151C / A171C; A157C / Q176C; A157C / D177C; Q159 C / A171C; P185C / A195C; I213C / I226C; I213C / G230C; G219C / E333C; L228C / V264C; D327C / P344C; F346C / T369C; F346C / S 370C;S361C / T444C;Q362C / N447C;P364C / N447C;T366C / V449C;N380C / G433C;G381C / K431C;G382C / G433C;T413C / A436C;V449C / I454C;V449C / D455C;V449C / I456C;V449C / S457C;S457C / V449C;S457C / I456C;K464C / V449C; and S470C / K471C.

3. The engineered protein of claim 1 or 2, further comprising at least one pair of cysteine ​​substitutions selected from the group consisting of: S186C / A195C.

4. The engineered protein of claim 2 or 3, wherein the paired cysteine ​​substitutions form disulfide bonds.

5. The engineered protein of any one of claims 1-4, comprising at least one cavity filling substitution or a group of cavity filling substitutions selected from the group consisting of: H27Y; V28M; V30I; L49F; L49W; I50W; S52L; I57F; K90Y; A140Y; I144W; A157F; V158L; V170I; V170M; A171V; V175L; V179L; E182F; E182W; G200E; I201F; I20 1W; L228F; L228W; R236Y; S246V; L256Y; V264F; V264W; V266F; V266W; S275F; S275M; T277F; T277L; T277W; L278F; L278W; V280F; V280W; R281Y; L282F; L282W; G345M; L356F; V384I; G433F; I443W; I443Y; A450F; I454F; and I456W.

6. The engineered protein of claim 5, wherein the substitution forms a salt bridge within a substitution pair or between a single substitution and a native amino acid in the protein.

7. The engineered protein of any one of claims 1-6, comprising at least one substitution or a group of substitutions selected from the group consisting of: T117P; A123P; T124P; S125P; A126P; V175P; G191P; N349P; L451P; and D452P.

8. The engineered protein of any one of claims 1-7, comprising at least one substitution or a group of substitutions selected from the group consisting of: S125W; I128F; I128W; E209W; and R236W.

9. The engineered protein of any one of claims 1-8, comprising at least one substitution or a group of substitutions selected from the group consisting of: K173Q; A202T; A334S; T367R; D455K; K471A; K471L; and W473A.

10. The engineered protein of any one of claims 1-9, comprising an E at position 108.

11. The engineered protein of any one of claims 1-10, comprising a combination of at least one engineered disulfide bond and at least one cavity-filling substitution.

12. The engineered protein of any one of claims 1-10, comprising a combination of at least one engineered disulfide bond and at least one proline substitution.

13. The engineered protein of any one of claims 1-10, comprising a set of substitutions selected from the group consisting of: G64C / G196C / V28M; G64C / G196C / V175L; G64C / G196C / V158L; G64C / G196C / A123P; G64C / G196C / S125P; G64C / G196C / I201W; G64C / G196C / L282F; G64C / G196C / L228W; G64C / G196C / R281Y; G64C / G196C / L282W; G64C / G196C / N349P; G64C / G196C / T367R ;G64C / G196C / K471A; A137C / I267C / V28M; A137C / I267C / V175L; A137C / I2 67C / V158L; A137C / I267C / A123P; A137C / I267C / S125P; A137C / I267C / I201 W; A137C / I267C / L282F; A137C / I267C / L228W; A137C / I267C / R281Y; A137C / I267C / L282W; A137C / I267C / N349P; A137C / I267C / T367R; A137C / I267C / K4 71A; L147C / A171C / V28M; L147C / A171C / V175L; L147C / A171C / V158L; L147 C / A171C / A123P; L147C / A171C / S125P; L147C / A171C / I201W; L147C / A171C / L282F; L147C / A171C / L228W; L147C / A171C / R281Y; L147C / A171C / L282W; L 147C / A171C / N349P; L147C / A171C / T367R; L147C / A171C / K471A; G64C / G196 C / A137C / I267C / K471A; G64C / G196C / A137C / I267C / V175L; G64C / G196C / A 137C / I267C / S125P; G64C / G196C / A137C / I267C / S125P / V175L; G64C / G196C / A137C / I267C / T367R; G64C / G196C / A137C / I267C / K471A / S125P; G64C / G19 6C / A137C / I267C / K471A / S125P / T367R / V175L; G64C / G196C / A137C / I267C;G64C / G196C / A137C / I267C / K471A / S125P / L282F / V175L;G64C / G196C / L147C / A171C / K471A / S125P / L282F / V175L;G64C / G196C / L147C / A171C / A137C / I267C / K471A / S125P / L282F / V175L;G64C / G196C / L147C / A171C;G64C / G196C / L147C / A171C / V28M / V175L / I201W / L228W / S125P / T367R / K471A;G64C / G196C / A137C / I267C / V28M / V175L / I201W / L228W / S125P / T367R / K471A;G64C / G196C / L147C / A171C / A137C / I267C / V28M / V175L / I201W / L228W / S125P / T367R / K471A;G64C / G196C / I151C / A171C / A137C / I267C;G64C / G196C / I151C / A171C / A137C / I267C / I231C / G230C;G64C / G196C / A137C / I267C / V28M / V175L / I201W / L228W / R281Y;G64C / G196C / A137C / I267C / V28M / V175L / I201W / L282F / L228W / R281Y;I151C / A171C / V449C / S457C;L168Q / G64C / G196C / A137C / I267C / K471A;L168Q / G64C / G196C / A137C / I267C / V175L;L168Q / G64C / G196C / A137C / I267C / S125P;L168Q / G64C / G196C / A137C / I267C / S125P / V175L;L168Q / G64C / G196C / A137C / I267C / T367R;L168Q / G64C / G196C / A137C / I267C / K471A / S125P;L168Q / G64C / G196C / A137C / I267C / K471A / S125P / T367R / V175L;L168Q / G64C / G196C / A137C / I267C;L168Q / G64C / G196C / A137C / I267C / K471A / S125P / L282F / V175L;L168Q / G64C / G196C / I151C / A171C / A137C / I267C;L168Q / G64C / G196C / I151C / A171C / A137C / I267C / I231C / G230C; L168Q / G64C / G19 6C / A137C / I267C / V28M / V175L / I201W / L228W / R281Y; L168Q / G64C / G196C / A137C / I267C / V28M / V175L / I201W / L282F / L228W / R281Y; L168Q / I151C / A171C / V449C / S4 57C; L168Q / I151C / A171C / V449C / S457C / V28M / V175L / R281Y; I151C / A171C / S186 C / A195C / V28M / V175L / R281Y; L168Q / I151C / A171C / L228W / L282F / R821Y / V175L; I151C / A171C / L228W / L282F / R821Y / V175L; I151C / A171C / L228W / L282F / R821Y / V 175L / G200E / I57F; I151C / A171C / L228W / L282F / R821Y / V175L / G200E / I57F / V449C / S457C; I151C / A171C / G200E / I57F; and I151C / A171C / G200E / I57F / V449C / S457C. ; 14. The engineered protein of any one of claims 1-13, wherein the substitution or substitution group is selected from any one of the substitutions and substitution groups of Tables 1 and 2.

15. The engineered protein of any one of claims 1-14, comprising an L168Q substitution.

16. The engineered protein of any one of claims 1-15, wherein the PIV F extracellular domain is a human PIV (hPIV) F extracellular domain.

17. The engineered protein of any one of claims 1-16, wherein the human PIV F extracellular domain is the hPIV3 F extracellular domain.

18. The engineered protein of any one of claims 1-17, wherein the protein does not comprise the cytoplasmic tail of PIV F.

19. The engineered protein of any one of claims 1-18, wherein the protein is fused to or conjugated to a trimerization domain.

20. The engineered protein of claim 19, wherein the protein is fused to a trimerization domain.

21. The engineered protein of claim 19 or 20, wherein the trimerization domain comprises a T4 fibrin trimerization domain, a GCN4 domain, a 4J4A domain, or a combination thereof.

22. The engineered protein of any one of claims 19-21, wherein the trimerization domain comprises a sequence selected from the group consisting of: LKQIVLRIMEIEARIAKI AKIEGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG,LKQIVLRIMEI EARIAKIEGSEFNSLKQIVLRIMEIEARIAKIE,LKQIVLRIMEIEARIAKIEGSLKQIVLRIMEIEARIAKIE,LKQIVLRIMEIEARIAKIEGSLE LIKLRIMEIEARIAKIEKDRAIL.

23. The engineered protein of any one of claims 1-22, wherein the protein is fused to or conjugated to a transmembrane domain.

24. The engineered protein of claim 23, wherein the protein is fused to a transmembrane domain.

25. The engineered protein of claim 23 or 24, wherein the transmembrane domain comprises a PIV F protein transmembrane domain.

26. The engineered protein of claim 25, wherein the PIV F protein transmembrane domain comprises the sequence IIIILIMMIILFIINITIITI.

27. The engineered protein of claim 23 or 24, wherein the transmembrane domain does not comprise a PIV F protein transmembrane domain.

28. The engineered protein of any one of claims 1-27, comprising an N-terminal signal sequence.

29. The engineered protein of any one of claims 1-28, wherein the protein exhibits improved solubility or stability compared to native PIV F in a post-fusion conformation.

30. The engineered protein of any one of claims 1-29, wherein the protein is immunogenic.

31. An engineered parainfluenza virus fusion protein (PIV F) trimer comprising three engineered proteins according to any one of claims 1 to 30.

32. The engineered trimer of claim 31 , wherein the trimer is stabilized in a prefusion conformation relative to a trimer of native PIV F protein subunits.

33. The engineered trimer of claim 31 or 32, wherein the trimer comprises at least one engineered disulfide bond between subunits.

34. The engineered trimer of claim 33, wherein the trimer comprises at least one engineered disulfide bond between subunits selected from the group consisting of: I118C / G381C; A119C / G381C; L120C / G381C; S125C / P374C; G219C / E333C; F346C / T369C; F346C / S370C; and V449C / S457C.

35. A nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of the engineered protein of any one of claims 1-30.

36. The nucleic acid molecule of claim 35, wherein the nucleic acid molecule further comprises a DNA expression vector.

37. The nucleic acid molecule of claim 35, wherein the nucleic acid molecule is mRNA.

38. The nucleic acid molecule of claim 35, wherein the nucleic acid molecule is a self-replicating RNA molecule.

39. The nucleic acid molecule of any one of claims 35-38, further comprising at least one chemical modification.

40. The nucleic acid molecule of claim 39, wherein the at least one chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thiol-1-methyl-1-deaza-pseudouridine, 2-thiol-1-methyl-pseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxy-pseudouridine, 4-thiol-1-methyl-pseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and 2'-0-methyluridine.

41. A pharmaceutical composition comprising (i) the engineered protein of any one of claims 1-30, (ii) the engineered trimer of any one of claims 31-34, or (iii) the nucleic acid molecule of any one of claims 35-40; and a pharmaceutically acceptable carrier.

42. The pharmaceutical composition of claim 41, further comprising an adjuvant.

43. The pharmaceutical composition of claim 41 or 42, comprising another PIV antigen.

44. The pharmaceutical composition of any one of claims 41-43, wherein the composition is formulated within cationic lipid nanoparticles.

45. A method for preventing parainfluenza virus (PIV) infection or a disease associated with PIV infection in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of any one of claims 41-44.

46. ​​A method of eliciting an immune response in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition of any one of claims 41-44.

47. A method for reducing parainfluenza virus (PIV) viral shedding in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition of any one of claims 41-44.

48. The pharmaceutical composition of any one of claims 41-44, for use in treating or preventing parainfluenza virus (PIV) infection or a disease associated with PIV infection in a subject.

49. The method of any one of claims 45-47 or the pharmaceutical composition of claim 48, wherein the subject is a mammal.

50. The pharmaceutical composition of any one of claims 41-44, for use in eliciting an immune response against parainfluenza virus (PIV).

51. Use of the pharmaceutical composition of any one of claims 41 to 44 in the manufacture of a medicament for treating or preventing parainfluenza virus (PIV) infection or a disease associated with PIV infection.

52. A composition comprising (i) the engineered protein of any one of claims 1-30 or (ii) the engineered trimer of any one of claims 31-34 bound to an antibody.

53. The composition of claim 52, wherein the antibody specifically binds to the PIV F extracellular domain in the prefusion conformation.

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