Prefusion-stabilized human parainfluenza virus 3F protein

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 for using the engineered PIV F polypeptides as diagnostics, in screening platforms, and / or in vaccine compositions.

Description

[Technical Field]

[0001] REFERENCE TO RELATED APPLICATIONS 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.

[0002] Sequence Listing Reference This application contains an electronically submitted Sequence Listing XML, which is incorporated herein by reference in its entirety. The Sequence Listing XML, created on January 5, 2024, is named UTFBP1325WO_ST26.xml and is 32,897 bytes in size.

[0003] 1.Technical Field 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. [Background technology]

[0004] 2. Description of Related Technology Parainfluenza viruses 1, 2, 3, and 4 (PIV1-4) cause mild to severe respiratory illness in humans. PIVs belong to the Paramyxoviridae family and are enveloped, single-stranded, negative-strand RNA (ssRNA) viruses. Seasonal PIV infections are associated with approximately 40% of children hospitalized with lower respiratory tract infections and approximately 75% of diagnosed cases of croup. PIV infections can occur throughout life. While most adults are at low risk for severe illness from PIV infection, infection in immunocompromised individuals and the elderly can lead to severe or life-threatening lower respiratory tract illness. The development of a vaccine against PIV is important for public health, but no vaccines have been licensed to date.

[0005] Long-term surveillance of respiratory viruses in the United States has identified PIV3 as the most common PIV serotype associated with symptomatic disease in children and adults, followed by PIV1, 2, and 4. The PIV genome encodes several envelope glycoproteins, one of which is the fusion glycoprotein (F). The F glycoprotein is a fusogen required for viral entry into cells and is an important target for the neutralizing antibody (nAb) response to infection. PIV F proteins share varying degrees of sequence conservation with exemplary F proteins from the Respirovirus and Orthorubulavirus genera, ranging from 20-50% identity and 35-65% similarity. While PIV vaccines incorporating the F subunit antigen are under development, no PIV vaccines or antiviral therapeutics are approved for human use. Therefore, vaccines that provide durable protection and therapies that avoid mortality and morbidity are desirable.

[0006] Therefore, there is a need for safe and effective immunogenic compositions and therapeutics to protect against PIV infection and its associated sequelae. There is also a need for diagnostic reagents to detect immune responses to PIV, to guide the design of F-based PIV vaccines, and to aid in the development of therapeutic or prophylactic antibodies against PIV. Summary of the Invention

[0007] overview Thus, provided herein are engineered proteins (i.e., SEQ ID NOS: 1-7) having at least one amino acid substitution compared to the amino acid sequence of a native respirovirus or orthorubulavirus F protein, wherein the engineered proteins are stabilized in the respirovirus or orthorubulavirus F prefusion conformation. The engineered proteins can specifically bind to a respirovirus or orthorubulavirus F protein prefusion-specific antibody.

[0008] In one embodiment, provided herein is an engineered protein comprising a respirovirus or orthorubulavirus fusion protein (preferably a parainfluenza virus fusion protein (PIV F)) ectodomain having at least 90% sequence identity to amino acids 19-481 of SEQ ID NO: 1 or 2, the engineered protein comprising one of the following sequences: 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 / K17 3C, S52C / S275C, S52L, L53C / S174C, P55C / V175C, P55C / Q176C, K56C / N155C, I57F, E58C / I183C, G64C / G196C, G64C / G200C, Q67C / L199 C, Q67C / G200C, Y71C / L203C, L86C / V266C, Q89C / A131C, K90Y, I93C / G116C, V94C / G116C, T95C / G116C, T117P, I118C / G381C, A119C / G38 1C, L120C / G381C, A123P, T124P, S125C / P374C, S125P, S125W, A126P, I128F, I128W, L134C / I267C, A137C / I267C, I144W, L147C / A171C , I151C / A171C, A157C / Q176C, A157C / D177C, A157F, V158L, Q159C / A171C, L168Q, V170I, V170M, A171V, K173Q, V175L, V175P, V179L, E 182F, E182W, P185C / A195C, G191P, G200E, I201F, I201W, A202T, E209W, I213C / I226C, I213C / G230C, G219C / E333C, L228C / V264C, L22 8F, L228W, R236W, R236Y, S246V, L256Y, V264F, V264W, V266F, V266W, S275F, S275M, T277F, T277L, T277W, L278F, L278W, V280F, V280W,R281Y, L282F, L282W, D327C / P344C, A334S, G345M, F346C / T369C, F346C / S370C, N349P, L356F, S361C / T444C, Q362C / N447C, P 364C / N447C, T366C / V449C, T367R, N380C / G433C, G381C / K431C, G382C / G433C, V384I, T413C / A436C, G433F, I443W, I443Y, V4 and at least one substitution or set of substitutions selected from the group consisting of 49C / I454C, V449C / D455C, V449C / I456C, V449C / S457C, A450F, L451P, D452P, I454F, D455K, I456W, S457C / V449C, S457C / I456C, K464C / V449C, S470C / K471C, K471A, K471L, W473A, wherein the positions are relative to SEQ ID NO: 1 or 2.

[0009] The engineered proteins were 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, and P55C / Q177C. 6C, K56C / N155C, E58C / I183C, G64C / G196C, G64C / G200C, Q67C / L199C, Q67C / G200C, Y71C / L203C, L86C / V266C, Q89C / A131C , I93C / G116C, V94C / G116C, T95C / G116C, I118C / G381C, A119C / G381C, L120C / G381C, S125C / P374C, L134C / I267C, A137C / I2 67C, L147C / A171C, I151C / A171C, A157C / Q176C, A157C / D177C, Q159C / A171C, P185C / A195C, I213C / I226C, I213C / G230C, G 219C / E333C, L228C / V264C, D327C / P344C, F346C / T369C, F346C / S370C, S361C / T444C, Q362C / N447C, P364C / N447C, T366C / The engineered protein may comprise, or may further comprise, at least one set of paired cysteine ​​substitutions selected from the group consisting of 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. The engineered protein may further comprise the paired cysteine ​​substitutions S186C / A195C. The paired cysteine ​​substitutions preferably form a disulfide bond.

[0010] The engineered proteins were H27Y, V28M, V30I, L49F, L49W, I50W, S52L, I57F, K90Y, A140Y, I144W, A157F, V158L, V170I, V170M, A171V, V175L, V179L, E182F, E182W, G200E, I201F, I201W, L228F, L228W, R236Y, S246V, L256Y, V264F, V264W, V266F, The substitutions may comprise, or may further comprise, at least one cavity-filling substitution or set of cavity-filling substitutions selected from the group consisting of: V266W, S275F, S275M, T277F, T277L, T277W, L278F, L278W, V280F, V280W, R281Y, L282F, L282W, G345M, L356F, V384I, G433F, I443W, I443Y, A450F, I454F, and I456W. The substitutions may form salt bridges within the pair of substitutions or between the single substitution and the native amino acid in the protein.

[0011] The engineered protein may comprise, or may further comprise, at least one substitution or set of substitutions selected from the group consisting of T117P, A123P, T124P, S125P, A126P, V175P, G191P, N349P, L451P, and D452P.

[0012] The engineered protein may comprise, or may further comprise, at least one substitution or set of substitutions selected from the group consisting of S125W, I128F, I128W, E209W, and R236W.

[0013] The engineered protein may comprise, or may further comprise, at least one substitution selected from the group consisting of K173Q, A202T, A334S, T367R, D455K, K471A, K471L, and W473A.

[0014] The engineered protein may contain, or may further contain, an E at position 108.

[0015] 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.

[0016] The engineered proteins were 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, and A137C / I267C / V28M. 137C / I267C / V175L, A137C / I267C / V158L, A137C / I267C / A123P, A137C / I26 7C / S125P, A137C / I267C / I201W, A137C / I267C / L282F, A137C / I267C / L228W, A137C / I267C / R281Y, A137C / I267C / L282W, A137C / I267C / N349P, A137C / I2 67C / T367R, A137C / I267C / K471A, L147C / A171C / V28M, L147C / A171C / V175L, L147C / A171C / V158L, L147C / A171C / A123P, L147C / A171C / S125P, L147C / A1 71C / I201W, L147C / A171C / L282F, L147C / A171C / L228W, L147C / A171C / R281Y , L147C / A171C / L282W, L147C / A171C / N349P, L147C / A171C / T367R, L147C / A 171C / K471A, G64C / G196C / A137C / I267C / K471A, G64C / G196C / A137C / I267C / V175L, G64C / G196C / A137C / I267C / S125P, G64C / G196C / A137C / I267C / S125 P / V175L, G64C / G196C / A137C / I267C / T367R, G64C / G196C / A137C / I267C / K47 1A / S125P, G64C / G196C / A137C / I267C / K471A / S125P / T367R / V175L, G64C / G1 96C / 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 / G196C / A137C / I267C / V28M / V175L / I201W / L228W / R281Y, L168Q / G64C / G196C / A137C / I267C / V28M / V175L / I201W / L282F / L228W / R281Y, L168 Q / I151C / A171C / V449C / S457C, L168Q / I151C / A171C / V449C / S457C / V28M / V1 75L / R281Y, I151C / A171C / S186C / A195C / V28M / V175L / R281Y, L168Q / I151C / A and I151C / A171C / G200E / I57F, and I151C / A171C / G200E / I57F, and I151C / A171C / G200E / I57F, and I151C / A171C / G200E / I57F, and I151C / A171C / G200E / I57F, and I151C / A171C / G200E / I57F, and I151C / A171C / G200E / I57F, and I151C / A171C / G200E / I57F, and I151C / A171C / G200E / I57F, and I151C / A171C / G200E / I57F, and I151C / A171C / G200E / I57F / V449C / S457C, and

[0017] The engineered protein may include a substitution or set of substitutions selected from any one of the substitutions and sets of substitutions in Tables 1 and 2. Any substitution or set of substitutions may be further combined with the L168Q substitution.

[0018] The ectodomain of a respirovirus or orthorubulavirus fusion protein (preferably a parainfluenza virus fusion protein (PIV F)) of the engineered protein can be the ectodomain of human PIV (hPIV) F. The human PIV F ectodomain can be an hPIV3 F ectodomain. The hPIV3 F ectodomain 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.

[0019] The engineered protein may not include the PIV F cytoplasmic tail.

[0020] The engineered protein can be fused or conjugated to a trimerization domain. The trimerization domain can include a T4 fibritin trimerization domain, a GCN4 domain, a 4J4A domain, or a combination thereof. The trimerization domain can include a sequence selected from the group consisting of: TIFF2026503061000002.tif57146.

[0021] The engineered protein can be fused or conjugated to a transmembrane domain. The transmembrane domain can include the transmembrane domain of the PIV F protein. The transmembrane domain of the PIV F protein can include the sequence IIIILIMMIILFIINITIITI. The transmembrane domain may not include the transmembrane domain of the PIV F protein.

[0022] The engineered protein may include an N-terminal signal sequence.

[0023] The engineered protein may exhibit improved solubility or stability compared to native PIV F in the post-fusion conformation. The engineered protein may be immunogenic.

[0024] In one embodiment, provided herein is an engineered respirovirus or orthorubulavirus fusion protein (preferably, parainfluenza virus fusion protein (PIV F)) trimer comprising three engineered proteins disclosed herein. The trimer may be stabilized in a prefusion conformation compared to a trimer of native PIV F protein subunits. The trimer may contain at least one engineered disulfide bond between the subunits. The trimer may contain at least one engineered disulfide bond between the subunits selected from the group consisting of I118C / G381C, A119C / G381C, L120C / G381C, S125C / P374C, G219C / E333C, F346C / T369C, F346C / S370C, and V449C / S457C.

[0025] In one embodiment, the present disclosure provides a nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of the engineered protein disclosed herein.The nucleic acid molecule may further comprise 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-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-0-methyluridine.

[0026] 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 an additional PIV antigen. The pharmaceutical composition may be formulated in cationic lipid nanoparticles. The pharmaceutical composition may be for use in treating or preventing parainfluenza virus (PIV) infection or a disease associated with PIV infection in a subject, or for use in eliciting an immune response against parainfluenza virus (PIV). The subject may be a mammal, such as a human.

[0027] In one embodiment, provided herein is a method for preventing parainfluenza virus (PIV) infection or a disease associated with PIV infection in a subject, or for inducing an immune response in a subject, or for reducing PIV viral shedding in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition provided herein. The subject may be a mammal, such as a human.

[0028] 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 the treatment or prevention of a parainfluenza virus (PIV) infection or a disease associated with PIV infection.

[0029] In one embodiment, provided herein is a composition comprising (i) an engineered protein provided herein, or (ii) an engineered trimer provided herein, bound to an antibody, wherein the antibody can specifically bind to the PIV F ectodomain in a prefusion conformation.

[0030] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that this detailed description and specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0031] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The 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.

[0032] [Figure 1] Matrix of sequence identity and similarity for representative Respiroviruses and Orthorubulaviruses. 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 Orthorubulavirus genera. Points in each matrix are colored by a percentage value ranging from 0% to 100% [right scale]. Similarity and identity were scored using the SIAS server provided by the Immunomedicine Group at the Universidad Complutense de Madrid, Secretariat General de Ciencia, Tecnologia e Innovation of Spain [http: / / imed.med.ucm.es / Tools / sias.html]. [Figure 2] Clustal sequence alignment of the fusion proteins of human respiroviruses 1 and 3, also known as HPIV1 and HPIV3. The sequence identity in the soluble ectodomain is 44%, with sequence similarity exceeding 60%. Identical residues at homologous positions are colored white on a black background, while similar amino acids are colored black on a gray background. Dissimilar residues at homologous sites are colored black on a white background. [Figure 3] Cryo-EM structure of PIV3 F-based (L168Q) with a C-terminal GCN4 tag in the post-fusion conformation. (Top left panel) Representative cryo-EM image. (Top right panel) Representative 2D class average. (Bottom left panel) Gold standard Fourier shell correlation plot. GSFSC is shown in Angstroms as a function of resolution. Resolution values ​​correspond to an FSC cutoff of 0.143. (Bottom right panel) Coulomb potential map of PIV3 F-based trimer in the post-fusion conformation. [Figure 4] SDS-PAGE of single-substitution PIV3 F protein purified by affinity chromatography and analyzed by reducing SDS-PAGE (Coomassie stain). Variant IDs are indicated at the top of each lane. Integrated band intensities were quantified using either ImageJ / Fiji or Licor Odyssey CLx. Molecular weight markers are included on the left side of each gel. The bottom panel shows both the affinity chromatography flow-through [FT] and elution [E]. [Figure 5] Biolayer interferometry quantification of HPIV3 F variant binding to the prefusion-specific antibody PIA174 IgG. Each panel shows the response (nm) as a function of time (sec) for PIV3 F variant binding to the AHC tip. The AHC tip was functionalized with PIA174 IgG, which recognizes prefusion PIV3 F. [Figure 6] Negative stain electron microscopy of disulfide variants JM-17 (I50C / A171C) and JM-20 (A137C / I1267C) in prefusion conformations. The top of each panel provides representative 2D class averages from negative stain EM images of JM-17 [left] and JM-20 [right]. Box sizes are indicated below the 2D classes and equal 230 Å. The bottom of each panel provides 3D reconstructions of JM-17 [left] and JM-20 [right] in prefusion conformations. [Figure 7]Quantification of relative expression yields of HPIV3 F variants by biolayer interferometry. Each graph shows the response (nm) as a function of time (seconds) for binding of HPIV3 F variants to the AHC tip. The AHC tip was functionalized with MF5 IgG, which recognizes the foldon tag. Binding curves were fitted to a line over the initial portion of the curve, which corresponds to a 60-second window after immersion in medium containing each variant derived from small-scale HEK293F expression cultures. [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 using a Licor Odyssey CLx. [Figure 9] Size-exclusion chromatography of PIV3 F variants. Each graph shows mAU as a function of elution volume for a set of PIV3 F variants. All samples shown in this figure were transfected and purified in parallel under identical conditions. Briefly, PIV3 F variants were purified by streptactin Sepharose, then concentrated and flash-frozen in liquid nitrogen. Samples were thawed and run sequentially on the same Superose 6 size-exclusion chromatography (SEC) column. For ease of comparison, the UV trace of the base construct sample, L168Q, is reproduced in each graph. [Figure 10] Additional SDS-PAGE quantification of combination variants containing tandem GCN4 / foldon tags at the C-terminus. Integrated band intensities for groups of gels of individual and combination variants are plotted and quantified with a Licor Odyssey CLx. [Figure 11] SDS PAGE after size exclusion chromatography of PIV3F combination variants 41 and 43. Large-scale expression (500 mL) of the two combination variants 41 and 43 was first purified by streptactin affinity chromatography followed by size exclusion chromatography (Superose 6). [Figure 12] Size-exclusion chromatography of PIV3F combination variants 43, 56, 57, and 58. Superose 6 size-exclusion elution profile of the set of combination variants, plotting absorbance units at 280 nanometers [mAU] as a function of elution volume in milliliters [mL]. In the inserted table, the expressed protein yield [mg / mL] and apparent melting temperature (Tmapp, in °C) are shown next to each variant ID. Tmapp was measured by differential scanning fluorimetry. The combination variant mutations are listed in Table 2. [Figure 13] Figures 13A-13B: Size-exclusion chromatography and cryo-EM of 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. Panels show a 2D class average, a 3D reconstruction at 3.3 Å, and a close-up of the model within the Coulomb potential map. [Figure 14] Cryo-EM structure of PIV3 F base (L168Q) with C-terminal tandem GCN4 / foldon tags in the prefusion conformation without any prefusion-specific antibodies. (Top left panel) Representative cryo-EM image. (Top right panel) Representative 2D class average. (Bottom left panel) Gold standard Fourier shell correlation plot. GSFSC is shown in Angstroms as a function of resolution. Resolution values ​​correspond to an FSC cutoff of 0.143. (Bottom right panel) Coulomb potential map or Coulomb potential map of PIV3 F base with C-terminal tandem GCN4 / foldon tags in the trimeric prefusion conformation. PIV3 F protomers are colored blue, red, and green. The extended portion of the structure stabilized by the C-terminal tag is colored gray. [Figure 15]Cryo-EM structure of PIV3 F combination variant 43 (Combo 43) in the prefusion conformation complexed with the prefusion-specific antibody PIA174. (Top left panel) Representative cryo-EM image. (Top right panel) Representative 2D class average. (Middle left panel) Gold standard Fourier shell correlation plot. GSFSC is shown in Angstroms as a function of resolution. Resolution values ​​correspond to an FSC cutoff of 0.143. (Middle right panel) Coulomb potential map of PIV3 F variant Combo 43 (I151C / A171C) with C-terminal tandem GCN4 / foldon tags in the trimeric prefusion conformation. PIV3 F Combo 43 protomers are colored blue, red, and green. The extended portion of the structure stabilized by the C-terminal tag is colored gray. (Bottom panel) A magnified view containing all-atom models of exemplary substitutions in the Coulomb potential map (shown in gray). Atoms are colored according to substitution type: disulfide, proline, and cavity filling. [Figure 16] Cryo-EM of PIV variant I93C / G116C. The panels show representative micrographs, 2D class averages, and 6 Å reconstructions of PIV F variant I93C / G116C using wild-type PIV F as background (Leu at position 168). [Figure 17] Biochemical and structural characterization of PIV3 F variant PB-68, V449C / S457C, which forms interprotomer disulfide bonds. (Top left panel) Non-reducing SDS-PAGE analysis of PIV3 variant PB-68, V449C / S457C, which behaves as a disulfide-bonded trimer. A control PIV3 variant that does not form interprotomer disulfide bonds and behaves as a single protomer was included. (Top right panel) Representative 2D class average of PIV3 variant PB-68 obtained from negative stain electron microscopy. (Bottom right panel) 3D negative stain EM reconstruction of PB-68. (Bottom left panel) Close-up of modeled substitution sites near heptad repeats. [Figure 18]Figures 18A-18B: Sample stability study of PIV F variant PB-68, 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) Separate preparations of V449C / S457C protein were 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-reduced trimer. A small amount of the sample ran at the size expected for protomers under non-reducing conditions (low molecular weight band, near the 71 kDa MW marker), which may also form intra-protomer disulfide bonds. [Figure 19] Figures 19A-19B: Four cryo-EM structures of PIV F Combo variants 41 and 58, each in two oligomeric states. Representative class-averaged and Coulomb potential maps for the trimer and dimer of trimers of (Figure 19A) Combo variant 41 and (Figure 19B) Combo variant 58. The resolution of each structure is indicated next to the map. [Figure 20] Figures 20A-20B: Variants exhibit a mixture of closed and open conformations at the PIA174 binding site, which can be biased toward the closed conformation by substitutions at the central trimer interface. (Figure 20A) Combo41 Coulomb potential maps of four unique conformations, varying from open to closed, showing conformational heterogeneity in the central vertex region (black arrows). (Figure 20B) Combo58 Coulomb potential map (3.4 Å) in the closed conformation. Despite extensive efforts to classify based on conformational heterogeneity in the central vertex region (arrow), no classes were observed in the open conformation. Atomic model and zoom of Combo58 with substitution position 201. [Figure 21]SDS-PAGE of wild-type, single, and combination variants of PIV3 F. 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. Both variants containing the V449C / S457C substitutions (Combo61-0-2 and Combo62-0-1) show the expected interprotomer disulfide-bonded trimer bands. [Figure 22] Size-exclusion chromatographs of wild-type, single, and combination variants of PIV3 F. Each graph shows mAU as a function of elution volume for a set of PIV3 F variants. All samples shown in this figure were transfected and purified in parallel under identical conditions. Briefly, PIV3 F variants were purified by streptactin Sepharose, then concentrated and flash-frozen in liquid nitrogen. Samples were thawed and run sequentially on the same Superose 6 column. For ease of comparison, the UV trace of the wild-type PIV3 F sample is reproduced in each panel. [Figure 23] Analysis of thermostability of wild-type, single, and combination variants of PIV3 F. Purified PIV F proteins, purified in parallel under identical conditions, were analyzed by differential scanning fluorimetry. Plots show the change in fluorescence versus temperature on the y-axis and temperature on the x-axis. Variants are plotted in groups, and wild-type DSF traces are reproduced in each panel for ease of comparison. DETAILED DESCRIPTION OF THE INVENTION

[0033] Detailed Description Provided herein are engineered parainfluenza virus (PIV) fusion (F) proteins with one or more amino acid substitutions that stabilize the PIV F protein in a prefusion conformation. The prefusion PIV F can be used as a vaccine antigen or a reagent for detecting and / or isolating antibodies in serum. The prefusion PIV F proteins described herein, and nucleic acids encoding the proteins, can be used, among other uses, for example, in methods of inducing an immune response in a subject, as potential immunogens in immunogenic compositions or vaccines against PIV, and as diagnostic tools.

[0034] I. Natural hPIV F The envelope glycoprotein of hPIV1, hPIV2, hPIV3, or hPIV4 promotes fusion of the viral membrane with the cellular membrane. In nature, the F proteins from hPIV1, hPIV2, hPIV3, and hPIV4 are initially synthesized as a single polypeptide precursor, approximately 550 amino acids long, designated F0. F0 contains an N-terminal signal peptide that directs localization to the endoplasmic reticulum, where the signal peptide is proteolytically cleaved. The remaining F0 residues can oligomerize to form trimers and be proteolytically processed by cellular proteases to generate 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 (approximately residues 22-113 in hPIV1, approximately residues 22-106 in hPIV2, approximately residues 19-109 in hPIV3, and approximately residues 21-103 in hPIV4). The larger of these fragments, F1, contains the C-terminal portion of the F0 precursor (approximately residues 114-555 in hPIV1, approximately residues 107-551 in hPIV2, approximately residues 110-539 in hPIV3, and approximately residues 104-544 in hPIV4), the extracellular / luminal region (approximately residues 114-497 in hPIV1, approximately residues 107-493 in hPIV2, approximately residues 110-493 in hPIV3, and approximately residues 104-486 in hPIV4), the transmembrane domain (approximately residues 498-518 in hPIV1, approximately residues 494-514 in hPIV2, approximately residues 494-514 in hPIV3, and approximately residues 487-507 in hPIV4), and the C-terminal cytoplasmic tail. The extracellular portion of the hPIV F protein is the hPIV F ectodomain, which includes the F2 protein and the F1 ectodomain.

[0035] The hPIV F protein shows remarkable sequence conservation within hPIV subtypes and other members of the Respirovirus and Orthorubulavirus genera (Figure 1). Given this conservation, one of skill in the art can readily compare amino acid positions between different hPIV F proteins of the same subtype or with F proteins from other members of the Respirovirus and Orthorubulavirus genera. Unless the context dictates otherwise, the numbering of amino acid substitutions disclosed herein is done with reference to SEQ ID NO: 1 (GenBank AGW51052.1) or 2 (SWISS-PROT: P06828.2) for hPIV3 (also known as human respirovirus 3) F, unless otherwise indicated.

[0036] Thus, the term PIV F polypeptide as used herein should be understood to refer to native PIV F polypeptides from any PIV strain (including but not limited to the human PIV3 strain), as well as any F protein from other members of the Respirovirus and Orthorubulavirus genera. Actual residue position numbers may need to be adjusted for F proteins from other strains depending on the actual sequence alignment. Additional viral F proteins to which the amino acid substitutions of the present disclosure can be applied include, for example, hPIV1 F (also known as human respirovirus 1) (GenBank BAS30410.1, SEQ ID NO: 3), hPIV2 (also known as human orthorubulavirus 2) F (GenBank AAA46842.1, SEQ ID NO: 4), hPIV4 (also known as human orthorubulavirus 4) F (GenBank AGU90035.1, SEQ ID NO: 5), Mumps orthorubulavirus F (GenBank BAA94388.1, SEQ ID NO: 6), and PIV5 (also known as mammalian orthorubulavirus 5) F (GenBank AAC95515.1, SEQ ID NO: 7).

[0037] II. PIV F protein stabilized in the prefusion conformation The three PIV F promoters oligomerize into the mature F protein, which adopts a metastable prefusion conformation that undergoes a conformational change to a postfusion conformation upon contact with the target cell membrane. This conformational change exposes a hydrophobic sequence known as the fusion peptide, which is located at the N-terminus of the F1 ectodomain and associates with the host cell membrane, promoting fusion of the viral or infected cell membrane with the target cell membrane.

[0038] Provided herein are engineered PIV3 F ectodomain trimers that include protomers containing one or more amino acid substitutions that stabilize the F ectodomain trimer in the prefusion conformation.

[0039] As used herein, "prefusion conformation" refers to a structural form adopted by a polypeptide that differs from the PIV F postfusion conformation, at least in terms of molecular dimensions or three-dimensional coordinates. The prefusion conformation refers to the structural form adopted by PIV F prior to the induction of a fusogenic event that results in the transition of F to the postfusion conformation. Isolating PIV F in a stable prefusion conformation can be useful in providing insight and directing the development of improved vaccines and immunogenic compositions to address the important public health problem of PIV infection. The prefusion conformation can be a conformation that is capable of binding to a prefusion-specific antibody.

[0040] A PIV F ectodomain trimer "stabilized in the prefusion conformation" contains one or more amino acid substitutions, deletions, or insertions compared to the corresponding native PIV F sequence that result in greater retention of the prefusion conformation compared to a PIV F ectodomain trimer formed from the corresponding native hPIV F sequence. "Stabilization" of the prefusion conformation can be, for example, energetic stabilization (e.g., reducing the energy of the prefusion conformation compared to the postfusion open conformation) and / or kinetic stabilization (e.g., reducing the rate of transition from the prefusion conformation to the postfusion conformation). Furthermore, stabilization of a PIV F ectodomain trimer in the prefusion conformation can include increased resistance to denaturation compared to the corresponding native PIV F sequence. Methods for determining whether an hPIV F ectodomain trimer is in the prefusion conformation are provided herein, including (but not limited to) negative stain electron microscopy and antibody binding assays using a prefusion conformation-specific antibody, e.g., the PIA3 or PIA174 antibody in the case of hPIV3.

[0041] The present disclosure provides engineered proteins that contain amino acid substitutions compared to the amino acid sequence of a corresponding native hPIV3 F protein (e.g., SEQ ID NO: 1 or 2). The amino acid mutations include amino acid substitutions, deletions, or additions compared to the native hPIV3 F protein. Thus, the engineered proteins are variants of the native hPIV3 F protein.

[0042] The engineered PIV F ectodomain trimer can be derived from a human strain of PIV other than hPIV3, such as hPIV1, hPIV2, or hPIV4. Based on the high sequence identity between the hPIV3 F sequence and other hPIV F sequences, residues in other hPIV F sequences that correspond to residues in hPIV3 F 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 for prefusion stabilization.

[0043] The engineered PIV F ectodomain trimer can be derived from a non-human strain of PIV, such as a bovine or caprine strain of PIV. Based on the high sequence identity between the human and non-human PIV F sequences, residues in the non-human PIV F sequence corresponding to those in hPIV F 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 a non-human PIV3 F sequence (e.g., GENBANK: AHZ90086.1 or AIW42876.1) for prefusion stabilization.

[0044] The engineered hPIV3 F ectodomain trimer comprises protomers that are "single-chain" proteins, and the F2 polypeptide and F1 ectodomain of each protomer are linked directly or via a peptide linker to form a continuous polypeptide chain. Some examples of native hPIV3 F proteins (e.g., GENBANK:AGW51052.1) do not contain a consensus furin cleavage site between the F1 and F2 proteins, and hPIV3 F immunogens based on such native hPIV3 F proteins generally do not need to be modified to generate single-chain F proteins. However, other native hPIV3 F proteins (e.g., SWISS-PROT:P06828.2) contain a consensus furin cleavage site between the F1 and F2 proteins, and hPIV3 F immunogens based on such native hPIV3 F proteins can be modified to generate single-chain F proteins. Exemplary modifications include amino acid substitutions, such as K108E substitutions, to remove the consensus furin cleavage site.

[0045] Engineered PIV F ectodomain trimer promoters include PIV F positions 19-481 and can contain any of the following: an amino acid substitution (e.g., K108E) to remove the consensus furin cleavage site between F2 and F1 (if the consensus site is present in the native sequence), either K or R at position 87, either T or S at position 95, either K or R at position 141, either V or I at position 165, either L or Q at position 168, either K or R at position 295, either T or V at position 267, either T or K at position 369, and either D or N at position 441.

[0046] The engineered PIV F ectodomain trimer may be a soluble protein complex for use, for example, as a recombinant subunit vaccine. In some such embodiments, the engineered PIV F ectodomain trimer promoters may each include a C-terminal linkage to a trimerization domain, such as the GCN4 trimerization domain. The trimerization domain promotes trimer formation and stabilization of the membrane-proximal surface of the engineered PIV F ectodomain trimer. For example, the C-terminal residue of the engineered PIV F ectodomain trimer promoter (e.g., a residue in the stem region of the trimer) may be directly linked to the trimerization domain or indirectly linked to the trimerization domain via 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 the GCN4 leucine zipper, trimerization motifs from pulmonary surfactant proteins (Hoppe et al. 1994 FEBS Lett 344:191-195), and collagen (McAlinden et al. 2003 J Biol Chem 278:42200-42207), any of which can be linked to the C-terminus of an engineered PIV F ectodomain promoter to promote trimerization, as long as the recombinant PIV F ectodomain trimer retains its prefusion conformation. In some examples, the promoters of the engineered PIV F ectodomain trimer can be linked to a GCN4 trimerization domain, e.g., each promoter in the trimer can include a C-terminal linkage to a GCN4 trimerization domain, e.g., a linkage to any one of positions 475-485 of hPIV3 F, e.g., position 481 of hPIV3 F.

[0047] The engineered PIV F ectodomain trimer may be a membrane-anchored protein complex, for example, for use in attenuated virus or virus-like particle vaccines. Membrane anchoring can be achieved, for example, by C-terminal linkage of the engineered PIV F ectodomain trimer protomer to a transmembrane domain and, optionally, a cytoplasmic tail, e.g., a PIV F transmembrane domain and cytoplasmic tail. The engineered PIV F ectodomain trimer protomer can be linked to the transmembrane domain using one or more peptide linkers (e.g., a 10-amino acid glycine-serine peptide linker, such as a gly-ser linker). Non-limiting examples of transmembrane domains for use in the disclosed embodiments include the hPIV3 F transmembrane domain.

[0048] The engineered protein may have certain beneficial properties, such as being immunogenic. The engineered protein may have increased immunogenicity or improved stability of the pre-fusion conformation compared to the corresponding native hPIV3 F protein. Stability refers to the degree 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 described herein, which may also result in improved stability of the pre-fusion conformation. Amino acid mutations introduced into the hPIV3 F 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 compared to the native hPIV3 F ectodomain.

[0049] Some ways to stabilize the conformation of the engineered protein compared to the native hPIV3 F protein include, but are not limited to, introducing disulfide bonds (both intra- and inter-protomer), modifying salt bridges, introducing electrostatic interactions, introducing hydrogen bonds, introducing prolines, filling cavities, amino acid substitutions that alter residue packing, and combinations thereof.

[0050] 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% isolated, at least 90% isolated, at least 95% isolated, at least 98% isolated, at least 99% isolated, or at least 99.9% isolated from the hPIV3 F polypeptide in the post-fusion conformation. The engineered protein can specifically bind to an hPIV3 F pre-fusion specific antibody.

[0051] It will be understood that a homogeneous population of engineered proteins of a particular conformation may include variations (variations in polypeptide modifications, such as glycosylation status) that do not change the conformational state of the engineered protein. The population of engineered proteins may maintain homogeneity over time. For example, when dissolved in aqueous solution, the engineered protein may form a population of proteins stabilized in the prefusion conformation for at least 12 hours, at least 24 hours, at least 48 hours, at least one week, at least two weeks, or longer. Those skilled in the art will understand that the engineered proteins provided herein are useful for eliciting an immune response against hPIV3 in mammals.

[0052] The engineered protein can contain introduced cysteine ​​substitutions compared to the native PIV F protein. The engineered protein can contain any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cysteine ​​substitutions. Without being bound by theory or mechanism, it is believed that the cysteine ​​substitutions described herein promote the stability of the polypeptide in a conformation other than the 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 positions of the cysteines can be within a sufficiently close distance to form disulfide bonds in the pre- but not post-fusion conformation of the PIV F protein.

[0053] The engineered protein can include a recombinant PIV F protein stabilized in a prefusion conformation by a disulfide bond between cysteines introduced at paired amino acid positions that are close to each other in the prefusion conformation and more distant from each other in the postfusion conformation. The paired cysteines can be present together in a single protomer, thus forming an intraprotomer disulfide bond, or the paired cysteines can be present in different protomers, thus forming an interprotomer disulfide bond.

[0054] Exemplary cysteine ​​substitutions relative to the native PIV F protein include any of the disulfide bond substitutions in Table 1, where the numbering is based on the numbering of SEQ ID NO: 1. The engineered protein can contain a combination of two or more of the disulfide bonds between paired cysteine ​​residues listed in Table 1.

[0055] The engineered protein may comprise 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 comprise at least one disulfide bond mutation and at least one proline mutation. The engineered protein may comprise at least one cysteine ​​substitution and at least one cavity-filling substitution. The engineered protein may comprise at least one cysteine ​​substitution and at least one charge-reducing substitution. The engineered protein may comprise at least one mutation or set of mutations selected from any one of the mutations in Table 1 or 2.

[0056] Table 1. Engineered hPIV3 F proteins (all positions correspond to SEQ ID NOs: 1, 2, or 8-11) TIFF2026503061000003.tif213134TIFF2026503061000004.tif213141TIFF2026503061000005.tif213141TIFF2026503061000006.tif213141TIFF2026503061000007.tif213141TIFF2026503061000008.tif21347a All constructs can also optionally include the L168Q substitution.

[0057] Table 2: Exemplary substitution combinations (all positions correspond to SEQ ID NOs: 1 or 2 or 8-11) TIFF2026503061000009.tif21374TIFF2026503061000010.tif213145TIFF2026503061000011.tif213136TIFF2026503061000012.tif213136TIFF2026 503061000013.tif213136TIFF2026503061000014.tif213136TIFF2026503061000015.tif213145TIFF2026503061000016.tif213135TIFF20265030610 00017.tif213144TIFF2026503061000018.tif213140TIFF2026503061000019.tif213144TIFF2026503061000020.tif213144TIFF2026503061000021.t if213144TIFF2026503061000022.tif213144TIFF2026503061000023.tif213144TIFF2026503061000024.tif213144TIFF2026503061000025.tif21364

[0058] III. Protein Preparation The proteins described herein can be prepared by conventional methods known in the art, for example, by expression in recombinant host systems using appropriate vectors. Suitable recombinant host cells include, for example, insect cells, mammalian cells, avian cells, bacteria, and yeast cells. Examples of suitable insect cells include, for example, Sf9 cells, Sf21 cells, Tn5 cells, Schneider S2 cells, and HIGH FIVE cells (clonal isolates derived from the parent Trichoplusia ni BTI-TN-5B1-4 cell line). Examples of suitable mammalian cells include Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK293 or Expi293 cells, typically transformed with fragmented 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., EBx® 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 known in the art.

[0059] Several suitable vectors for expressing recombinant proteins in insect cells or mammalian cells are well known and have been used in the art. Suitable vectors may contain several components, including, but not limited to, one or more of the following: an origin of replication; a selectable marker gene; one or more expression control elements, such as transcriptional control elements (e.g., promoters, enhancers, terminators), and / or one or more translation signals; and a signal or leader sequence for targeting to the secretory pathway in a selected host cell (e.g., of mammalian origin, or from a heterologous mammalian or non-mammalian species). For example, for expression in insect cells, a suitable baculovirus expression vector, such as PFASTBAC, is used to generate recombinant baculovirus particles. The baculovirus particles are amplified and used to infect insect cells to express the recombinant protein. For expression in mammalian cells, a vector that drives the expression of the construct in the desired mammalian host cell (e.g., Chinese hamster ovary cells) is used.

[0060] Proteins can be purified using any suitable method. For example, methods for purifying proteins by immunoaffinity chromatography are known in the art. Suitable methods for purifying desired proteins are known in the art, including precipitation and various types of chromatography, such as hydrophobic interaction, ion exchange, affinity, chelation, and size exclusion. A suitable purification scheme can be created using two or more of these or other suitable methods. Optionally, the protein can contain a "tag" that facilitates purification, such as an epitope tag or a histidine tag. Such tagged proteins can be purified, for example, from conditioned medium by chelation or affinity chromatography.

[0061] IV. Protein-Encoding Nucleic Acids Nucleic acid molecules encoding the proteins described herein are also provided. These nucleic acid molecules include DNA sequences, cDNA sequences, and RNA sequences. Nucleic acid molecules encoding only the ectodomain of a protein are also contemplated. The nucleic acid molecules can be incorporated into a vector, such as an expression vector.

[0062] The nucleic acid may be a self-replicating RNA molecule. The nucleic acid may comprise a modified RNA molecule. Also provided are compositions comprising the nucleic acids described herein.

[0063] V. Formulations and Methods of Use Provided herein are methods for inducing an immune response against PIV in a mammal, the method comprising administering to the mammal an immunological composition effective to induce an immune response, the composition comprising an engineered PIV F pre-fusion protein or a polynucleotide encoding the engineered PIV F pre-fusion protein. The induced immune response can be a protective immune response, i.e., the response reduces the risk or severity of PIV infection or its clinical consequences. The immune response can include a humoral immune response, a cellular immune response, or both. The immune response can include a T cell response or a B cell response. The cellular immune response can include a helper T cell (Th) response, a CD8+ cytotoxic T cell (CTL) response, or both. The humoral immune response can include antibody-presenting B cells, and the antibody can be a neutralizing antibody against PIV. Neutralizing antibodies block viral infection of cells. The immune response can reduce or prevent infection of cells. The neutralizing antibody response can be complement-dependent or complement-independent. The neutralizing antibody response can be complement-independent. The neutralizing antibody response may be cross-neutralizing, i.e., antibodies generated against the administered composition neutralize related PIV virus strains other than the strain used in the composition.

[0064] The method may include administering a single dose of the composition. The method may further include administering a booster dose of the composition to the subject.

[0065] The desired response is to inhibit, reduce, or prevent PIV infection. The desired response is to reduce PIV viral shedding. The method may reduce PIV viral shedding in saliva. The reduction in PIV viral shedding in a mammal is compared to viral shedding in a mammal that has not been administered the engineered PIVF protein. The term "viral shedding" is used herein according to its plain and ordinary meaning in medicine and virology to refer to the production and release of virus from infected cells. Virus may be released from mammalian cells. Virus may be released from an infected mammal into the environment. Virus may be released from cells within a mammal. The desired response is to reduce PIV viral titer. The method may reduce PIV nucleic acid in serum.

[0066] PIV infection, viral shedding, or viral titer need not be completely eliminated, reduced, or prevented for the method to be effective. For example, administration of an effective amount of an agent can reduce PIV infection (e.g., as measured by infection of cells or by the number or percentage of subjects infected with PIV), viral shedding, or viral titer by a desired amount, such as 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.

[0067] The engineered proteins described herein can be delivered directly as components of immunogenic compositions or vaccines. Alternatively, nucleic acids encoding the proteins described herein can be administered to produce the proteins or immunogenic fragments in vivo. Protein formulations, recombinant nucleic acids (e.g., DNA, RNA, mRNA, self-replicating RNA, or any variant thereof), and / or viral vectors (e.g., live, monoinfectious, non-replicating assembled virions or other virus-like particles, or alphavirus VRPs) containing sequences encoding the engineered proteins provided herein can be included in immunogenic compositions or vaccines. Such compositions can produce the proteins described herein by translation of an open reading frame, which may be codon-optimized. The compositions can include at least one RNA polynucleotide encoding at least one PIV F antigenic polypeptide or immunogenic fragment thereof and at least one 5'-end cap. The 5'-end cap can be 7mG(5')ppp(5')NImpNp.

[0068] When 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 their analogs covalently linked together. The nucleic acid molecules described herein generally contain phosphodiester linkages, but optionally include nucleic acid analogs, which may have at least one different linkage, such as a phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoramidite linkage, as well as peptide nucleic acid backbones and linkages. Mixtures of naturally occurring polynucleotides and analogs can be produced; alternatively, mixtures of different polynucleotide analogs, as well as mixtures of naturally occurring polynucleotides and analogs, may be produced. The nucleic acid molecule may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, for example, by conjugation with a labeling component. The term also includes both double-stranded and single-stranded molecules. Unless otherwise specified or required, the term polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms known or predicted to constitute the double-stranded form. Nucleic acid molecules are composed of a specific sequence of the four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), and, if the polynucleotide is RNA, uracil (U) in place of thymine. Thus, the term "nucleic acid sequence" is the alphabetical representation of a nucleic acid molecule. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses not only the sequence explicitly indicated, but also its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues.

[0069] At least one polynucleotide may have at least one chemical modification. At least one polynucleotide may further have a second chemical modification. The polynucleotide may be RNA. At least one polynucleotide having at least one chemical modification may have a 5'-end cap. The at least one chemical modification can be selected from pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l-l-methyl-l-deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-O-methyluridine. At least 80% (e.g., 85%, 90%, 95%, 98%, 99%, 100%) of the uracils of the open reading frame may have a chemical modification, and optionally, the composition is formulated in a lipid nanoparticle. All uracils of the open reading frame may have a chemical modification. The chemical modification may be at the 5-position of the uracil. The chemical modification may be N1-methylpseudouridine.

[0070] Nucleic acids of the present disclosure may contain one or more modified nucleosides containing modified sugar moieties. Such compounds containing one or more sugar-modified nucleosides may have desirable properties, such as enhanced nuclease stability or increased binding affinity to target nucleic acids, compared to oligonucleotides containing only nucleosides containing natural sugar moieties. In some embodiments, the modified sugar moiety is a substituted sugar moiety. In some embodiments, the modified sugar moiety is a sugar surrogate. Such sugar surrogates may contain one or more substitutions corresponding to the substitutions of the substituted sugar moiety.

[0071] 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' and / or 5' positions. Examples of suitable sugar substituents at the 2' position include, but are not limited to, 2'-F, 2'-OCH ("OMe" or "O-methyl"), and 2'-O(CH)OCH ("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, OCF, O(CH)SCH, O(CH)-O-N(Rm)(Rn), and O-CH-C(=O)-N(Rm)(Rn), where each Rm and Rn is independently H or a substituted or unsubstituted C1-C10 alkyl. Examples of 5'-position sugar substituents 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., a TF-5'-methyl sugar moiety (see, e.g., PCT International Application WO2008 / 101157 for additional 5',2'-bis-substituted sugar moieties and nucleosides).

[0072] Nucleosides containing a 2'-substituted sugar moiety are referred to as 2'-substituted nucleosides. In some embodiments, a 2'-substituted nucleoside comprises a 2'-substituent selected from halo, allyl, amino, azido, SH, CN, OCN, CF, OCF, O, S, or N(Rm)-alkyl; O, S, or N(Rm)-alkenyl; O, S, or N(Rm)-alkynyl; O-alkylenyl-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), where each Rm and Rn is independently H, an amino-protecting group, or a substituted or unsubstituted C1-C10 alkyl. These 2'-substituents may be further substituted with one or more substituents independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl, and alkynyl.

[0073] In some embodiments, the 2'-substituted nucleoside comprises a 2'-substituent selected from F, NH, N, OCF, O--CH, O(CH)NH, CH-CH=CH, O--CH-CH=CH, OCHCHOCH, O(CH)SCH, O--(CH)--O--N(R)(R), O(CH)O(CH)N(CH), and N-substituted acetamide (O--CH--C(=O)--N(R)(R), where each R and R is independently H, an amino-protecting group, or a substituted or unsubstituted C-C alkyl.

[0074] In some embodiments, a 2'-substituted nucleoside comprises a sugar moiety that includes a 2'-substituent selected from 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.

[0075] In some embodiments, a 2'-substituted nucleoside comprises a sugar moiety that includes a 2'-substituent selected from F, O--CH3, and OCH2CH2OCH3.

[0076] In some embodiments, nucleosides of the present disclosure comprise one or more unmodified nucleobases. In certain embodiments, nucleosides of the present disclosure comprise one or more modified nucleobases.

[0077] In some embodiments, the modified nucleobase is selected from 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 include, as defined herein, 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-propynylCH3)uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo Included are uracil, cytosine, and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo, especially 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, promiscuous bases, size-expanded bases, and fluorinated bases. Further 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 can also include those in which the purine or pyrimidine base is replaced with other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone. Additional nucleobases include those disclosed in U.S. Patent No. 3,687,808, The Concise Encyclopedia of Polymer Science and Engineering, Kroschwitz, JI, Ed., John Wiley & Sons, 1990, 858-859, Englisch et al., 1991, and Sanghvi, YS, 1993.

[0078] Representative United States patents that teach the preparation of certain of the above-described modified nucleobases, as well as other modified nucleobases, include, but are not limited to, U.S. Pat. Nos. 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, Nos. 5,502,177, 5,525,711, 5,552,540, 5,587,469, 5,594,121, 5,596,091, 5,614,617, 5,645,985, 5,681,941, 5,750,692, 5,763,588, 5,830,653, and 6,005,096, each of which is incorporated herein by reference in its entirety.

[0079] Additional modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of the 5' terminal nucleotide. For example, one additional modification of the ligand-conjugated oligonucleotides of the present disclosure involves chemically linking to the oligonucleotide one or more additional non-ligand moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. Such moieties include 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), aliphatic chains, such as dodecanediol or undecyl residues (Saison-Behmoaras et al., 1991; Kabanov et al., 1990; Svinarchuk et al., 1993), phospholipids, such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., 1995; Shea et al., 1996). These include, but are not limited to, nucleotides such as nucleotides containing ...In some embodiments, (m)RNA molecules used herein may have uracils replaced with pseudouracils, such as 1-methyl-3'-pseudouridylyl bases. In some embodiments, some of the uracils are replaced, while in other embodiments, all of the uracils are replaced. (m)RNA may include 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.

[0080] Nucleic acid molecules, whether native or modified, may be delivered as naked nucleic acid molecules or in a delivery vehicle such as a lipid nanoparticle. The lipid nanoparticle may contain one or more nucleic acids present in a weight ratio of about 5:1 to about 1:100 relative 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.

[0081] In some embodiments, the lipid nanoparticles used herein may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 lipids. These lipids may include triglycerides, phospholipids, steroids or sterols, PEGylated lipids, or groups having an ionizable group (such as an alkylamine) and one or more hydrophobic groups (such as a C6 or higher alkyl group).

[0082] In some aspects of the present disclosure, lipid nanoparticles are mixed with one or more steroids or steroid derivatives.In some embodiments, steroids or steroid derivatives include any steroid or steroid derivative.As used herein, in some embodiments, the term "steroid" refers to a class of compounds that have a 4-ring, 17-carbon ring structure, which may further include one or more substitutions, including alkyl groups, alkoxy groups, hydroxy groups, oxo groups, acyl groups, or double bonds between two or more carbon atoms.

[0083] In some aspects of the present disclosure, the lipid nanoparticles are mixed with one or more PEGylated lipids (or PEG-lipids). In some embodiments, the present disclosure includes using any lipid with a PEG group 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 attached to a linker group bearing a PEG chain. Some non-limiting examples of PEG-lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates, PEG-modified dialkylamines and PEG-modified 1,2-diacyloxypropan-3-amines, PEG-modified diacylglycerols and dialkylglycerols. In some embodiments, PEG-modified diastearoyl phosphatidylethanolamine or PEG-modified dimyristoyl-sn-glycerol. In some embodiments, the PEG modification is measured by the molecular weight of the PEG component of the lipid. In some embodiments, the PEG modification has a molecular weight of about 100 to about 15,000. In some embodiments, the molecular weight is about 200 to about 500, about 400 to about 5,000, about 500 to about 3,000, or about 1,200 to about 3,000. The molecular weight of the PEG modification is 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 that can be used in the present disclosure are taught by U.S. Pat. No. 5,820,873, WO2010 / 141069, or U.S. Pat. No. 8,450,298, which are incorporated herein by reference.

[0084] In some aspects of the present disclosure, the lipid nanoparticles are mixed with one or more phospholipids. In some embodiments, any lipid that also contains a phosphate group. In some embodiments, the 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 optionally, a small organic molecule. In some embodiments, the small organic molecule is an amino acid, a sugar, or an amino-substituted alkoxy group such as choline or ethanolamine. In some embodiments, the phospholipid is a phosphatidylcholine. In some embodiments, the phospholipid is distearoylphosphatidylcholine or dioleoylphosphatidylethanolamine. In some embodiments, other zwitterionic lipids are used, where zwitterionic lipids are defined as lipids and lipid-like molecules that have both positive and negative charges.

[0085] Some aspects of the present disclosure provide lipid nanoparticles containing a compound containing a lipophilic component and a cationic component, wherein the cationic component is ionizable. In some embodiments, the ionizable cationic lipid contains one or more groups that are protonated at physiological pH but can be deprotonated at pHs above 8, 9, 10, 11, or 12 and have no charge. The ionizable cationic group may contain one or more protonatable amines capable of forming a cationic group at physiological pH. The ionizable cationic lipid compound may also further contain one or more lipid components, such as two or more fatty acids having C6-C24 alkyl or alkenyl carbon groups. These lipid groups may be linked via ester bonds or further attached to a sulfur atom via a Michael addition reaction. In some embodiments, these compounds may be dendrimers, dendrons, polymers, or combinations thereof.

[0086] In some aspects of the present disclosure, compositions are provided that contain a compound containing a lipophilic component and a cationic component, wherein the cationic component is ionizable. In some embodiments, ionizable cationic lipids refer to lipids and lipid-like molecules that have a nitrogen atom capable of acquiring an electric charge (pKa). These lipids may be known in the literature as cationic lipids. These molecules containing amino groups typically have two to six hydrophobic chains, often alkyl or alkenyl, such as C6-C24 alkyl or alkenyl groups, but may also have at least one or more than six tails.

[0087] In some embodiments, the amount of lipid nanoparticles with encapsulated nucleic acid molecules in the pharmaceutical composition is about 0.1 w / w% to about 50 w / w%, about 0.25 w / w% to about 25 w / w%, about 0.5 w / w% to about 20 w / w%, about 1 w / w% to about 15 w / w%, about 2 w / w% to about 10 w / w%, about 2 w / w% to about 5 w / w%, or about 6 w / w% to about 10 w / w%. In some embodiments, the amount of lipid nanoparticles with encapsulated 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.

[0088] In some aspects, the present disclosure includes one or more sugars incorporated into the pharmaceutical composition. In some embodiments, the sugar used herein is a sugar. These sugars may be used to act as cryoprotectants to protect the pharmaceutical composition from destabilization during the drying process. These water-soluble excipients include carbohydrates or sugars, such as disaccharides such as sucrose, trehalose, or lactose; trisaccharides such as fructose, glucose, or galactose, which constitute raffinose; 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, maltotetritoitol, or polyglycitol.

[0089] In some embodiments, the amount of sugar in the pharmaceutical composition is about 25% to about 98% w / w, 40% to about 95% w / w, 50% to about 90% w / w, 50% to about 70% w / w, or about 80% to about 90% w / w. In some embodiments, the amount of sugar in the pharmaceutical composition is from about 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%, 52.5 w / w%, 55 w / w%, 57.5 w / w%, 60 w / w%, 62.5 w / w%, 65 w / w%, 67.5 w / w%, 70 w / w%, 75 w / w%, 80 w / w%, 82.5 w / w%, 85 w / w%, 87.5 w / w%, 90 w / w% to about 95 w / w%, or any range derivable therein.

[0090] In some embodiments, the pharmaceutically acceptable polymer is a copolymer. The pharmaceutically acceptable polymer may further comprise 1, 2, 3, 4, 5, or 6 subunits of different individual polymer subunits. These polymer subunits may comprise 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 that is polyoxyethylene and a hydrophobic subunit that is polyoxypropylene.

[0091] In some embodiments, expression cassettes are used to express PIV F proteins, either for subsequent purification and delivery to cells / subjects or for direct use in viral-based delivery approaches. Provided herein are expression vectors containing one or more nucleic acids encoding PIV F proteins.

[0092] Expression requires that appropriate signals be provided within the vector, including various regulatory elements such as enhancers / promoters from both viral and mammalian sources that drive expression of the engineered PIV 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, where part or all of the nucleic acid's coding sequence can be 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 required to initiate the 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 the initiation of RNA polymerase and expression of the gene. An "expression vector" is intended to include an expression cassette contained in a genetic construct that is capable of replication and thus includes one or more of an origin of replication, a transcription termination signal, a poly-A region, a selectable marker, and a multipurpose cloning site.

[0093] The term promoter will be used herein to refer to a group of transcriptional control modules clustered around an initiation site for RNA polymerase II. Most of the ideas about how promoters are organized come from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent efforts, have shown that promoters are composed of discrete functional modules, each of which consists of approximately 7-20 bp of DNA and contains one or more recognition sites for transcriptional activator or repressor proteins.

[0094] At least one module in each promoter functions to position the start site for RNA synthesis. The best-known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoters for the mammalian terminal deoxynucleotidyl transferase genes and the SV40 late genes, individual elements overlapping the start site themselves help to anchor the start point.

[0095] Additional promoter elements regulate the frequency of transcription initiation. Typically, these are located in the region 30–110 bp upstream of the start site, although several promoters have recently been shown to contain functional elements downstream of the start site. The spacing between promoter elements is often flexible, allowing promoter function to be maintained even when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart, after which activity begins to decline. Depending on the promoter, individual elements appear to function either cooperatively or independently to activate transcription.

[0096] In certain embodiments, viral promoters such as the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, the rat insulin promoter, and the glyceraldehyde-3-phosphate dehydrogenase promoter can be used to obtain high levels of expression of the coding sequence of interest. The use of other viral or mammalian cell or bacterial phage promoters known in the art to achieve expression of the coding sequence of interest is also contemplated, so long as the expression level is sufficient for the given purpose. By using a promoter with well-known properties, the expression level and pattern of the protein of interest after transfection or transformation can be optimized. Furthermore, selection of a promoter that is regulated in response to specific physiological signals may allow for inducible expression of the gene product.

[0097] Enhancers are genetic elements that increase transcription from promoters located at distant locations on the same DNA molecule. Enhancers are organized much like promoters; that is, they are composed of many individual elements, each of which binds one or more transcriptional proteins. The fundamental distinction between enhancers and promoters is operational. While the enhancer region as a whole must be capable of stimulating transcription from a distance, this need not be true of the promoter region and its components. On the other hand, promoters must have one or more elements at specific sites and in specific orientations that direct the initiation of RNA synthesis; enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, and often appear to have very similar modular organizations.

[0098] Below is a list of promoters / enhancers and inducible promoters / enhancers that can be used in combination with a nucleic acid encoding a gene of interest in an expression construct. Additionally, any promoter / enhancer combination (according to the Eukaryotic Promoter Database EPDB) can also be used to drive expression of the gene. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional gene expression construct.

[0099] The promoter and / or enhancer may be, for example, an immunoglobulin light chain, an immunoglobulin heavy chain, a T-cell receptor, HLA DQ a and / or DQ β, β-interferon, interleukin-2, interleukin-2 receptor, MHC class II 5, MHC class II HLA-Dra, β-actin, muscle creatine kinase (MCK), prealbumin (transthyretin), elastase I, metallothionein (MTII), collagenase, albumin, α-fetoprotein, t-globin, β-globin, c-fos, c-HA-ras, insulin, neuronal cell adhesion molecule (NCAM), α1-antitrypsin, H2B (TH2B) histone, mouse and / or type I collagen, glucose-regulated protein (GRP94 and GRP78), rat growth hormone, human serum amyloid A (SAA), troponin I (TN I), platelet-derived growth factor (PDGF), SV40, polyoma, retrovirus, papillomavirus, hepatitis B virus, human immunodeficiency virus, cytomegalovirus (CMV), and gibbon ape leukemia virus.

[0100] When a cDNA insert is used, it is typically desirable to include a polyadenylation signal to achieve proper polyadenylation of the gene transcript. Any polyadenylation sequence may be used, such as the human growth hormone and SV40 polyadenylation signals. Terminators are also contemplated as elements of the expression cassette. These elements may function to enhance message levels and minimize readthrough of other sequences from the cassette.

[0101] There are several methods by which expression vectors can be introduced into cells. In certain embodiments, the expression construct comprises a virus or an engineered construct derived from a viral genome. Certain viruses have the ability to enter cells via receptor-mediated endocytosis and integrate into the host cell genome, resulting in stable and efficient expression of viral genes, making them attractive candidates for the transfer of foreign genes into mammalian cells. They have a relatively low capacity for foreign DNA sequences and a limited host spectrum. Furthermore, their carcinogenic potential and cytopathic effects in permissive cells raise safety concerns. Although they can only accommodate up to 8 kB of foreign genetic material, they can be easily introduced into a variety of cell lines and experimental animals.

[0102] One method for in vivo delivery is the use of an adenovirus expression vector. "Adenovirus expression vector" is intended to include a construct containing sufficient adenovirus sequences (a) to support packaging of the construct and (b) to express an engineered PIV F protein cloned therein. In this context, expression does not require that the gene product be synthesized.

[0103] Expression vectors include genetically engineered forms of adenovirus. Knowledge of the genetic organization of adenovirus, a 36 kB linear, double-stranded DNA virus, allows for the replacement of large segments of adenoviral DNA with foreign sequences up to 7 kB. In contrast to retroviruses, adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate episomal without potential genotoxicity. Furthermore, adenoviruses are structurally stable, and no genome rearrangements have been detected after extensive amplification. Adenoviruses can infect virtually all epithelial cells, regardless of their cell cycle stage. Thus far, adenoviral infection appears to be associated only with mild illness, such as acute respiratory illness, in humans.

[0104] Adenoviruses are particularly suitable for use as gene transfer vectors due to their moderate genome size, ease of manipulation, high titer, wide target cell range, and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis-receptors necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain distinct transcription units that are separated by the initiation of viral DNA replication. The E1 region (E1A and E1B) encodes proteins responsible for regulating the transcription of the viral genome and a few cellular genes. Expression of the E2 region (E2A and E2B) results in the synthesis of these proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression, and host cell shutoff. Late gene products, including most of the viral capsid proteins, are expressed only after significant processing of a single primary transcript driven by the major late promoter (MLP). The MLP (located at 16.8 mu) is particularly efficient during the late stages of infection; all mRNAs produced from this promoter possess the 5'-triplet leader (TPL) sequence, making it the preferred mRNA for translation. In one system, recombinant adenovirus is generated from homologous recombination between a shuttle vector and a proviral vector. Due to the possibility of recombination between two proviral vectors, wild-type adenovirus can be generated from this process. Therefore, it is essential to isolate single clones of virus from individual plaques and examine their genomic structure.

[0105] The generation and propagation of current replication-deficient adenoviral vectors relies on a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells with Ad5 DNA fragments and constitutively expresses the E1 protein. Because the E3 region is not essential to the adenoviral genome, current adenoviral vectors rely on 293 cells to carry foreign DNA in either the E1 or D3 region, or both. Adenoviruses can naturally package approximately 105% of the wild-type genome, providing an extra capacity of approximately 2 kb of DNA. Combined with the approximately 5.5 kb of DNA that is replaceable 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. More than 80% of the adenoviral genome remains in the vector backbone, representing a source of vector-derived cytotoxicity. Furthermore, E1-deleted viruses are incompletely replication-deficient.

[0106] Helper cell lines can be derived from human cells, such as human fetal kidney cells, muscle cells, hematopoietic cells, or other mesenchymal or epithelial human fetal cells. Alternatively, helper cells can be derived from cells of other mammalian species that are permissive for human adenovirus. Such cells include, for example, Vero cells or other mesenchymal or epithelial monkey fetal cells. As noted above, a preferred helper cell line is 293.

[0107] The adenoviruses of the present disclosure are replication-deficient, or at least conditionally replication-deficient. The adenovirus may 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 a conditionally replication-deficient adenovirus vector for use in the present invention.

[0108] Other viral vectors may also be used as expression constructs in the present disclosure. Vectors derived from viruses such as vaccinia virus, adeno-associated virus (AAV), and herpes virus may also be used. They offer several attractive features for various mammalian cells.

[0109] In certain embodiments, the vector is an AAV vector. AAV is a small virus that infects humans and some other primate species. AAV is not currently known to cause disease. The virus induces a very mild immune response, further supporting its apparent lack of pathogenicity. In many cases, AAV vectors integrate into the host cell genome, which may be important for certain applications but may also have undesirable consequences. AAV-based gene therapy vectors can infect both dividing and quiescent cells and persist extrachromosomally without integrating into the host cell genome, whereas native viruses do indeed undergo some integration of virally delivered genes into the host genome. These characteristics make AAV a very attractive candidate for generating viral vectors for gene therapy and for generating isogenic human disease models. Recent human clinical trials using AAV for gene therapy in the retina have shown promise. AAV belongs to the genus Dependoparvovirus, which in turn belongs to the family Parvoviridae. The virus is a small (20 nm), non-enveloped, replication-deficient virus.

[0110] Wild-type AAV has attracted considerable interest from gene therapy researchers due to several characteristics. Chief among these is the virus's apparent lack of pathogenicity. It can also infect non-dividing cells and has the ability to stably integrate into the host cell genome at a specific site on human chromosome 19 (designated AAVS1). This characteristic makes it somewhat more predictable than retroviruses, which pose the threat of random insertion and mutagenesis, sometimes leading to the development of cancer. While the AAV genome most frequently integrates at the designated site, random integration into the genome occurs at a very low frequency. However, the development of AAV as a gene therapy vector eliminated this integration capability by removing the rep and cap sequences from the vector DNA. The desired gene, along with a promoter to drive gene transcription, is inserted between inverted terminal repeats (ITRs), which support concatemer formation in the 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 life of the host cell. In dividing cells, AAV DNA is lost with cell division because 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 very low frequency. AAVs also exhibit very low immunogenicity, which appears to be limited to the generation of neutralizing antibodies, while they do not induce a clearly defined cytotoxic response. This feature, along with their ability to infect quiescent cells, gives them an advantage over adenoviruses as vectors for human gene therapy.

[0111] The AAV genome is composed of either positive- or negative-stranded single-stranded deoxyribonucleic acid (ssDNA) and is approximately 4.7 kilobases long. The genome contains inverted terminal repeats (ITRs) at both ends of the DNA strand and two open reading frames (ORFs), rep and cap. The former consists of four overlapping genes encoding the Rep proteins required throughout the AAV life cycle, while the latter contains overlapping nucleotide sequences for the capsid proteins VP1, VP2, and VP3, which interact together to form the icosahedral capsid.

[0112] Inverted terminal repeat (ITR) sequences contain 145 bases each. They are so named because of their symmetry, which has been shown to be required for efficient replication of the AAV genome. The feature of these sequences that confers this property is their ability to form hairpins, which contribute to so-called self-priming, allowing primase-independent synthesis of the second DNA strand. ITRs have also been shown to be required for both integration of AAV DNA into and rescue from the host cell genome (chromosome 19 in humans) and efficient encapsidation of AAV DNA combined with the generation of fully assembled, deoxyribonuclease-resistant AAV particles.

[0113] With regard to gene therapy, the ITRs appear to be the only sequences required in cis next to the therapeutic gene, while the structural (cap) and packaging (rep) proteins can be delivered in trans. Based on this assumption, many methods have been established for the efficient production of recombinant AAV (rAAV) vectors containing reporter or therapeutic genes. However, it has also been demonstrated that the ITRs are not the only elements required in cis for efficient replication and encapsidation. A few research groups have identified sequences termed cis-acting Rep-dependent elements (CAREs) located within the coding sequence of the rep gene. CAREs have been shown to enhance replication and encapsidation when present in cis.

[0114] Each of the immunogenic compositions discussed herein can be used alone or in combination with one or more other antigens, whether from the same viral pathogen or from different or multiple pathogen sources. These compositions can be used for prophylactic (to prevent infection) or therapeutic (to treat disease after infection) purposes.

[0115] The term "pharmaceutically acceptable" can mean approved by a federal or state regulatory agency for use in animals, more particularly humans, or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia. The term "carrier" refers to a diluent, excipient, or vehicle with which a therapeutic is administered. Such a pharmaceutical carrier can be a sterile liquid, such as water, and preferably includes an adjuvant. Water is a particular carrier when the pharmaceutical composition is administered by injection, such as intramuscularly. Saline solutions and aqueous dextrose and glycerol solutions may 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, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like.

[0116] The immunogenic composition may contain a diluent, such as water, saline, glycerol, ethanol, or the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. The immunogenic composition may contain one or more salts. The salts may be inorganic potassium or sodium salts, such as potassium chloride, sodium chloride, dibasic potassium phosphate, monobasic potassium phosphate, dibasic sodium phosphate, or monobasic sodium phosphate. The immunogenic composition may also contain one or more phosphate salts, such as to form a phosphate buffer. The phosphate buffer may contain each of the phosphate salts 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.

[0117] The immunogenic composition may include an adjuvant. Exemplary adjuvants for enhancing the effectiveness of the compositions 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 muramyl peptides (see below), or bacterial cell wall components), such as (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-based block polymer L121, and thr-MDP, microfluidized or vortexed into a submicron emulsion to produce a larger particle size emulsion); and (c) RIBI™ Adjuvant System (RAS), (Ribi Immunochem, Hamilton, Mont.) (containing 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (DETOX™)); (3) saponin adjuvants that may be used, such as QS-21, STIMULON™ (Cambridge (3) complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA), (4) cytokines such as interleukins (IL-1, IL-2, etc.), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor (TNF), (5) toll-like receptor agonists, and (6) other substances that act as adjuvants to enhance the effectiveness of the composition. The composition may be adjuvant-free. The composition may further comprise lipid nanoparticles. The composition may be formulated in nanoparticles.The composition may further comprise cationic or polycationic compounds, including protamine or other cationic peptides or proteins, such as poly-L-lysine (PLL).

[0118] Generally, the components of the compositions of the present disclosure are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the amount of active ingredient.When the composition is to be administered by inhalation, it can be dispensed by an inhalation bottle containing pharmaceutical-grade sterile water or saline.When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed before administration.

[0119] The compositions described herein can optionally include an immunologically effective amount of the polypeptide or polynucleotide, as well as any other of the components described above. By "immunologically effective amount," it is meant that administration of that amount to an individual, either as a single dose or as part of a series, is effective to elicit an immune response. The elicited immune response can be sufficient, for example, to treat and / or prevent and / or reduce the incidence of an illness, infection, or disease. This amount will vary depending on the health and physical condition of the individual being treated, the taxonomic group of the individual being treated (e.g., non-human primate, primate, etc.), the ability of the individual's immune system to synthesize antibodies, the degree of protection desired, the vaccine formulation, the treating physician's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine testing.

[0120] Any suitable route of administration can be used. For example, the engineered proteins of the present disclosure described herein, or nucleic acids encoding the engineered proteins, can be formulated for parenteral administration, for example, formulated for injection via intradermal, intravenous, intraarterial, intramuscular, subcutaneous, intratumoral, or even intraperitoneal routes. Particularly preferred routes of administration include intramuscular, intradermal, and subcutaneous injection. The formulations can alternatively be administered by topical routes directly to mucous membranes, for example, by nasal drops, inhalation, or by nebulizer.

[0121] The composition can be administered according to any suitable schedule. Dosage treatment can be by a single-dose schedule or a multiple-dose schedule. Multiple doses may be used in a primary immunization schedule and / or a booster immunization schedule. In a multiple-dose schedule, various doses may be given by the same or different routes. Multiple doses are typically administered at least one week apart (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.). The immunogenic composition may be administered in combination with other immunomodulatory agents.

[0122] The compositions disclosed herein can be used to treat both children and adults. Thus, a human subject can be under 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.

[0123] VI. Antibodies and Diagnostic Uses The above-described polypeptides can be used to generate both polyclonal and monoclonal antibodies. If polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit, goat, guinea pig, horse, etc.) is immunized with an immunogenic polypeptide bearing a PIV F pre-fusion epitope(s). Serum from the immunized animal is collected and processed according to known procedures. If serum containing polyclonal antibodies against the PIV F pre-fusion epitope contains antibodies against other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for generating and processing polyclonal antisera are known in the art.

[0124] Monoclonal antibodies against PIV F prefusion epitopes can also be readily produced by those skilled in the art. General methodologies for producing monoclonal antibodies by hybridomas are known. Immortal antibody-producing cell lines can be created by cell fusion or other techniques, such as direct transformation of B lymphocytes with oncogenic DNA or transfection with Epstein-Barr virus. Panels of monoclonal antibodies produced against PIV F prefusion epitopes can be screened for various properties, i.e., isotype, epitope affinity, etc.

[0125] Antibodies, both monoclonal and polyclonal, directed against PIV F prefusion epitopes are particularly useful for diagnosis, and those that are neutralizing are useful for passive immunotherapy. In particular, monoclonal antibodies can be used to generate anti-idiotypic antibodies.

[0126] Both polypeptides immunologically reactive with serum containing PIV F antibodies and antibodies raised against these polypeptides can be useful 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. The design of immunoassays is subject to numerous variables, a variety of which are known in the art. For example, an immunoassay can utilize a polypeptide having the sequence set forth in any one of SEQ ID NOS: 1-7.

[0127] Alternatively, immunoassays may use a combination of viral antigens derived from the polypeptides described herein. For example, monoclonal antibodies against at least one polypeptide described herein, a combination of monoclonal antibodies against the polypeptides described herein, monoclonal antibodies against different viral antigens, polyclonal antibodies against the polypeptides described herein, or polyclonal antibodies against different viral antigens may be used. Protocols may be based, for example, on competitive or direct reactions, or may be sandwich-type assays. Protocols may also use, for example, solid supports or may involve immunoprecipitation. Most assays involve the use of labeled antibodies or polypeptides; the labels may be, for example, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays that amplify the signal from the probe are also known, examples of which include assays utilizing biotin and avidin, as well as enzyme-labeled and mediated immunoassays, such as ELISA assays.

[0128] Kits suitable for immunodiagnosis and containing appropriately labeled reagents are constructed by packaging the appropriate materials, including the engineered PIV F protein containing the PIV F prefusion epitope or antibodies to the epitope, together with the remaining reagents and materials required to perform the assay in a suitable container, along with a suitable set of assay instructions.

[0129] The polynucleotide probes can also be packaged into diagnostic kits. The diagnostic kits include probe DNA, which may be labeled, or the probe DNA may be unlabeled and components for labeling may be included in the kit. The kits may also include other appropriately packaged reagents and materials required for the particular hybridization protocol, such as standards, as well as instructions for performing the test.

[0130] VII. Immunodetection Methods The present disclosure relates to immunodetection methods for binding, purifying, extracting, quantifying, and otherwise generally detecting PIV F protein. While such methods can be applied in a conventional sense, another application is in the quality control and monitoring of vaccine stocks, where antibodies according to the present disclosure can be used to assess antigen quantity or integrity (i.e., long-term stability). Alternatively, the methods can be used to screen various antibodies for appropriate / desired reactivity profiles.

[0131] Some immunodetection methods include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), immunoradiometric assays, fluorescent immunoassays, chemiluminescence assays, bioluminescence assays, and Western blots, to name a few. Competitive assays for the detection and quantification of PIV F protein are also provided. The steps of various useful immunodetection methods are described in the scientific literature, for example, in Doolittle and Ben-Zeev (1999), Gulbis and Galand (1993), De Jager et al. (1993), and Nakamura et al. (1987). Generally, immunobinding methods involve obtaining a sample suspected of containing PIV F protein and contacting the sample with a first antigen according to the present disclosure, optionally under conditions effective to allow the formation of an immune complex.

[0132] These methods include methods for detecting or purifying PIV F protein from a sample. The antibody is preferably linked to a solid support, such as in the form of a column matrix, and a sample suspected of containing PIV F protein is applied to the immobilized antibody. Unwanted components are washed from the column, leaving PIV F protein-expressing cells immunocomplexed to the immobilized antibody, which are then collected by removing the organism or antigen from the column.

[0133] Immunobinding methods also include methods for detecting and quantifying the amount of PIV F protein or related components in a sample, as well as detecting and quantifying any immune complexes formed during the binding process. Here, a sample suspected of containing PIV F protein would be obtained, the sample would be contacted with an antibody that binds to PIV F protein or its components, and the amount of immune complexes formed under specific conditions would then be detected and quantified. 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 including blood and serum (e.g., a nasal swab), or a secretion such as feces or urine.

[0134] Contacting a selected biological sample with antibodies under conditions effective and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) generally involves simply adding the antibody composition to the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with (i.e., bind to) the PIV F protein. After this time, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot, or Western blot, is generally washed to remove any nonspecifically bound antibody species so that only the specifically bound antibodies within the primary immune complexes are detected.

[0135] In general, detection of immune complex formation is well known in the art and can be achieved by applying a number of approaches. These methods are generally based on the detection of labels or markers, such as radioactive, fluorescent, biological, and enzymatic tags. Patents relating to the use of such labels 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 is known in the art, additional advantages may be found through the use of secondary binding ligands, such as secondary antibodies and / or biotin / avidin ligand binding arrangements.

[0136] The antibody used for detection may itself be linked to a detectable label, and then this label can simply be detected, thereby determining the amount of primary immune complexes in the composition. Alternatively, the first antibody that becomes bound in the primary immune complex may be detected using a second binding ligand that has binding affinity for that antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand is often itself an antibody, and therefore may be referred to as a "secondary" antibody. The primary immune complex is contacted with a labeled secondary binding ligand or antibody under conditions effective to allow the formation of secondary immune complexes and for a period of time sufficient for this. The secondary immune complex is then generally washed to remove any non-specifically bound labeled secondary antibody or ligand, and then the label remaining in the secondary immune complex is detected.

[0137] Further methods involve detecting primary immune complexes using a two-step approach. As described above, a second binding ligand, such as an antibody, having binding affinity for the antibody is used to form secondary immune complexes. After washing, the secondary immune complexes are again contacted with a third binding ligand or antibody having binding affinity for the second antibody under conditions effective and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes). The third ligand or antibody is linked to a detectable label so that the tertiary immune complexes thus formed can be detected. This system can allow for signal amplification, if desired.

[0138] One immunodetection method uses two different antibodies. A first biotinylated antibody is used to detect the target antigen, followed by a second antibody to detect biotin bound to the complexed biotin. In this method, the sample to be tested is first incubated in a solution containing the first-step antibody. If the target antigen is present, a portion of the antibody binds to the antigen, forming a biotinylated antibody / antigen complex. The antibody / antigen complex is then amplified by incubation in sequential solutions of streptavidin (or avidin), biotinylated DNA, and / or complementary biotinylated DNA, with each step adding additional biotin moieties to the antibody / antigen complex. The amplification steps are repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing a second-step antibody directed against biotin. This second-step antibody is labeled with an enzyme that can be used to detect the presence of the antibody / antigen complex, for example, by histoenzymology using a chromogenic substrate. By suitable amplification, macroscopically visible conjugates can be produced.

[0139] Another known immunodetection method utilizes immuno-PCR (polymerase chain reaction) techniques. The PCR method is similar to the Cantor method up to the incubation with biotinylated DNA, but instead of using multiple streptavidin and biotinylated DNA incubations, the DNA / biotin / streptavidin / antibody complex is washed with a low pH or high salt buffer, which releases the antibody. The resulting wash solution is then used to perform a PCR reaction using suitable primers along with appropriate controls. At least in theory, the enormous amplification power and specificity of PCR can be exploited to detect single antigen molecules.

[0140] A. ELISA Immunoassays, in their simplest and most straightforward sense, are binding assays. Certain preferred immunoassays are the various types of enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs) 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 blotting, dot blotting, FACS analysis, and the like may also be used.

[0141] In one exemplary ELISA, the antibodies of the present disclosure are immobilized on a selected surface exhibiting protein affinity, such as the wells of 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 nonspecifically bound immune complexes, the bound antigen can be detected. Detection may be achieved by the addition of another anti-PIV F protein antibody linked to a detectable label. This type of ELISA is a simple "sandwich ELISA." Detection may also be achieved by the addition of a second anti-PIV F protein antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, where the third antibody is linked to a detectable label.

[0142] 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 nonspecifically bound immune complexes, the bound anti-PIV F protein antibody is detected. If the first anti-PIV F protein antibody is linked to a detectable label, the immune complex may be detected directly. Again, the immune complex may be detected using a second antibody that has binding affinity for the first anti-PIV F protein antibody, where the second antibody is linked to a detectable label.

[0143] Regardless of the format used, ELISAs have certain features in common, such as coating, incubating, and binding, washing to remove non-specifically bound species, and detecting bound immune complexes, which are described below.

[0144] When coating a plate with either an antigen or an antibody, the wells of the plate are generally incubated with a solution of the antigen or antibody, either overnight or for a specified period of time. The wells of the plate are then washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with respect to the test antiserum. These include solutions of bovine serum albumin (BSA), casein, or milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface, thus reducing the background caused by nonspecific binding of the antiserum to the surface.

[0145] In ELISA, it is perhaps more common to use secondary or tertiary detection means rather than a direct procedure. Thus, after binding of protein or antibody to the well, reducing background by coating with a non-reactive material, and removing unbound material by washing, the immobilizing surface is contacted with the biological sample to be tested under conditions effective to allow immune complex (antigen / antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, and the secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or third binding ligand.

[0146] "Under conditions effective to allow immune complex (antigen / antibody) formation" means conditions that 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 tend to assist in reducing nonspecific background.

[0147] "Suitable" conditions also mean that the incubation is at a temperature or for a period of time sufficient to allow effective binding. The incubation step is typically at a temperature of preferably about 25°C to 27°C for about 1 to 2 to 4 hours, or overnight at about 4°C.

[0148] All incubation steps in an ELISA are followed by washing the contact surface to remove uncomplexed material. A preferred washing procedure involves washing with a solution such as PBS / Tween or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the presence of even minute amounts of immune complexes can be determined.

[0149] To provide a means of detection, the second or third antibody will have a label associated therewith to permit detection. Preferably, this will be an enzyme that will generate color development upon incubation with an appropriate chromogenic substrate. Thus, for example, it may be desirable to contact or incubate the first and second immune complexes with urease, glucose oxidase, alkaline phosphatase, or hydrogen peroxidase-conjugated antibodies for a period of time and under conditions that favor the development of further immune complex formation (e.g., a 2-hour incubation at room temperature in a PBS-containing solution such as PBS-Tween).

[0150] After incubation with the labeled antibody and subsequent washing to remove unbound material, the amount of label is quantified by incubation with a chromogenic substrate, such as, for example, urea, or bromocresol purple, or 2,2'-azino-di-(3-ethyl-benzothiazoline-6-sulfonic acid (ABTS), or HO (if peroxidase is the enzyme label). Quantitation is then achieved by measuring the degree of color produced, for example, using a visible spectrum spectrophotometer.

[0151] B. Western Blot Western blot (alternatively, protein immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by polypeptide length (denaturing conditions) or by the protein's 3D structure (native / non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

[0152] Samples may be taken from whole tissues or cell cultures. In most cases, solid tissues are first mechanically disrupted using a blender (for larger sample volumes), a homogenizer (for smaller volumes), or by sonication. Cells may also be disrupted by one of the above mechanical methods. Various combinations of detergents, salts, and buffers may be used to promote cell lysis and solubilize proteins. Protease and phosphatase inhibitors are often added to prevent digestion of the sample by its own enzymes. Tissue preparation is often performed at low temperatures to avoid protein denaturation.

[0153] Gel electrophoresis is used to separate proteins from a sample. Protein separation can be by isoelectric point (pI), molecular weight, charge, or a combination of these factors. The nature of the separation depends on the sample treatment and the nature of the gel. This is a very useful method for determining proteins. It is also possible to use two-dimensional (2D) gels, which spread proteins from a single sample in two dimensions. Proteins are separated according to their isoelectric point (the pH at which their net charge is neutral) in the first dimension and according to their molecular weight in the second dimension.

[0154] To make proteins accessible to antibody detection, they are transferred from the gel onto a membrane made of nitrocellulose or polyvinylidene fluoride (PVDF). A membrane is placed on top of the gel, and a stack of filter paper is placed on top of it. The entire stack is then immersed in a buffer solution, which carries the proteins along with it and moves them up the filter paper by capillary action. Another method for transferring proteins, called electroblotting, uses an electric current to draw the proteins from the gel onto a PVDF or nitrocellulose membrane. The proteins migrate from the gel onto the membrane while maintaining the structure they had in the gel. As a result of this blotting process, the proteins are exposed on a thin surface layer for detection (see below). Both types of membranes are chosen for their nonspecific protein-binding properties (i.e., they bind all proteins equally well). Protein binding is based on hydrophobic interactions as well as charge interactions between the membrane and the protein. Nitrocellulose membranes are less expensive than PVDF but are much more fragile and do not withstand repeated probing as well. The uniformity and overall effectiveness of protein transfer from the gel to the membrane can be confirmed by staining the membrane with Coomassie Brilliant Blue or Ponceau S dye. Once transferred, the protein is detected using a labeled primary antibody, or an unlabeled primary antibody followed by indirect detection using labeled Protein A or a secondary labeled antibody that binds to the Fc region of the primary antibody.

[0155] C. Immunohistochemistry The antibodies of the present disclosure may also be used in conjunction with both fresh-frozen and / or formalin-fixed, paraffin-embedded tissue blocks prepared for immunohistochemical (IHC) studies. Methods for preparing tissue blocks from these particulate specimens have been used successfully 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).

[0156] Briefly, frozen sections can be prepared by rehydrating 50 ng of frozen "ground" tissue in phosphate-buffered saline (PBS) at room temperature in small plastic capsules; pelleting the particles by centrifugation; resuspending them in viscous embedding medium (OCT); mixing by inversion and / or pelleting again by centrifugation; flash-freezing in -70°C isopentane; cutting off the plastic capsule and / or removing the cylinder of frozen tissue; securing the tissue cylinder on a cryostat microtome chuck; and / or cutting 25-50 serial sections from the capsule. Alternatively, frozen whole tissue samples can be used for serial sectioning.

[0157] Permanent sections may be prepared by a similar method, which involves rehydrating a 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin and fixing for 4 hours; washing / pelleting; resuspending in warm 2.5% agar; pelleting; chilling 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 permanent serial sections. Again, whole tissue samples may be substituted.

[0158] D. Immunodetection Kit In yet a further embodiment, the present disclosure relates to an immunodetection kit for use with the immunodetection method described above. Because antibodies can be used to detect PIV F protein, the kit may include an antibody. The immunodetection kit would therefore contain, in suitable container means, a first antibody that binds to PIV F protein, and optionally, an immunodetection reagent.

[0159] In certain embodiments, the antibody may 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 may take any one of a variety of forms, including a detectable label associated with or linked to a given antibody. Detectable labels associated with or bound to a secondary binding ligand are also contemplated. An exemplary secondary ligand is a secondary antibody that has binding affinity for the first antibody.

[0160] Further suitable immunodetection reagents for use in the present kits include two-component reagents comprising a second antibody having binding affinity for the first antibody, together with a third antibody having binding affinity for the second antibody, where the third antibody is linked to a detectable label. As noted above, several exemplary labels are known in the art, and all such labels may be used in connection with the present disclosure.

[0161] The kit may further include a suitably aliquoted composition of PIV F protein, whether labeled or unlabeled, that can be used to generate a standard curve for the detection assay. The kit may contain the antibody-label conjugate either in fully conjugated form, in the form of an intermediate, or as a separate moiety to be conjugated by the user of the kit. The components of the kit may be packaged either in aqueous medium or in lyophilized form.

[0162] The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe, or other container means, into which the antibody may be placed, or preferably, suitably aliquoted. The kits of the present disclosure will also typically include a means for containing the antibody, antigen, and any other reagent containers in close confinement for commercial sale. Such containers may include injection- or blow-molded plastic containers into which the desired vials may be retained.

[0163] E. Flow Cytometry and FACS The antibodies of the present disclosure may also be used in flow cytometry or FACS. Flow cytometry is a laser or impedance-based technology used in many detection assays, including cell counting, cell sorting, biomarker detection, and protein engineering. This technology suspends cells in a flowing fluid and passes them through an electrical detection device, allowing for simultaneous multiparameter analysis of the physical and chemical properties of up to thousands of particles per second. Flow cytometry is routinely used in the diagnosis of disorders, particularly blood cancers, but has many other applications in basic research, clinical practice, and clinical trials.

[0164] Fluorescence-activated cell sorting (FACS) is a specialized 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 properties. Generally, this technique involves drawing a cell suspension into the center of a rapidly flowing, narrow liquid stream. The flow is arranged so that the cells are separated by a large distance relative to their diameter. A vibrating mechanism breaks the stream into individual droplets. Just before the stream splits into droplets, it passes through a fluorescence measurement station, where the fluorescence of each cell is measured. A charged ring is placed exactly where the stream splits into droplets. A charge is placed on the ring just before the fluorescence intensity is measured, and an opposite charge is captured on the droplets as they split from the stream. The charged droplets then fall through an electrostatic deflection system, which deflects them into containers based on their charge.

[0165] In certain embodiments, for use in flow cytometry or FACS, antibodies of the present disclosure are labeled with a fluorophore and then bound to cells of interest, which are analyzed in a flow cytometer or sorted by a FACS machine.

[0166] VIII.Definitions It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. As used herein, the use of the singular includes the plural unless expressly stated otherwise. As used herein, the use of "or" means "and / or" unless expressly stated otherwise. Furthermore, the use of the term "comprising" and other forms, such as "comprises" and "included," is not limiting. Furthermore, terms such as "element" or "component" encompass both elements and components comprising one unit and elements and components comprising more than one subunit, unless expressly stated otherwise. Furthermore, the use of the term "portion" can include a portion of a moiety or an entire portion.

[0167] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. As used herein, the terms "a" or "an" can mean one or more. As used herein, in the claim(s), when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more than one.

[0168] As used herein, the term "about" when referring to a measurable value, such as an amount, time duration, and the like, is intended to encompass a variation of up to ±10% from the specified value. Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and the like used in the specification and claims are to be understood as being modified in all occurrences by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the disclosed subject matter. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0169] As used herein, the term "essentially free" with respect to a specified component is used herein to mean that none of the specified components are intentionally incorporated into the composition and / or are present only as contaminants or in trace amounts. Thus, the total amount of the specified component resulting from any unintentional contamination of the composition is well below 0.05%, preferably below 0.01%. Most preferred are compositions in which no amount of the specified component can be detected by standard analytical methods.

[0170] The term "antibody" refers to an intact immunoglobulin of any isotype, or a fragment thereof that can compete with the intact antibody for specific binding to a target antigen, including, for example, chimeric, humanized, fully human, and bispecific antibodies. An "antibody" is a species of antigen-binding protein. Intact antibodies generally contain at least two full-length heavy chains and two full-length light chains, although in some cases they may contain fewer chains, such as naturally occurring antibodies in camelids, which may contain only heavy chains. An antibody may be derived from only a single source or may be "chimeric," i.e., different portions of the antibody may be derived from two different antibodies, as described further below. Antigen-binding proteins, antibodies, or binding fragments are produced in hybridomas, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise indicated, the term "antibody" includes antibodies containing two full-length heavy chains and two full-length light chains, as well as derivatives, variants, fragments, and muteins thereof, examples of which are described below. Additionally, unless expressly excluded, antibodies include monoclonal antibodies, bispecific antibodies, minibodies, 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.

[0171] The structural unit of a natural antibody comprises a tetramer. Each such tetramer typically consists of two identical pairs of polypeptide chains, each pair having one full-length "light" chain (in certain embodiments, approximately 25 kDa) and one full-length "heavy" chain (in certain embodiments, approximately 50-70 kDa). The amino-terminal portion of each chain typically contains a variable region of approximately 100-110 or more amino acids that is typically responsible for antigen recognition. The carboxy-terminal portion of each chain typically defines a constant region that may be responsible for effector function. Human light chains are typically classified as kappa and lambda light chains. Heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including, but not limited to, IgM1 and IgM2. IgA is similarly subdivided into subclasses, including, but not limited to, IgA1 and IgA2. Within full-length light and heavy chains, the variable and constant regions are typically connected by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 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 / heavy chain pair typically form the antigen-binding site.

[0172] The term "variable region" or "variable domain" refers to a portion of an antibody light and / or heavy chain that typically comprises approximately the amino-terminal 120-130 amino acids in heavy chains and about 100-110 amino-terminal amino acids in light chains. In certain embodiments, the variable regions of different antibodies vary significantly in amino acid sequence, even among antibodies of the same species. The variable regions of an antibody typically determine the specificity of a particular antibody for its target.

[0173] Variable regions typically share the same general structure, with relatively conserved framework regions (FRs) connected by three hypervariable regions (also called complementarity-determining regions or CDRs). The CDRs from the two chains of each pair are typically aligned by the framework regions, which may enable binding to a specific epitope. From the N-terminus to the C-terminus, both light chain and heavy chain variable regions typically contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain typically follows the definitions of the 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).

[0174] In certain embodiments, an antibody heavy chain binds to an antigen in the absence of an antibody light chain. In certain embodiments, an antibody light chain binds to an antigen in the absence of an antibody heavy chain. In certain embodiments, an antibody binding region binds to an antigen in the absence of an antibody light chain. In certain embodiments, an antibody binding region binds to an antigen in the absence of an antibody heavy chain. In certain embodiments, an individual variable region specifically binds to an antigen in the absence of other variable regions.

[0175] Definitive delimitation of CDRs and identification of the residues that constitute the antibody binding site are accomplished by solving the structure of the antibody and / or the structure of the antibody-ligand complex, which may be accomplished by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. Various analytical methods can be used to identify or approximately predict CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition, and the contact definition.

[0176] The Kabat definition is a standard for numbering residues in antibodies and is typically used to identify CDR regions. See, e.g., Johnson & Wu, Nucleic Acids Res., 28:214-8 (2000). The Chothia definition is similar to the Kabat definition, but takes into account the location of certain structural loop regions. See, e.g., Chothia et al., J. Mol. Biol., 196:901-17 (1986); Chothia et al., Nature, 342:877-83 (1989). The AbM definition uses a suite of integrated computer programs produced by the Oxford Molecular Group to model antibody structure. See, e.g., Martin et al., Proc Natl Acad Sci (USA), 86:9268-9272 (1989), "AbM TM See, for example, "A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd. The AbM definition models the tertiary structure of antibodies from the primary sequence using a combination of knowledge databases and ab initio methods such as those described in Samudrala et al., "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198 (1999). The contact definition is based on an analysis of available complex crystal structures. See, for example, MacCallum et al., J. Mol. Biol., 5:732-45 (1996).

[0177] By convention, the CDR regions of the heavy chain are typically referred to as H1, H2, and H3, numbered sequentially from the amino terminus to the carboxy terminus, and the CDR regions of the light chain are typically referred to as L1, L2, and L3, numbered sequentially from the amino terminus to the carboxy terminus.

[0178] The term "light chain" includes full-length light chains and fragments thereof containing sufficient variable region sequence to confer binding specificity. A full-length light chain contains a variable region domain, VL, and a constant region domain, CL. The variable region domain of a light chain is at the amino-terminus of the polypeptide. Light chains include kappa chains and lambda chains.

[0179] The term "heavy chain" includes full-length heavy chains and fragments thereof containing sufficient variable region sequence to confer binding specificity. A full-length heavy chain contains a variable region domain, VH, and three constant region domains, CH1, CH2, and CH3. The VH domain is at the amino-terminus of the polypeptide, and the CH domain is at the carboxyl-terminus, with CH3 being closest to the carboxy-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.

[0180] Bispecific or bifunctional antibodies are typically artificial hybrid antibodies having two different heavy / light chain pairs 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, e.g., Songsivilai et al., Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol., 148:1547-1553 (1992).

[0181] The term "antigen" refers to a substance that can induce an adaptive immune response. Specifically, an antigen is a substance that serves as a target for a receptor of the adaptive immune response. Typically, an antigen is a molecule that binds to an antigen-specific receptor but is not capable of inducing an immune response in the body by itself. Antigens are usually proteins and polysaccharides, and less frequently lipids. As used herein, antigens also include immunogens and haptens.

[0182] The "Fc" region comprises two heavy chain fragments comprising the CH1 and CH2 domains of an antibody, held together by two or more disulfide bonds and hydrophobic interactions of the CH3 domain.

[0183] The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[0184] An antibody that "specifically binds to" or is "specific for" a particular polypeptide or epitope on a particular polypeptide is an antibody that binds to a particular polypeptide or an epitope on a particular polypeptide without substantially binding to any other polypeptides or epitopes of polypeptides. For example, a PIV F protein-specific antibody of the present disclosure is specific for PIV F protein. An antibody that binds to PIV F protein has 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 less, e.g., 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13 The antibody may have a dissociation constant (Kd) of 0.05 M.

[0185] The term "compete" when used in the context of antigen binding proteins (e.g., antibodies or antigen-binding fragments thereof) competing for the same epitope refers to competition between the antigen binding proteins as determined by an assay in which the antigen binding protein (e.g., antibody or antigen-binding fragment thereof) being tested blocks or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., a ligand, or reference antibody) to a common antigen (e.g., a PIV F protein or fragment thereof). Whether one antigen-binding protein competes with another antigen-binding protein can be determined by numerous types of competitive binding assays, such as solid-phase direct or indirect radioimmunoassays (RIAs), solid-phase direct or indirect enzyme immunoassays (EIAs), sandwich competition assays (see, e.g., Stahl et al., 1983, Methods in Enzymology 9:242-253); solid-phase direct biotin-avidin EIAs (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619); solid-phase direct labeled assays; solid-phase direct labeled sandwich assays (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct labeled RIAs using 1-125 labels (see, e.g., Morel et al., 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); 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-labeled 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 cells bearing any of these, 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 cells in the presence of the test antigen-binding protein. Typically, the test antigen-binding protein is present in excess.Antigen-binding proteins identified by competitive 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 adjacent epitope that is sufficiently close to the epitope bound by the reference antigen-binding protein to create steric hindrance. Additional details regarding 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) specific binding of the reference antigen-binding protein to a 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.

[0186] The term "epitope" as used herein refers to a specific atom or group of amino acids on an antigen to which an antibody binds. An epitope can be either a linear epitope or a conformational epitope. A linear epitope is formed by a continuous amino acid sequence from an antigen and interacts with an antibody based on its primary structure. On the other hand, a conformational epitope is composed of a discontinuous section of the amino acid sequence of an antigen and interacts with an antibody based on the antigen's 3D structure. Generally, an epitope is approximately 5 or 6 amino acids long. If two antibodies exhibit competitive binding to an antigen, they may bind to the same epitope within the antigen.

[0187] A useful measure of antibody potency in the art is the "50% neutralization titer." Another useful measure of antibody potency is any one of the following: "60% neutralization titer," "70% neutralization titer," "80% neutralization titer," and "90% neutralization titer." For example, to determine the 50% neutralization titer, serum from an immunized animal is diluted to assess how well the diluted serum retains its ability to block 50% of infectious viruses from entering cells. For example, a titer of 700 means that the serum retains its ability to neutralize 50% of infectious viruses after being diluted 700 times. Therefore, a higher titer indicates a stronger neutralizing antibody response. The titer may be a range having 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 50%, 60%, 70%, 80%, or 90% neutralization titer ranges are 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 11000, The upper limit can be 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, the 50% neutralization titer can be about 3,000 to about 6,500. "About" means plus or minus 10% of the stated value.

[0188] The term "host cell" means a cell that has been transformed or is capable of being transformed with a nucleic acid sequence and thereby expresses a gene of interest. The term includes the progeny of a parent cell, whether or not the progeny is identical in morphology or genetic make-up to the original parent cell, so long as the gene of interest is present.

[0189] The term "identity" refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. "Percent identity" refers to the percent of identical residues between amino acids or nucleotides in the compared molecules, and is calculated based on the size of the smallest molecule being compared. For these calculations, gaps in the alignment (if any) are preferably handled 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, A.M., ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D.W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A.M., and Griffin, H.G., 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.

[0190] When calculating percent identity, the sequences being compared are typically aligned to maximize the sequence identity.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 align the two polypeptides or polynucleotides that are to determine percent sequence identity.Sequences are aligned to optimize their corresponding amino acid or nucleotide match (the "matched range" as determined by the algorithm). A gap opening penalty (which is calculated as 3 × average diagonal element, where "average diagonal element" is the average of the diagonal elements of the comparison matrix being used and a "diagonal element" is the score or number assigned to each perfect amino acid match according to a particular comparison matrix) and gap extension penalty (which is usually 1 / 10 × gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62, can be used in conjunction 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 and 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.

[0191] Examples of parameters that can be used in determining 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.

[0192] A particular alignment scheme for aligning two amino acid sequences may result in matching of only a short region of the two sequences, and this small aligned region may have very high sequence identity even if there is no significant relationship between the two full-length sequences. Thus, the selected alignment method (GAP program) can be adjusted to result in an alignment spanning at least 50 or other number of consecutive amino acids of the target polypeptide, if so desired.

[0193] As used herein, the term "linked" refers to an association through intramolecular interactions, such as covalent, metallic, and / or ionic bonds, or through intermolecular interactions, such as hydrogen bonds or non-covalent bonds.

[0194] The term "operably linked" refers to an arrangement of elements such that the components so described are configured to perform their normal function. Thus, a given signal peptide operably linked to a polypeptide directs the secretion of the polypeptide from a cell. In the case of a promoter, a promoter operably linked to a coding sequence directs the expression of the coding sequence. A promoter or other control elements need not be contiguous with the coding sequence, so long as they function to direct the expression of the coding sequence. For example, untranslated but transcribed intervening sequences can be present between the promoter sequence and the coding sequence, and the promoter sequence would still be considered "operably linked" to the coding sequence.

[0195] Use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to alternatives only or the alternatives are not mutually exclusive, even if the disclosure supports a definition that refers only to alternatives and "and / or." As used herein, "another" can mean at least a second or more.

[0196] The terms "polynucleotide" or "nucleic acid" include both single-stranded and double-stranded nucleotide polymers. The nucleotides that make up a polynucleotide can be ribonucleotides or deoxyribonucleotides, or modified forms of either type of nucleotide. Such 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, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoroaniladate, and phosphoramidate.

[0197] The terms "polypeptide" and "protein" refer to a polymer having the amino acid sequence of a native protein, i.e., a protein produced by a non-recombinant cell in the natural state, or include molecules produced by genetic engineering or recombinant cells that have the amino acid sequence of a native protein or that have one or more amino acid deletions, additions, and / or substitutions of 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 that have one or more amino acid deletions, additions, and / or substitutions of antigen-binding proteins. The term "polypeptide fragment" refers to a polypeptide that has an amino-terminal deletion, a carboxyl-terminal deletion, and / or an internal deletion compared to the full-length native protein. Such fragments may also contain modified amino acids compared to the native protein. Fragments may be between about 5 and 500 amino acids in length. For example, fragments can be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids in length. Useful polypeptide fragments include immunologically functional fragments of antibodies that contain the binding domain. In the case of PIV F protein-binding antibodies, useful fragments include, but are not limited to, CDR regions, heavy and / or light chain variable domains, portions of antibody chains containing two CDRs, or simply the variable regions thereof.

[0198] Pharmaceutically acceptable carriers are conventional. Remington's Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the fusion proteins disclosed herein. Generally, the nature of the carrier will depend on the particular mode of administration being employed. For example, parenteral formulations usually comprise injectable fluids that contain pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered may contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents, for example, sodium acetate or sorbitan monolaurate.

[0199] As used herein, the term "subject" refers to a human or any non-human animal (e.g., a mouse, rat, rabbit, dog, cat, cow, goat, pig, sheep, horse, or primate). Human includes prenatal and postnatal forms. A subject may be a human. A subject may be a patient, which refers to a human who visits a healthcare 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 disorder, but may or may not exhibit symptoms of the disease or disorder.

[0200] As used herein, the term "therapeutically effective amount" or "effective dosage" refers to a dosage or concentration of a drug effective to treat a disease or condition, e.g., with respect to the use of a monoclonal antibody or antigen-binding fragment thereof disclosed herein to treat a viral infection.

[0201] As used herein, "treating" or "treatment" of a condition includes preventing or alleviating the condition, slowing the onset or rate of development of the condition, reducing the risk of developing the condition, preventing or delaying the onset of symptoms associated with the condition, reducing or terminating symptoms associated with the condition, causing complete or partial regression of the condition, curing the condition, or any combination thereof.

[0202] As used herein, a "vector" refers to a nucleic acid molecule that is introduced into a host cell, thereby producing a transformed host cell. A vector may contain nucleic acid sequences that enable it to replicate in the host cell, such as an origin of replication. A vector may also contain one or more therapeutic genes and / or selectable marker genes, as well as 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 other than those native to the cell. A vector optionally includes materials that aid in achieving entry of the nucleic acid into the cell, such as a viral particle, liposome, protein coat, etc. [Example]

[0203] IX. Working Example The following examples are included to demonstrate preferred embodiments of the invention. It should be understood by those of skill in the art that the techniques disclosed in the examples which follow are representative of techniques discovered by the inventors to function well in the practice of the invention, and therefore can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, understand that many changes can be made in the specific embodiments which are disclosed and still obtain like or similar results, without departing from the spirit and scope of the invention.

[0204] Example 1 The sequence used for hPIV3 F ectodomain design contains residues 19-481 of SEQ ID NO:1. In screening single-substitution designs, the L168Q substitution was included to increase solubility and provide more consistent purification. The GCN4 CC tri2 trimerization domain was included. The construct further contained an HRV3C protease recognition site, an octa-histidine tag, and a tandem Twin-Strep tag, which were cloned into the mammalian expression plasmid pαH. The sequence of this base construct with the L168Q substitution is shown in SEQ ID NO:8, with a lysine at position 141.

[0205] Using structure-based design, substitutions aimed at prioritizing the stability of the prefusion conformation were introduced into the base construct (PIV3F L168Q ectodomain with a GCN4 trimerization tag that adopts a postfusion conformation, Figure 3; lysine 141 in SEQ ID NO:8). Pairs of core-facing residues separated by less than 5 Å were replaced with aromatic side chains or pairs of aromatic and positively charged side chains to favor π-π or π-cation interactions, respectively. Alternatively, residues were replaced with elongated or bulkier hydrophobic side chains to fill existing internal cavities. Disulfide bonds were engineered to increase overall stability or prevent the formation of postfusion conformations. Charged or polar substitutions were aimed at forming hydrogen bonds or salt bridges with native residues predicted to be within 4.0 Å.

[0206] The single substitution designs were carried forward to a combinatorial round of screening, in which the L168Q substitution from the first round was changed back to leucine. For some combinatorial variants, the L168Q substitution was later reintroduced. Additionally, the T4 fibritin foldon trimerization motif was included between the GCN4 CC tri2 trimerization domain and the HRV3C protease recognition site (SEQ ID NO: 11, lysine at position 141).

[0207] Exemplary single substitution designs are provided in Table 1. Exemplary substitution combinations are provided in Table 2.

[0208] 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. Cultures were grown for 4–6 days, and the culture supernatant was isolated by centrifugation and passage through a 0.22 μm filter. Proteins were purified from the supernatant using StrepTactin resin (IBA). hPIV3 F variants were 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. Protein purity, monodispersity, and expression levels were determined by SDS-PAGE (e.g., Figures 4, 8, 10, 11, 17, 18, and 21) and SEC (e.g., Figures 9, 12, 13A, and 22). The first peak corresponds to multimers of trimeric post-fusion hPIV3 F, and the second peak corresponds to monomeric hPIV3 F trimers.

[0209] Negative staining electron microscopy (nsEM) analysis was performed on a portion of the hPIV3 F variant. The purified hPIV3 F variant was 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 a CF-400-CU grid (Electron Microscopy Sciences) that had been plasma-cleaned for 30 seconds with a 4:1 O2 / H2 ratio in a Solarus 950 plasma cleaning device (Gatan) and stained with 2% (w / v) uranyl acetate. The grid was imaged at 60,000X magnification (corresponding to a calibrated pixel size of 3.6 Å / pixel) in a 2010f TEM (Japan Electron Optics Laboratory) operated at 200 kV and equipped with a OneView camera (Gatan). Exemplary data are shown in Figures 6 and 17.

[0210] To confirm that the stabilizing substitutions did not result in any unintended conformational changes, cryo-EM structures of various designs were determined (Figures 3, 13B, 14-16, 19, and 20). Purified hPIV3 F variants were diluted to concentrations ranging from 1 to 3 mg / mL in 2 mM Tris pH 8.0, 200 mM NaCl, and 0.02% NaN3 and applied to plasma-cleaned CF-400 1.2 / 1.3 grids or UltrAuFoil 1.2 / 1.3 grids. They were then blotted for 3–6 s with a Vitrobot Mark IV (ThermoFisher) and plunge-frozen in liquid ethane. Micrographs were collected from single grids using either (i) a Titan Krios (ThermoFisher) equipped with a K3 direct electron detector (Gatan) or (ii) a Glacios (ThermoFisher) equipped with a Falcon IV. Data were collected at a calibrated magnification of 0.83 Å / pixel for Krios and 0.94 Å / pixel for Falcon 4. ***

[0211] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations may be applied to the methods and in the steps or sequence of steps of the methods described herein without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain agents that are chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.

[0212] References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. International Patent Publication No. WO2018 / 081289 International Patent Publication No. WO2022 / 207839 U.S. Patent Publication No. 2022 / 0024987

Claims

1. 1. An engineered protein comprising a parainfluenza virus fusion protein (PIV F) ectodomain having at least 90% sequence identity to amino acids 19-481 of SEQ ID NO: 1 or 2, wherein the engineered protein is 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, S 52L, L53C / S174C, P55C / V175C, P55C / Q176C, K56C / N155C, I57F, E58C / I183C, G64C / G196C, G64C / G200C, Q67C / L199C, Q67C / G200C, Y71C / L203 C, L86C / V266C, Q89C / A131C, K90Y, I93C / G116C, V94C / G116C, T95C / G1 16C, T117P, I118C / G381C, A119C / G381C, L120C / G381C, A123P, T124P, S 125C / P374C, S125P, S125W, A126P, I128F, I128W, L134C / I267C, A137C / I267C, I144W, L147C / A171C, I151C / A171C, A157C / Q176C, A157C / D17 7C, A157F, V158L, Q159C / A171C, L168Q, V170I, V170M, A171V, K173Q, V 175L, V175P, V179L, E182F, E182W, P185C / A195C, G191P, G200E, I201F, I201W, A202T, E209W, I213C / I226C, I213C / G230C, G219C / E333C, L228 C / 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, G38 1C / K431C, G382C / G433C, V384I, T413C / A436C, G433F, I443W, I443Y, V449C / I454C, V449C / D455C, V449C / I456C, the engineered protein comprising at least one substitution or set of substitutions selected from the group consisting of V449C / S457C, A450F, L451P, D452P, I454F, D455K, I456W, S457C / V449C, S457C / I456C, K464C / V449C, S470C / K471C, K471A, K471L, W473A, the position of which is relative to SEQ ID NO: 1 or 2.

2. H27C / F437C, H27C / T439C, H27C / P440C, H27C / I443C, N33C / K295C, G37C / S337C, S41C / P283C, Y48C / I169C, Y48C / I169C / A 140Y, L49C / L278C, I50C / A171C, I50C / T277C, S52C / K173C, S52C / S275C, L53C / S174C, P55C / V175C, P55C / Q176C, K56C / N1 55C, E58C / I183C, G64C / G196C, G64C / G200C, Q67C / L199C, Q67C / G200C, Y71C / L203C, L86C / V266C, Q89C / A131C, I93C / G11 6C, V94C / G116C, T95C / G116C, I118C / G381C, A119C / G381C, L120C / G381C, S125C / P374C, L134C / I267C, A137C / I267C, L147 C / A171C, I151C / A171C, A157C / Q176C, A157C / D177C, Q159C / A171C, P185C / A195C, I213C / I226C, I213C / G230C, G219C / E3 33C, L228C / V264C, D327C / P344C, F346C / T369C, F346C / S370C, S361C / T444C, Q362C / N447C, P364C / N447C, T366C / V449C, N 2. The engineered protein of claim 1, comprising at least one set of pairwise cysteine ​​substitutions selected from the group consisting of 380C / 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. 3. The engineered protein of claim 1 or 2, further comprising at least one set of paired cysteine ​​substitutions selected from the group consisting of S186C / A195C.

4. 4. The engineered protein of claim 2 or 3, wherein the pair of cysteine ​​substitutions form a disulfide bond.

5. H27Y, V28M, V30I, L49F, L49W, I50W, S52L, I57F, K90Y, A140Y, I144W, A157F, V158L, V170I, V170M, A171V, V175L, V1 79L, E182F, E182W, G200E, I201F, I201W, L228F, L228W, R236Y, S246V, L256Y, V264F, V264W, V266F, V266W, S275F, S 5. The engineered protein of any one of claims 1 to 4, comprising at least one cavity filling substitution or set of cavity filling substitutions selected from the group consisting of: 275M, T277F, T277L, T277W, L278F, L278W, V280F, V280W, R281Y, L282F, L282W, G345M, L356F, V384I, G433F, I443W, I443Y, A450F, I454F, and I456W.

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

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

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

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

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

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

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

13. 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 / I267C / V158L、A137C / 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 / A123P、L147C / A171C / S125P、L147C / A171C / I201W、L147C / A171C / L282F、L147C / A171C / L228W、L147C / A171C / R281Y、L147C / A171C / L282W、L147C / A171C / N349P、L147C / A171C / T367R、L147C / A171C / K471A、G64C / G196C / A137C / I267C / K471A、G64C / G196C / A137C / I267C / V175L、G64C / G196C / A137C / I267C / S125P、G64C / G196C / A137C / I267C / S125P / V175L、G64C / G196C / A137C / I267C / T367R、G64C / G196C / A137C / I267C / K471A / S125P、G64C / G196C / 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 / G196C / A137C / I267C / V28M / V175L / I201W / L228W / R281Y, L168Q / G6 4C / G196C / A137C / I267C / V28M / V175L / I201W / L282F / L228W / R281Y, L168Q / I151C / A171C / V449C / S457C, L168Q / I151C / A171C / V449C / S457C / V28M / V175L / R281Y, I 151C / A171C / S186C / A195C / V28M / V175L / R281Y, L168Q / I151C / A171C / L228W / L28 11. The engineered protein of any one of claims 1 to 10, comprising a set of substitutions selected from the group consisting of: I151C / A171C / L228W / L282F / R821Y / V175L, I151C / A171C / L228W / L282F / R821Y / V175L / G200E / I57F, I151C / A171C / L228W / L282F / R821Y / V175L / G200E / I57F / V449C / S457C, I151C / A171C / G200E / I57F, and I151C / A171C / G200E / I57F / V449C / S457C.

14. 14. The engineered protein of any one of claims 1 to 13, wherein the substitution or set of substitutions is selected from any one of the substitutions and sets of substitutions in Tables 1 and 2.

15. 15. The engineered protein of any one of claims 1 to 14, comprising a L168Q substitution.

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

17. 17. The engineered protein of any one of claims 1 to 16, wherein the human PIV F ectodomain is a hPIV3 F ectodomain.

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

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

20. 20. The engineered protein of claim 19 fused to a trimerization domain.

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

22. the trimerization domain is 22. The engineered protein of any one of claims 19 to 21, comprising a sequence selected from the group consisting of:

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

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

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

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

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

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

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

30. 30. The engineered protein of any one of claims 1 to 29, which 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. 32. The engineered trimer of claim 31, which is stabilized in the prefusion conformation compared to a native PIV F protein subunit trimer.

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

34. 34. The engineered trimer of claim 33, comprising 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 an engineered protein according to any one of claims 1 to 30.

36. 36. The nucleic acid molecule of claim 35, further comprising a DNA expression vector.

37. 36. The nucleic acid molecule of claim 35, which is mRNA.

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

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

40. 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-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and 2'-O-methyluridine.

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

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

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

44. 44. The pharmaceutical composition of any one of claims 41 to 43, formulated in cationic lipid nanoparticles.

45. 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 to 44.

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

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

48. 45. The pharmaceutical composition of any one of claims 41 to 44 for use in the treatment or prevention of a parainfluenza virus (PIV) infection or a disease associated with PIV infection in a subject.

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

50. A pharmaceutical composition according to any one of claims 41 to 44 for use in eliciting an immune response against parainfluenza viruses (PIV).

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

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

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

Images