CN120569211A - Human parainfluenza virus 3F protein stabilized before fusion - Google Patents

Abstract

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

Description

Human parainfluenza virus 3F protein stabilized before fusion

Citation of related application

The present application claims priority from U.S. provisional application No. 63/479,127, filed on 1 month 9 of 2023, the entire contents of which are incorporated herein by reference.

Reference to sequence Listing

The present application contains sequence Listing XML that has been electronically submitted and is hereby incorporated by reference in its entirety. The sequence Listing XML was created at 2024, 1/5, under the name UTFBP1325WO_ST26.XML, and was 32,897 bytes in size.

Background

1. Technical field

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

2. Description of related Art

Parainfluenza viruses 1,2,3 and 4 (PIV 1-4) cause mild to severe respiratory disease in humans. PIV is an enveloped single stranded antisense RNA [ ssRNA (-) ] virus as a member of the paramyxoviridae family. Seasonal PIV infections are associated with about 40% of hospitalized cases of lower respiratory tract infections in children and about 75% of definite croup cases. PIV infections may occur throughout a person's lifetime. Most adults are at low risk of suffering from serious disease after infection with PIV, but infections in immunocompromised individuals and the elderly sometimes lead to serious or life threatening lower respiratory tract disease. Although development of a vaccine against PIV is critical to public health, no vaccine has been licensed to date.

Long term monitoring of respiratory viruses in the united states has found PIV3 to be the most common PIV serotype associated with symptomatic diseases in children and adults, followed by PIVs 1, 2 and 4. The genome of PIV encodes several envelope glycoproteins, one of which is the fusion glycoprotein (F). The F glycoprotein is a fusion agent necessary for the entry of the virus into cells and is also an important target for neutralizing antibody (nAb) infection reactions. PIV F proteins have varying degrees of sequence conservation, with the exemplary F proteins of the respiratory and mumps genera ranging from 20-50% identical and 35-65% similar. Although PIV vaccines incorporating F subunit antigens are under development, no PIV vaccine or antiviral therapy has been approved for use in humans. Thus, a vaccine that provides long lasting protection and therapies that avoid death and morbidity are desirable.

Thus, there is a need for safe and effective immunogenic compositions and therapies to protect against PIV infection and its associated sequelae. Diagnostic reagents are also needed to detect immune responses to PIV, guide the design of F-based PIV vaccines, and support the development of therapeutic or prophylactic antibodies against PIV.

Disclosure of Invention

Thus, provided herein are engineered proteins having at least one amino acid substitution relative to the amino acid sequence of the natural respiratory tract virus or mumps virus F protein (i.e., SEQ ID NOS: 1-7), wherein the engineered proteins are stable in the pre-fusion conformation of the respiratory tract virus or mumps virus F. The engineered proteins may specifically bind to antibodies specific for respiratory tract virus or mumps virus F protein pre-fusion.

In one embodiment, provided herein is an engineered protein comprising an extracellular domain of a respiratory tract virus or a mumps virus fusion protein (preferably, a parainfluenza virus fusion protein (PIV F)) having at least 90% sequence identity to amino acids 19-481 of SEQ ID No. 1 or 2, said engineered protein comprising at least one substitution or a set of substitutions ：H27C/F437C;H27C/T439C;H27C/P440C;H27C/I443C;H27Y;V28M;V30I;N33C/K295C;G37C/S337C;S41C/P283C;Y48C/I169C;Y48C/I169C/A140Y;L49C/L278C;L49F;L49W;I50C/A171C;I50C/T277C;I50W;S52C/K173C;S52C/S275C;S52L;L53C/S174C;P55C/V175C;P55C/Q176C;K56C/N155C;I57F;E58C/I183C;G64C/G196C;G64C/G200C;Q67C/L199C;Q67C/G200C;Y71C/L203C;L86C/V266C;Q89C/A131C;K90Y;I93C/G116C;V94C/G116C;T95C/G116C;T117P;I118C/G381C;A119C/G381C;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;E182F;E182W;P185C/A195C;G191P;G200E;I201F;I201W;A202T;E209W;I213C/I226C;I213C/G230C;G219C/E333C;L228C/V264C;L228F;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;P364C/N447C;T366C/V449C;T367R;N380C/G433C;G381C/K431C;G382C/G433C;V384I;T413C/A436C;G433F;I443W;I443Y;V449C/I454C;V449C/D455C;V449C/I456C;V449C/S457C;A450F;L451P;D452P;I454F;D455K;I456W;S457C/V449C;S457C/I456C;K464C/V449C;S470C/K471C;K471A;K471L;W473A, selected from the group consisting of and wherein said position is relative to SEQ ID No. 1 or 2.

The engineered protein may or may not further comprise at least one set of paired cysteine substitutions ：H27C/F437C;H27C/T439C;H27C/P440C;H27C/I443C;N33C/K295C;G37C/S337C;S41C/P283C;Y48C/I169C;Y48C/I169C/A140Y;L49C/L278C;I50C/A171C;I50C/T277C;S52C/K173C;S52C/S275C;L53C/S174C;P55C/V175C;P55C/Q176C;K56C/N155C;E58C/I183C;G64C/G196C;G64C/G200C;Q67C/L199C;Q67C/G200C;Y71C/L203C;L86C/V266C;Q89C/A131C;I93C/G116C;V94C/G116C;T95C/G116C;I118C/G381C;A119C/G381C;L120C/G381C;S125C/P374C;L134C/I267C;A137C/I267C;L147C/A171C;I151C/A171C;A157C/Q176C;A157C/D177C;Q159C/A171C;P185C/A195C;I213C/I226C;I213C/G230C;G219C/E333C;L228C/V264C;D327C/P344C;F346C/T369C;F346C/S370C;S361C/T444C;Q362C/N447C;P364C/N447C;T366C/V449C;N380C/G433C;G381C/K431C;G382C/G433C;T413C/A436C;V449C/I454C;V449C/D455C;V449C/I456C;V449C/S457C;S457C/V449C;S457C/I456C;K464C/V449C; and S470C/K471C selected from the group consisting of. The engineered protein may also comprise cysteine substitutions in the S186C/A195C pair. The paired cysteine substitutions preferably form disulfide bonds.

The engineered protein may or may not further comprise at least one cavity-filling substitution or a set of cavity-filling substitutions ：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;V266W;S275F;S275M;T277F;T277L;T277W;L278F;L278W;V280F;V280W;R281Y;L282F;L282W;G345M;L356F;V384I;G433F;I443W;I443Y;A450F;I454F; and I456W selected from the group consisting of. Substitutions may form a salt bridge within a substitution pair or between a single substitution and a natural amino acid in the protein.

The engineered protein may or may not further comprise at least one substitution or a set of substitutions selected from the group consisting of T117P, A123P, T124P, S125P, A126P, V175P, G191P, N349P, L451P, and D452P.

The engineered protein may or may not further comprise at least one substitution or a set of substitutions selected from the group consisting of S125W, I128F, I128W, E209W, and R236W.

The engineered protein may or may not further comprise at least one substitution selected from the group consisting of K173Q, A202T, A334S, T367R, D455K, K471A, K471L, and W473A.

The engineered protein may or may not also comprise E at position 108.

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.

The engineered protein may comprise a set of substitutions ：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/G64C/G196C/A137C/I267C/V28M/V175L/I201W/L282F/L228W/R281Y;L168Q/I151C/A171C/V449C/S457C;L168Q/I151C/A171C/V449C/S457C/V28M/V175L/R281Y;I151C/A171C/S186C/A195C/V28M/V175L/R281Y;L168Q/I151C/A171C/L228W/L282F/R821Y/V175L;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 selected from the group consisting of.

The engineered protein may comprise 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.

The respiratory virus or mumps virus fusion protein (preferably parainfluenza virus fusion protein (PIV F)) ectodomain of the engineered protein may be the human PIV (hPIV) F ectodomain. The human PIV F ectodomain may be the hPIV 3F ectodomain. The hPIV 3F extracellular domain may 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.

The engineered protein may not comprise the cytoplasmic tail of PIV F.

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

MKQIEDKIEEILSKIYHIENEIARIKKLIGEAP、IEDKIEEILSKIY HIENEIARIKKLIGEAP、MKQVEDKIEEILSKIYHIENEIARIKKLIGEAP、VEDKIEEILSKIYHIENEIARIKKLIGEAP、DTYLSAIEDKIEEIL SKIYHIENEIARI、LKQIVLRIMEIEARIAKIE、LKQIVLRIMEIEARI AKIEGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG、LKQIVLRIMEI EARIAKIEGSEFNSLKQIVLRIMEIEARIAKIE、LKQIVLRIMEIEARI AKIEGSLKQIVLRIMEIEARIAKIE And LKQIVLRIMEIEARIAKIEGSL ELIKLRIMEIEARIAKIEKDRAIL.

The engineered protein may be fused to or conjugated to the transmembrane domain. The transmembrane domain may comprise a PIV F protein transmembrane domain. The PIV F protein transmembrane domain may comprise sequence IIIILIMMIILFIINITIITI. The transmembrane domain may not comprise a PIV F protein transmembrane domain.

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

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

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

In one embodiment, provided herein are nucleic acid molecules comprising a nucleotide sequence encoding an amino acid sequence of an engineered protein disclosed herein. The nucleic acid molecule may also include a DNA expression vector. The nucleic acid molecule may be an 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 may be selected from the group consisting of pseudouridine, N1-methyl pseudouridine, N1-ethyl pseudouridine, 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-dihydro-pseudouridine, 2-thio-dihydro-uridine, 2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydro-pseudouridine, 5-methoxy-uridine and 2' -0-methyl-uridine.

In one embodiment, provided herein is a pharmaceutical composition comprising (i) an engineered protein disclosed herein, (ii) an engineered trimer disclosed herein, or (iii) a nucleic acid molecule disclosed herein, and a pharmaceutically acceptable carrier. The pharmaceutical composition may further comprise an adjuvant. The pharmaceutical composition may further comprise another PIV antigen. The pharmaceutical composition may be formulated within cationic lipid nanoparticles. The pharmaceutical compositions are useful for treating or preventing parainfluenza virus (PIV) infection or a disease associated with PIV infection in a subject, or for eliciting an immune response against parainfluenza virus (PIV). The subject may be a mammal, such as a human.

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

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 a PIV infection.

In one embodiment, provided herein are compositions comprising (i) an engineered protein provided herein or (ii) an engineered trimer provided herein in combination with an antibody. The antibodies can specifically bind to the PIV F ectodomain in the pre-fusion conformation.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the 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.

Drawings

The following drawings form a 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.

FIG. 1 shows a sequence identity and similarity matrix for representative respiratory viruses and mumps virus viruses. Multiple sequence alignment (Clustal Omega) was analyzed to determine the pair-wise sequence identity and similarity of a representative set of F proteins from respiratory viruses and mumps. The dots in each matrix are colored in their percent values from 0% to 100% [ right scale ]. Similarity and identity were scored by SIAS server 、SECRETARIA GENERAL DE CIENCIA、TECNOLOGIA EINNOVACION OF SPAIN[http://imed.med.ucm.es/Tools/sias.html], hosted by the university of mad compton s immunology group.

FIG. 2 alignment of clustered sequences of human respiratory virus type 1 and type 3 (also referred to as HPIV1 and HPIV 3) fusion proteins. The sequence identity of the soluble extracellular domain is 44% and the sequence similarity is >60%. The same residues at homologous positions are colored with white letters on a black background and similar amino acids are colored with black letters on a gray background. Non-similar residues at homologous sites are black in color with a white background.

FIG. 3. Low temperature EM structure of PIV 3F base with C-terminal GCN4 tag in post-fusion conformation (L168Q). (upper left panel) representative low temperature electron micrographs. (upper right panel) representative class 2D average. (bottom left) golden standard fourier shell correlation diagram showing GSFSC versus angstrom resolution. The resolution value corresponds to an FSC cut-off value of 0.143. (lower right panel) coulomb potential diagram or coulomb potential diagram of PIV 3F base trimer in post-fusion conformation.

FIG. 4 SDS-PAGE of single substitutions. PIV 3F protein was purified by affinity chromatography and analyzed by reducing SDS-PAGE [ Coomassie blue staining ]. Variant ID is indicated at the top of each lane. The integrated band intensities were quantified using ImageJ/Fiji or Licor Odyssey CLx. The left side of each gel included a molecular weight marker. For the bottom-most plot, the affinity chromatography flow-through [ FT ] and elution [ E ] are shown.

Figure 5 biological layer interferometry quantification of binding of hpiv 3F variants to pre-fusion specific antibody PIA174 IgG. Each graph shows the response (nm) versus time (seconds) of PIV 3F variants binding to the AHC tip. The AHC tip was functionalized with PIA174 IgG to recognize pre-fusion PIV 3F.

FIG. 6 negative staining electron micrographs of disulfide bond variants JM-17 (I50C/A171C) and JM-20 (A137C/I1267C) in the pre-fusion conformation. The top of each plot provides representative class 2D averages of JM-17 left and JM-20 right negatively stained EM images. The size of the box is shown below the 2D class and is equal to 230 angstroms. The bottom of each plot provides 3D reconstructions of JM-17[ left ] and JM-20[ right ] of the pre-fusion conformation.

FIG. 7 biological layer interferometry quantification of relative expression yield of HPIV3F variants. Each graph shows the response (nm) versus time (seconds) of PIV 3F variants binding to the AHC tip. The AHC tip was functionalized with MF5 IgG, recognizing the foldon tag. The binding curve was fitted to a straight line at the initial part of the curve, which corresponds to a 60 second window after immersion in medium containing each variant from the small-scale HEK293F expression culture.

FIG. 8.C SDS-PAGE quantification of combinatorial variants containing tandem GCN4/Foldon tags. Individual variant IDs and combined variant IDs are indicated above each lane. The integrated band intensities were plotted under each gel and quantified in Licor Odyssey CLx.

Fig. 9. Size exclusion chromatography of PIV3F variants each graph shows the mAU versus elution volume for a PIV3F variant group. Each sample shown in this figure was transfected and purified in parallel under the same conditions. Briefly, PIV3F variants were purified by STREPTACTIN SEPHAROSE, then concentrated and flash frozen in liquid nitrogen. Samples were thawed and analyzed one by one through the same Superose 6 Size Exclusion Chromatography (SEC) column. For ease of comparison, the UV trace of the base construct sample L168Q is reproduced on each plot.

FIG. 10. Additional SDS-PAGE quantification of combinatorial variants containing the tandem GCN4/Foldon tag at the C-terminus. The integrated band intensities of a set of individual and combined variant gels were plotted and quantified in Licor Odyssey CLx.

Fig. 11 SDS PAGE after size exclusion chromatography of piv3f combination variants 41 and 43. Large scale expression (500 mL) of both combination variants 41 and 43 was first purified by Streptomyces affinity chromatography, followed by size exclusion chromatography (Superose 6).

Fig. 12 size exclusion chromatography of piv3f combinatorial variants 43, 56, 57 and 58. Superose 6 size exclusion elution curves for a set of combinatorial variants, wherein absorbance units at 280 nm [ mAU ] are plotted against elution volume in milliliters [ mL ]. In the inset, the yield [ mg/mL ] and apparent melting temperature (Tm _app,℃).Tm_app is determined by differential scanning fluorescence) of the expressed protein are indicated alongside each variant ID.

FIGS. 13A-13B. Size exclusion chromatography and cryoEM of PIV F variant L168 Q+I151C/A171C. (FIG. 13A) SEC of three variants, L168Q, L168Q+I151C/A171C and L186Q+I213C/G230C. The traces show characteristic trimers and dimer peaks of trimers. (FIG. 13B) Low temperature EM of the L168Q+I151C/A171C variant. The figure shows the 2D class average,An enlarged view of the model in the 3D reconstruction and coulomb potential diagram.

FIG. 14. Low temperature EM structure of PIV 3F base (L168Q) with tandem GCN4/Foldon tag at C-terminus of pre-fusion conformation without any pre-fusion specific antibodies. (upper left panel) representative low temperature electron micrographs. (upper right panel) representative class 2D average. (bottom left) golden standard fourier shell correlation diagram showing GSFSC versus angstrom resolution. The resolution value corresponds to an FSC cut-off value of 0.143. (lower right panel) coulomb potential or coulomb potential plot of PIV 3F bases with C-terminal tandem GCN4/foldon tag in pre-trimer fusion conformation. The PIV 3F source colors are blue, red and green. The color of the extended portion of the structure stabilized by the C-terminal tag is gray.

FIG. 15 Low temperature EM structure of PIV 3F combinatorial variant 43 (Combo 43) in pre-fusion conformation complexed with pre-fusion specific antibody PIA 174. (upper left panel) representative low temperature electron micrographs. (upper right panel) representative class 2D average. (left middle plot) golden standard fourier shell correlation plot, showing GSFSC versus angstrom resolution. The resolution value corresponds to an FSC cut-off value of 0.143. (right middle panel) coulomb potential or coulomb potential plot of PIV 3F variant combination 43 (I151C/a 171C) with C-terminal tandem GCN4/foldon tag in pre-trimer fusion conformation. The PIV 3F Combo 43 source colors are blue, red and green. The color of the extended portion of the structure stabilized by the C-terminal tag is gray. (lower panel) magnified views of all exemplary substituted atomic models within the coulomb potential diagram are shown in gray. Atoms are colored according to substitution type, disulfide bonds, proline and cavity filling.

FIG. 16 Low temperature EM of PIV variant I93C/G116C. The figure shows representative photomicrographs, class 2D averages and for PIV F variant I93C/G116CReconstruction, wild-type PIV F was used as background (Leu at position 168).

FIG. 17 biochemical and structural characterization of the PIV 3F variant PB-68, V449C/S457C, which forms an inter-protogenic disulfide bond. (upper left panel) non-reducing SDS-PAGE analysis of the PIV3 variant PB-68, V449C/S457C, which runs as disulfide-linked trimers. A control PIV3 variant was included that did not form inter-protomer disulfide bonds and was run as a single protomer. (upper right panel) representative 2D class average for PIV3 variant PB-68 under negative staining electron microscope. (lower right panel) 3D negative staining EM reconstruction of PB-68. (lower left panel) an enlarged view of the simulated substitution sites around the heptad repeat.

FIG. 18A-18B. Sample stability test of PIV F variant PB-68, V449C/S457C. (FIG. 18A) purified V449C/S457C protein was incubated at 4℃for 0, 7 and 30 days, and then analyzed by non-reducing SDS-PAGE. (FIG. 18B) separately prepared V449C/S457C proteins were incubated at 37℃for 1, 7 and 14 days and then analyzed by reducing and non-reducing SDS-PAGE. The bands indicated by the arrows indicate the presence of non-reducing trimers. A small portion of the sample was run at the original expected size under non-reducing conditions (low molecular weight band, near 71kDa MW marker), which may also form disulfide bonds in the original.

FIGS. 19A-19B. Four low temperature EM structures of PIV F Combo variants 41 and 58, each with two oligomeric states. Representative class averages and coulomb potential plots for trimer and dimer of trimer of Combo variant 41 (fig. 19A) and Combo variant 58 (fig. 19B). The resolution of each structure is indicated beside the figure.

FIGS. 20A-20B. Variants employ a mixture of closed and open conformations at the PIA174 binding site, which can be biased towards closing by substitution at the central trimer interface. (FIG. 20A) Combo41 coulomb potential plot for four unique conformations, varying from open to closed, indicating conformational heterogeneity of the central apical region (black arrow). (FIG. 20B) Combo58 Coulomb potential diagram in closed conformationAlthough we have made a lot of effort to classify based on conformational heterogeneity of the central apical region (arrow), no class was observed in the open conformation. Atomic model and enlarged view of Combo58, substitution position 201.

FIG. 21 SDS-PAGE of PIV 3F wild-type, single and combined variants. Wild-type PIV 3F and several variants (L168Q, I C/A171C, combo61, combo61-0-1, combo62-0 and Combo 62-0-1) were analyzed by reducing and non-reducing SDS-PAGE. Variants containing V449C/S457C substitutions (Combo 61-0-2 and Combo 62-0-1) each showed the expected inter-protomer disulfide trimeric bands.

Fig. 22 size exclusion chromatograms of wild-type, single and combined variants of piv 3F. Each graph shows the mAU versus elution volume for the PIV3F variant group. Each sample shown in this figure was transfected and purified in parallel under the same conditions. Briefly, PIV3F variants were purified by STREPTACTIN SEPHAROSE, then concentrated and flash frozen in liquid nitrogen. Samples were thawed and analyzed one by one on the same Superose 6 column. For ease of comparison, the UV trace of wild-type PIV3F samples was simulated in each figure.

Thermal stability analysis of wild-type, single and combinatorial variants of piv 3F. Purified PIV F protein purified in parallel under the same conditions was analyzed by differential scanning fluorescence. The y-axis in the graph represents fluorescence as a function of temperature, and the x-axis represents temperature. Variants are plotted in groups, with wild-type DSF trajectories simulated in each figure for comparison.

Detailed Description

Provided herein are engineered parainfluenza virus (PIV) fusion (F) proteins having one or more amino acid substitutions that stabilize the PIV F protein in a pre-fusion conformation. The pre-fusion PIV F may be used as a vaccine antigen or reagent for detection and/or isolation of antibodies in serum. The pre-fusion PIV F proteins and nucleic acids encoding the proteins described herein are useful, for example, as potential immunogens in immunogenic compositions or vaccines against PIV, in methods of inducing immune responses in a subject, and as diagnostic tools, among other uses.

I. Natural hPIV F

The envelope glycoprotein of hPIV1, hPIV2, hPIV3 or hPIV4 promotes fusion of the virus and cell membrane. In nature, the F proteins hPIV1, hPIV2, hPIV3 and hPIV4 were initially synthesized as single polypeptide precursors, approximately 550 amino acids in length, called F0. F0 comprises an N-terminal signal peptide which directs its localization to the endoplasmic reticulum, where it is proteolytically cleaved. The remaining F0 residues oligomerize to form trimers and can be proteolytically processed by cellular proteases to produce two disulfide-linked fragments F1 and F2. In hPIV 1F the cleavage site is located between about residues 112/113, in hPIV 2F the cleavage site is located between about residues 106/107, in hPIV 3F the cleavage site is located between about residues 109/110, and in hPIV 4F the cleavage site is located between about residues 103/104. The smaller of these fragments, F2, originates from the N-terminal part of the F0 precursor (hPIV 1, about residues 22-113; hPIV2, about residues 22-106; hPIV3, about residues 19-109; hPIV4, about residues 21-103). The larger of these fragments, F1, comprises the C-terminal portion of the F0 precursor (hPIV 1, about residues 114-555; hPIV2, about residues 107-551; hPIV3, about residues 110-539; hPIV4, about residues 104-544), and comprises the extracellular region/luminal region (hPIV 1, about residues 114-497; hPIV2, about residues 107-493; hPIV3, about residues 110-493; hPIV4, about residues 104-486), the transmembrane domain (hPIV 1, about residues 498-518; hPIV2, about residues 494-514; IVhPIV 3, about residues 494-514; hPIV4, about residues 487-507) and the cytoplasmic tail at the C-terminus. The extracellular portion of the hPIV F protein is the hPIV F extracellular domain, which includes the F2 protein and the F1 extracellular domain.

The hPIV F protein shows significant sequence conservation in the hPIV subtype and other members of the respiratory and mumps genera (fig. 1). In view of this conservation, one of ordinary skill in the art can readily compare the amino acid positions of different hPIV F proteins of the same subtype, or with the F proteins of other members of the respiratory and mumps genera. Unless the context indicates otherwise, the numbering of the amino acid substitutions disclosed herein is made with reference to SEQ ID NO:1 (GenBank AGW 51052.1) or 2 (SWISS-PROT: P06828.2) of hPIV3 (also known as human respiratory virus 3) F, unless otherwise indicated.

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

PIV F proteins stabilized in the pre-fusion conformation

Three PIV F-precursors oligomerize in the mature F-protein, which adopts a metastable pre-fusion conformation, triggering a conformational change upon contact with the target cell membrane to a post-fusion conformation. This conformational change exposes a hydrophobic sequence, called a fusion peptide, which is located N-terminal to the F1 extracellular domain and associates with the host cell membrane, facilitating fusion of the membrane of the virus or infected cell with the target cell membrane.

Provided herein are engineered PIV 3F ectodomain trimers comprising a precursor comprising one or more amino acid substitutions that stabilize the F ectodomain trimers in a pre-fusion conformation.

As used herein, a "pre-fusion conformation" refers to a structural conformation employed by a polypeptide that differs from a post-fusion conformation of PIV F, at least in terms of molecular size or three-dimensional coordinates. The pre-fusion conformation refers to the structural conformation that PIV F adopts prior to triggering a fusion event, resulting in the conversion of F into a post-fusion conformation. Isolation of PIV F in a stable pre-fusion conformation may help provide information and guidance for improved vaccine and immunogenic composition development to address the important public health issue of PIV infection. The pre-fusion conformation may be a conformation that binds to a pre-fusion specific antibody.

The PIV F ectodomain trimer "stable in the pre-fusion conformation" comprises one or more amino acid substitutions, deletions or insertions compared to the corresponding native PIV F sequence, which increases the retention of the pre-fusion conformation compared to PIV F ectodomain trimers formed from the corresponding native hPIV F sequence. The "stabilization" of the pre-fusion conformation may be, for example, energy stabilization (e.g., reducing the energy of the pre-fusion conformation relative to the post-fusion open conformation) and/or kinetic stabilization (e.g., reducing the rate of transition from the pre-fusion conformation to the post-fusion conformation). Furthermore, the stability of PIV F ectodomain trimers in the pre-fusion conformation may include an increase in resistance to denaturation compared to the corresponding native PIV F sequences. Provided herein are methods of determining whether a hPIV F ectodomain trimer is in a pre-fusion conformation and include, but are not limited to, negative staining electron microscopy and antibody binding assays using pre-fusion conformation specific antibodies (such as PIA3 or PIA174 antibodies in the case of hPIV 3).

The present disclosure provides engineered proteins comprising amino acid substitutions relative to the amino acid sequence of a corresponding native hPIV3F protein (e.g., SEQ ID NO:1 or 2). Amino acid mutations include amino acid substitutions, deletions or additions relative to the native hPIV3F protein. Thus, the engineered protein is a mutant of the native hPIV3F protein.

The engineered PIV F ectodomain trimer may be from a human PIV strain other than hPIV3, such as hPIV1, hPIV2 or hPIV4. Residues of other hPIV F sequences corresponding to hPIV3F sequences can be readily obtained based on the high degree of sequence identity between the hPIV3F sequence and the other hPIV F sequences. Any amino acid substitution (or combination of substitutions) described herein for stabilizing hPIV3F in its pre-fusion conformation can be introduced into another hPIV F sequence to achieve pre-fusion stabilization.

The engineered PIV F ectodomain trimer may be from a non-human PIV strain, such as a bovine or ovine PIV strain. Residues of the non-human PIV F sequence corresponding to the hPIV F sequence can be readily obtained based on the high degree of sequence identity between the human PIV F sequence and the non-human PIV F gene sequence. Any amino acid substitution (or combination of substitutions) described herein for stabilizing hPIV 3F in its pre-fusion conformation can be introduced into a non-human PIV 3F sequence (e.g., GENBANK: AHZ90086.1 or AIW 42876.1) to achieve pre-fusion stabilization.

The engineered hPIV3F ectodomain trimer comprises the pathogens as "single chain" proteins, wherein the F2 polypeptide and F1 ectodomain of each pathogen are linked directly or through a peptide linker to form a continuous polypeptide chain. Some examples of native hPIV3F proteins (such as GENBANK: AGW 51052.1) do not contain a consensus furin cleavage site between the F1 and F2 proteins, and hPIV3F immunogens based on such native hPIV3F proteins typically do not require modification to produce single chain F proteins. However, other native hPIV3F proteins (such as SWISS-PROT: P06828.2) do contain a consensus furin cleavage site between the F1 and F2 proteins, and hPIV3F immunogens based on such native hPIV3F proteins can be modified to produce single chain F proteins. Exemplary modifications include amino acid substitutions to remove consensus furin cleavage sites, such as the K108E substitution.

The protomer of the engineered PIV F ectodomain trimer comprises PIV F19-481 and may comprise any of the amino acid substitutions (such as K108E) to remove the consensus furin cleavage site between F2 and F1 (if the consensus site is present in the native sequence), K or R at position 87, T or S at position 95, K or R at position 141, V or I at position 165, L or Q at position 168, K or R at position 295, T or V at position 267, T or K at position 369, and D or N at position 441.

The engineered PIV F ectodomain trimer may be a soluble protein complex, for example, for use as a recombinant subunit vaccine. In several such embodiments, the precursors of the engineered PIV F ectodomain trimer may each comprise a C-terminal linkage to a trimerization domain, such as a GCN4 trimerization domain. The trimerization domain promotes trimerization and stability of the membrane proximal aspect of the engineered PIV F ectodomain trimer. For example, the C-terminal residue of the original (such as the residue of the stem region of the trimer) of the engineered PIV F ectodomain trimer may be directly linked to the trimerization domain or indirectly linked to the trimerization domain through a peptide linker. Exemplary linkers include glycine and glycine-serine linkers. Non-limiting examples of exogenous multimerization domains that promote stable trimerization of soluble recombinant proteins include GCN4 leucine zipper, trimerization motifs from pulmonary surfactant proteins (Hoppe et al 1994FEBS Lett344:191-195), collagen (MCALINDEN et al 2003J Biol Chem278:42200-42207), any of which may be attached to the C-terminus of an engineered PIV F ectodomain protomer to promote trimerization, so long as the recombinant PIV F ectodomain trimer retains the pre-fusion conformation. In some examples, the protomers of the engineered PIV F ectodomain trimer may be linked to the GCN4 trimerization domain, e.g., each protomer in the trimer may include a C-terminal linkage to the GCN4 trimerization domain, such as a linkage to any of hPIV 3F 475-485 (such as hPIV 3F 481).

The engineered PIV F ectodomain trimer may be a membrane anchored protein complex, for example, for use in an attenuated virus or virus-like particle vaccine. Membrane anchoring can be achieved, for example, by linking a source of an engineered PIV F ectodomain trimer to the C-terminus of the transmembrane domain and optionally the cytoplasmic tail (such as PIV F transmembrane domain and cytoplasmic tail). One or more peptide linkers (such as glycine-serine linkers, e.g., 10 amino acid glycine-serine peptide linkers) can be used to attach the protomer of the engineered PIV F ectodomain trimer to the transmembrane domain. Non-limiting examples of transmembrane domains for the disclosed embodiments include the hPIV 3F transmembrane domain.

Engineered proteins may have certain beneficial properties, such as immunogenicity. The engineered proteins may have enhanced immunogenicity or improved pre-fusion conformational stability compared to the corresponding native hPIV 3F protein. Stability refers to the degree to which the transition of hPIV 3F from the pre-fusion conformation to the post-fusion conformation is hindered or prevented. The engineered protein may exhibit one or more introduced mutations as described herein, which may also result in increased stability of the pre-fusion conformation. Amino acid mutations introduced into the hPIV 3F protein include amino acid substitutions, deletions and/or additions. Mutations in the amino acid sequence of the engineered proteins may be amino acid substitutions, insertions and/or deletions relative to the native hPIV3-F extracellular domain.

Several ways of stabilizing the engineered protein conformation compared to the native hPIV 3F protein include, but are not limited to, amino acid substitutions that introduce disulfide bonds (both in and between the precursors), modify salt bridges, introduce electrostatic interactions, introduce hydrogen bonds, introduce proline, fill cavities, alter the packaging of residues, and combinations thereof.

The engineered proteins can be isolated, i.e. from the hPIV 3F protein having a post-fusion conformation. Thus, the engineered protein may be, for example, at least 80% separated, at least 90% separated, at least 95% separated, at least 98% separated, at least 99% separated, or at least 99.9% separated from the hPIV 3F polypeptide in the post-fusion conformation. The engineered protein can specifically bind to the hPIV 3F pre-fusion specific antibody.

It will be appreciated that a homogeneous population of engineered proteins of a particular conformation may include a change in the conformational state of the engineered protein that does not change (such as a change in polypeptide modification, e.g., glycosylation state). The amount of engineered protein may remain homogenous over time. For example, when the engineered protein is dissolved in an aqueous solution, a population of proteins that stabilize in the pre-fusion conformation for at least 12 hours, at least 24 hours, at least 48 hours, at least one week, at least two weeks, or more can be formed. One of ordinary skill in the art will appreciate that the engineered proteins provided herein can be used to elicit an immune response in a mammal to hPIV 3.

The engineered protein may include an introduced cysteine substitution as compared to the native PIV F protein. The engineered protein may comprise any of 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 cysteine substitutions. Without being bound by theory or mechanism, the cysteine substitutions described herein are believed to promote stability of the polypeptide in a non-PIV F post-fusion conformation. The introduced cysteine substitutions may be introduced by protein engineering, for example, by including one or more disulfide-forming substituted cysteine residues. The amino acid positions of cysteines may be sufficiently close to form disulfide bonds in the PIV F protein conformation prior to fusion, but not after fusion.

The engineered protein may comprise a recombinant PIV F protein that is stabilised in a pre-fusion conformation by disulfide bonds between cysteines that are introduced into a pair of amino acid positions that are close to each other in the pre-fusion conformation and further apart in the post-fusion conformation. The cysteine pairs may be present in a single pathogen at the same time, thereby forming disulfide bonds within the pathogen, or the cysteine pairs may be present in different pathogens, thereby forming inter-pathogen disulfide bonds.

In contrast to the native PIV F protein, exemplary cysteine substitutions include any disulfide substitution in Table 1, the numbering of which is based on the numbering of SEQ ID NO: 1. The engineered proteins may include a combination of two or more disulfide bonds between pairs of cysteine residues listed in table 1.

The engineered protein may include a combination of two or more different types of mutations selected from the group consisting of engineered disulfide mutations, cavity filling mutations, and proline mutations. The engineered protein may include at least one disulfide mutation and at least one proline mutation. The engineered proteins may include at least one cysteine substitution and at least one cavity filling substitution. The engineered proteins may include at least one cysteine substitution and at least one charge reducing substitution. The engineered protein may include at least one mutation selected from any one of the mutations or groups of mutations in table 1 or 2.

III protein preparation

The proteins described herein can be prepared by conventional methods known in the art, such as by expression in a recombinant host system using a suitable vector. Suitable recombinant host cells include, for example, insect cells, mammalian cells, avian cells, bacterial and yeast cells. Examples of suitable insect cells include, for example, sf9 cells, sf21 cells, tn5 cells, SCHNEIDER S cells, and HIGH live cells (clonal isolates derived from the parental Trichoplusia ni BTI-Tn-5B1-4 cell line). Examples of suitable mammalian cells include Chinese Hamster Ovary (CHO) cells, human embryonic kidney cells (HEK 293 or Expi 293 cells, typically transformed by sheared adenovirus type 5 DNA), NIH-3T3 cells, 293-T cells, vero cells and HeLa cells. Suitable avian cells include for example chicken embryonic stem cells (e.g.,Cells), chicken embryo fibroblasts, chicken embryo germ cells, quail fibroblasts (e.g., ELL-O), and duck cells. Suitable insect cell expression systems, such as baculovirus vector systems, are known to those skilled in the art. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form. Avian cell expression systems are also known to those skilled in the art. Similarly, bacterial and mammalian cell expression systems are also known to those skilled in the art.

A variety of suitable vectors for expressing recombinant proteins in insect or mammalian cells are known and routinely used in the art. Suitable vectors may contain a variety of elements including, but not limited to, one or more of an origin of replication, a selectable marker gene, one or more expression control elements such as a transcriptional control element (e.g., promoter, enhancer, terminator) and/or one or more translational signals, and a signal sequence or leader sequence for targeting to a secretory pathway in a selected host cell (e.g., 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 produce recombinant baculovirus particles. Baculovirus particles are amplified and used to infect insect cells to express recombinant proteins. For expression in mammalian cells, vectors are used that will drive expression of the construct in the desired mammalian host cell (e.g., chinese hamster ovary cells).

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

Nucleic acids encoding proteins

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

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.

V. formulations and methods of use thereof

Provided herein are methods of inducing an immune response in a mammal against PIV, the methods comprising administering to the mammal an amount of an immune composition effective to induce an immune response, wherein the composition comprises an engineered PIV F pre-fusion protein or a polynucleotide encoding an engineered PIV F pre-fusion protein. The immune response induced may be a protective immune response, i.e. the response reduces the risk or severity of PIV infection or clinical outcome. The immune response may include a humoral immune response, a cell-mediated immune response, or both. The immune response may include a T cell response or a B cell response. The cell-mediated immune response may include a helper T cell (Th) response, a cd8+ cytotoxic T Cell (CTL) response, or both. The humoral immune response may include antibody presenting B cells, and the antibodies may be neutralizing antibodies to PIV. Neutralizing antibodies block virus-infected cells. The immune response may reduce or prevent cellular infection. The neutralizing antibody response may be complement-dependent or complement-independent. The neutralizing antibody response may be complement independent. The neutralizing antibody response may be cross-neutralizing, i.e., the antibodies raised against the administered composition neutralize the associated PIV virus of a strain different from the strain used in the composition.

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

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

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

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

In the case where a nucleic acid molecule encoding an engineered PIV F protein is used in a pharmaceutical composition, the nucleic acid molecule may comprise or consist of deoxyribonucleotides and/or ribonucleotides or analogs thereof covalently linked together. Nucleic acid molecules as described herein typically contain phosphodiester linkages, but in some cases may include nucleic acid analogs that may have at least one different linkage, for example, phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphite linkages, as well as peptide nucleic acid backbones and linkages. Mixtures of naturally occurring polynucleotides and analogs can be prepared, or mixtures of different polynucleotide analogs can be prepared, as well as mixtures of naturally occurring polynucleotides and analogs. The nucleic acid molecules may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. The nucleotide structure, if present, may be modified before or after assembly of the polymer. The nucleotide sequence may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. The term also includes both double-stranded and single-stranded molecules. Unless otherwise indicated or required, the term polynucleotide encompasses both a double stranded form and each of two complementary single stranded forms known or predicted to constitute the double stranded form. The nucleic acid molecule consists of a specific sequence of four nucleotide bases, adenine (A), cytosine (C), guanine (G), thymine (T), and when the polynucleotide is RNA, thymine is uracil (U). Thus, the term "nucleic acid sequence" is an alphabetical representation of a nucleic acid molecule. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. 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.

At least one polynucleotide may have at least one chemical modification. The at least one polynucleotide may further comprise a second chemical modification. The polynucleotide may be RNA. At least one polynucleotide having at least one chemical modification may have a 5'. The at least one chemical modification may be selected from pseudouridine, N1-methyl pseudouridine, N1-ethyl pseudouridine, 2-thiouridine, 4 '-thiouridine, 5-methylcytosine, 2-thiol-1-methyl-1-deaza-pseudouridine, 2-thio1-methyl-pseudouridine, 2-thio5-aza-uridine, 2-thiodihydro-pseudouridine, 2-thio-dihydro-uridine, 2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydro-pseudouridine, 5-methoxy-uridine and 2' -0-methyl uridine. At least 80% (e.g., 85%, 90%, 95%, 98%, 99%, 100%) of the uracils in the open reading frame may have chemical modifications, optionally wherein the composition is formulated in a lipid nanoparticle. All uracils in the open reading frame may have chemical modifications. Chemical modification may occur at the 5-position of uracil. The chemical modification may be N1-methyl pseudouridine.

The nucleic acids of the present disclosure may comprise one or more modified nucleosides that comprise a modified sugar moiety. Such compounds comprising one or more sugar-modified nucleosides may have desirable properties, such as enhanced nuclease stability or increased binding affinity to a target nucleic acid, relative to oligonucleotides comprising only nucleosides comprising naturally occurring sugar moieties. In some embodiments, the modified sugar moiety is a substituted sugar moiety. In some embodiments, the modified sugar moiety is a sugar substitute. Such sugar substitutes may comprise one or more substitutions corresponding to those of the substituted sugar moiety.

In some embodiments, the modified sugar moiety is a substituted sugar moiety comprising one or more non-bridging sugar substituents, including but not limited to substituents at the 2 'position and/or the 5' position. Examples of sugar substituents suitable for the 2 '-position include, but are not limited to, 2' -F, 2'-OCH3 ("OMe" or "O-methyl") and 2' -O (CH 2) 2OCH3 ("MOE"). In certain embodiments, the sugar substituent at the 2' position is selected from the group consisting of allyl, amino, azido, thio, O-allyl, O- -C1-C10 alkyl, O- -C1-C10 substituted alkyl, OCF3, O (CH 2) 2SCH3, O (CH 2) 2- -O- -N (Rm) (Rn), and O- -CH2- -C (=O) - -N (Rm) (Rn), wherein each Rm and Rn is independently H or substituted or unsubstituted C1-C10 alkyl. Examples of sugar substituents at the 5 '-position include, but are not limited to, 5' -methyl (R or S), 5 '-vinyl and 5' -methoxy. In some embodiments, the substituted saccharide comprises more than one non-bridging sugar substituent, e.g., a T-F-5' -methyl sugar moiety (for additional 5',2' -disubstituted sugar moieties and nucleosides, see, e.g., PCT international application WO 2008/101157).

Nucleosides comprising 2 '-substituted sugar moieties are referred to as 2' -substituted nucleosides. In some embodiments, the 2 '-substituted nucleoside comprises a 2' -substituent group selected from halo, allyl, amino, azido, SH, CN, OCN, CF, OCF3, O, S, or N (Rm) -alkyl, O, S or N (Rm) -alkenyl, O, S or N (Rm) -alkynyl, O-alkylene-O-alkyl, alkynyl, alkylaryl, aralkyl, O-alkylaryl, O-aralkyl, O (CH 2) 2SCH3, O (CH 2) 2- -O- -N (Rm) (Rn), or O- -CH2- -C (=O) - -N (Rm) (Rn), wherein each Rm and Rn is independently H, an amino protecting group, or a substituted or unsubstituted C1-C10 alkyl. These 2' -substituents may be further substituted with one or more substituents independently selected from the group consisting of hydroxy, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO 2), mercapto, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl, and alkynyl.

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

In some embodiments, the 2 '-substituted nucleoside comprises a sugar moiety comprising a 2' -substituent group selected from F, OCF, O- -CH3, OCH2CH2OCH3, O (CH 2) 2SCH3, O (CH 2) 2- -O- -N (CH 3) 2, - -O (CH 2) 2O (CH 2) 2N (CH 3) 2, and O- -CH2- -C (=O) - -N (H) CH3.

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

In some embodiments, the nucleosides of the present disclosure comprise one or more unmodified nucleobases. In certain embodiments, a nucleoside of the present disclosure comprises one or more modified nucleobases.

In some embodiments, the modified nucleobase is selected from the group consisting of a universal base, a hydrophobic base, a promiscuous base, a size-expanded base, and a fluorinated base, as defined herein. 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines including 2-aminopropyladenine, 5-propynyluracil, 5-propynylcytosine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynylCH 3) uracil and cytosine and other alkynyl derivatives of cytosine bases, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halo in particular 5-bromo, 5-trifluoromethyl and 5-substituted uracil, 7-methyl and guanine, 7-methyl and 7-adenine, 7-deaza, 2-azaadenine and 7-methyl, 7-deaza, 2-deaza and 7-azaadenine and 3-deaza, 2-deaza, 3-deaza and 2-azaadenine and 3-deaza and 2-azaadenine, as defined herein. Additional modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine ([ 5,4-b ] [1,4] benzoxazin-2 (3H) -one), phenothiazine cytidine (1H-pyrimido [5,4-b ] [1,4] benzothiazin-2 (3H) -one), G-clamps such as substituted phenoxazine cytidine (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. Other nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer SCIENCE AND ENGINEERING, kroschwitz, J.I. code, john Wiley & Sons,1990,858-859, those disclosed by Englisch et al, 1991, and those disclosed by Sanghvi, Y.S., 1993.

Representative U.S. patents teaching the preparation of certain of the above-described modified nucleobases, as well as other modified nucleobases, include, but are not limited to, U.S. patent 3,687,808;4,845,205;5,130,302;5,134,066;5,175,273;5,367,066;5,432,272;5,457,187;5,459,255;5,484,908;5,502,177;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 by reference herein in its entirety.

Additional modifications may also be made at other positions on the oligonucleotide, particularly at the 3 'position of the sugar on the 3' terminal nucleotide and the 5 'position of the 5' terminal nucleotide. For example, one additional modification of the ligand-conjugated oligonucleotides of the present disclosure involves chemically linking one or more additional non-ligand moieties or conjugates to the oligonucleotide, which enhances the activity, cell distribution, or cell uptake of the oligonucleotide. Such moieties include, but are not limited to, lipid moieties such as cholesterol moieties (Letsinger et al, 1989), cholic acid (Manoharan et al, 1994), thioethers such as hexyl-5-tritylthiol (Manoharan et al, 1992; manoharan et al, 1993), thiocholesterol (Oberhauser et al, 1992), fatty chains such as dodecyl glycol or undecyl residues (Saison-Behmoaras et al, 1991; kabanov et al, 1990; svinarchhuk et al, 1993), phospholipids such as hexacosa glycerol or triethylammonium 1, 2-di-O-hexadecyl-rac-glycerol-3-H-phosphonate (Manoharan et al, 1995; shea et al, 1990), polyamines or polyethylene glycol chains (Manoharan et al, 1995) or adamantaneacetic acid (Manoharan et al, 1995), palmityl moieties (Mishra et al, 1995) or octadecyl or hexylamino-carbonyl-oxy cholesterol moieties (Crooke et al, 1996). In some aspects, the nucleic acid molecule encoding the engineered PIV F protein is a modified RNA, such as, for example, a modified mRNA. Modified (m) RNAs contemplate certain chemical modifications that confer increased stability and low immunogenicity to mRNA, thereby facilitating the expression of therapeutically important proteins. For example, N1-methyl-pseudouridine (N1 mψ) is superior to several other nucleoside modifications and combinations thereof in terms of translational ability. In some embodiments, the (m) RNA molecules used herein may replace uracil with pseudouracil, such as 1-methyl-3'-pseudouridylyl base (1-methyl-3' -pseudouridylyl base). In some embodiments, some uracils are replaced, but in other embodiments, all uracils have been replaced. (m) the RNA may comprise a 5' cap, a 5' utr element, an optionally codon optimized open reading frame, a 3' utr element and a poly (a) sequence and/or polyadenylation signal.

Nucleic acid molecules, whether native or modified, can be delivered as naked nucleic acid molecules or in a delivery vehicle such as a lipid nanoparticle. The lipid nanoparticle may comprise one or more nucleic acids in a weight ratio to the lipid nanoparticle of about 5:1 to about 1:100. 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.

In some embodiments, the lipid nanoparticle used herein may contain one, two, three, four, five, six, seven, eight, nine, or ten lipids. These lipids may include triglycerides, phospholipids, steroids or sterols, pegylated lipids or groups having an ionizable group such as an alkyl amine and one or more hydrophobic groups such as a C6 or greater alkyl group.

In some aspects of the disclosure, the lipid nanoparticle is mixed with one or more steroids or steroid derivatives. In some embodiments, the steroid or steroid derivative includes any steroid or steroid derivative. As used herein, the term "steroid" is a class of compounds having a four-ring 17 carbocyclic ring structure, which may further comprise one or more substituents including alkyl, alkoxy, hydroxy, oxo, acyl, or double bonds between two or more carbon atoms.

In some aspects of the disclosure, the lipid nanoparticle is mixed with one or more pegylated lipids (or PEG lipids). In some embodiments, the present disclosure includes the use of any lipid to which a PEG group has been attached. In some embodiments, the PEG lipid is a diglyceride that further comprises 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 having a PEG chain. Some non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG ceramide conjugation, PEG-modified dialkylamines and PEG-modified 1, 2-diacyloxypropan-3-amines, PEG-modified diacylglycerols, and dialkylglycerols. In some embodiments, PEG-modified distearoyl phosphatidylethanolamine or PEG-modified dimyristoyl-sn-glycerol. In some embodiments, 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 from about 200 to about 500, from about 400 to about 5,000, from about 500 to about 3,000, or from about 1,200 to about 3,000. The PEG modified molecular weight is from about 100、200、400、500、600、800、1,000、1,250、1,500、1,750、2,000、2,250、2,500、2,750、3,000、3,500、4,000、4,500、5,000、6,000、7,000、8,000、9,000、10,000、12,500 to about 15,000. Some non-limiting examples of lipids that can be used in the present disclosure are taught in U.S. patent 5,820,873, WO 2010/141069, or U.S. patent 8,450,298, which are incorporated herein by reference.

In some aspects of the disclosure, the lipid nanoparticle is mixed with one or more phospholipids. In some embodiments, any lipid that also comprises 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, sugar, or amino-substituted alkoxy group, such as choline or ethanolamine. In some embodiments, the phospholipid is phosphatidylcholine. In some embodiments, the phospholipid is distearoyl phosphatidylcholine or dioleoyl phosphatidylethanolamine. In some embodiments, other zwitterionic lipids are used, where zwitterionic lipids define lipids and lipid-like molecules having both positive and negative charges.

In some aspects of the present disclosure, lipid nanoparticles comprising a compound comprising a lipophilic and a cationic component are provided, wherein the cationic component is ionizable. In some embodiments, the cationically ionizable lipid contains one or more groups that are protonated at physiological pH but deprotonated and uncharged at a pH above 8, 9, 10, 11, or 12. The ionizable cationic groups may contain one or more protonatable amines capable of forming cationic groups at physiological pH. The cationically ionizable lipid compound may further comprise one or more lipid components, such as two or more fatty acids having a C6-C24 alkyl or alkenyl carbon group. These lipid groups may be attached by ester linkages or may be further added to the sulfur atom by Michael addition (Michael addition). In some embodiments, these compounds may be dendrimers (dendrimers), dendrimers (dendron), polymers, or combinations thereof.

In some aspects of the present disclosure, compositions are provided that include a compound that includes a lipophilic and a cationic component, wherein the cationic component is ionizable. In some embodiments, ionizable cationic lipids refer to lipids and lipid-like molecules having a nitrogen atom with an available charge (pKa). These lipids may be referred to in the literature as cationic lipids. These molecules having amino groups typically have 2 to 6 hydrophobic chains, typically alkyl or alkenyl groups such as C6-C24 alkyl or alkenyl groups, but may have at least 1 or more than 6 tails (tails).

In some embodiments, the amount of lipid nanoparticle encapsulating nucleic acid molecules in the pharmaceutical composition is about 0.1% wt/wt to about 50% wt/wt, about 0.25% wt/wt to about 25% wt/wt, about 0.5% wt/wt to about 20% wt/wt, about 1% wt/wt to about 15% wt/wt, about 2% wt/wt to about 10% wt/wt, about 2% wt/wt to about 5% wt/wt, or about 6% wt/wt to about 10% wt/wt. In some embodiments, the amount of lipid nanoparticle encapsulating a nucleic acid molecule in a 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.

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

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

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

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

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

The term promoter will be used herein to refer to a set of transcriptional control modules clustered around the initiation site of RNA polymerase II. Many of the considerations regarding how promoters are organized stem from analysis of several viral promoters, including those of the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, complemented by more recent work, have shown that promoters consist of discrete functional modules, each consisting of approximately 7-20bp of DNA, containing one or more recognition sites for transcriptional activators or repressors.

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

Additional promoter elements regulate the frequency of transcription initiation. Typically, they are located in a region 30-110bp upstream of the start site, although many promoters have recently been found to contain functional elements downstream of the start site as well. The spacing between promoter elements is generally flexible, so that promoter function is preserved when the elements are inverted or moved relative to each other. In the tk promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements may act synergistically or independently to activate transcription.

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 glyceraldehyde-3-phosphate dehydrogenase may be used to obtain high levels of expression of the coding sequences of interest. It is also contemplated that expression of the coding sequence of interest may be achieved using other viral or mammalian cell or bacteriophage promoters well known in the art, provided that the level of expression is sufficient for a given purpose. By using promoters with known characteristics, the expression level and pattern of the protein of interest after transfection or transformation can be optimized. Furthermore, selection of promoters that are regulated in response to particular physiological signals may allow for inducible expression of the gene product.

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 made up of a number of individual elements, each of which binds to one or more transcriptional proteins. The basic distinction between enhancers and promoters is operational. The entire enhancer region must be capable of remotely stimulating transcription, which is not necessary for the promoter region or its constituent elements. On the other hand, promoters must have one or more elements that direct the initiation of RNA synthesis at specific sites and in specific directions, whereas enhancers lack these specificities. Promoters and enhancers are typically overlapping and contiguous, and appear to typically have very similar modular organization.

The following is a list of promoters/enhancers and inducible promoters/enhancers that can be used in combination with nucleic acids encoding a gene of interest in an expression construct. In addition, any promoter/enhancer combination (according to eukaryotic promoter database EPDB) may also be used to drive expression of the gene. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters, whether as part of a delivery complex or as an additional genetic expression construct, if provided with an appropriate bacterial polymerase.

Promoters and/or enhancers may be, for example, immunoglobulin light chain, immunoglobulin heavy chain, T cell receptor, HLA DQ a and/or DQ β, β -interferon, 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, neural Cell Adhesion Molecule (NCAM), α ₁ -antitrypsin, H2B (TH 2B) histone, mouse and/or I collagen, glucose regulatory proteins (GRP 94 and GRP 78), rat growth hormone, human Serum Amyloid A (SAA), troponin I (TN I), platelet Derived Growth Factor (PDGF), 40, polyoma virus, retrovirus, hepatitis B virus, cytomegalovirus, hepatitis B virus, human papilloma virus, and cytomegalovirus.

Where cDNA inserts are employed, one would normally desire to include polyadenylation signals to effect proper polyadenylation of the gene transcript. Any polyadenylation sequence may be used, such as human growth hormone and SV40 polyadenylation signals. Also contemplated as elements of the expression cassette are terminators. These elements can be used to enhance message levels and minimize read through (read through) into other sequences.

There are a variety of ways in which an expression vector can be introduced into a cell. In certain embodiments, the expression construct comprises a virus or an engineered construct derived from a viral genome. The ability of certain viruses to enter cells via receptor-mediated endocytosis, integrate into the host cell genome, and stably and efficiently express viral genes makes them attractive candidates for transferring foreign genes into mammalian cells. These viruses have a relatively low capacity for foreign DNA sequences and have a limited host profile. In addition, they raise safety concerns in allowing for oncogenic potential and cytopathic effects in the cell. They can only hold up to 8kB of foreign genetic material, but can be easily introduced into a variety of cell lines and laboratory animals.

One method for in vivo delivery involves the use of an adenovirus expression vector. "adenoviral expression vectors" are intended to include those constructs that contain sufficient adenoviral sequences to (a) support packaging of the construct and (b) express the engineered PIV F protein that has been cloned therein. In this case, the expression does not require a synthetic gene product.

The expression vector comprises an adenovirus in a genetically engineered form. Knowledge of the genetic organization of adenovirus (a 36kB linear double stranded DNA virus) allows substitution of large pieces of adenovirus DNA with up to 7kB foreign sequences. In contrast to retroviruses, adenovirus infection of host cells does not result in chromosomal integration, as adenovirus DNA can be replicated episomally without potential genotoxicity. In addition, adenoviruses are structurally stable and no genomic rearrangement is detected after extensive amplification. Adenoviruses can infect almost all epithelial cells, regardless of their cell cycle phase. To date, adenovirus infection appears to be associated with only mild diseases, such as acute respiratory illness in humans.

Adenovirus is particularly suitable for use as a gene transfer vector because it has a medium size genome, is easy to handle, high potency, broad target cell range, and high infectivity. The viral genome contains 100-200 base pair inverted repeats (ITRs) at both ends, which are cis-elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units, which are divided by the onset of viral DNA replication. The E1 region (E1A and E1B) encodes a protein responsible for regulating transcription of the viral genome and some cellular genes. Expression of the E2 region (E2A and E2B) results in the synthesis of proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression and host cell shutdown. The products of late genes, including most viral capsid proteins, are expressed only after significant treatment of a single primary transcript emitted by the Major Late Promoter (MLP). MLP (at 16.8 m.u.) is particularly effective at the late stage of infection, and all mRNAs emitted from this promoter have 5' -tripartite leader (TPL) sequences, which makes them the preferred mRNAs for translation. In one system, the recombinant adenovirus is produced by homologous recombination between a shuttle vector and a proviral vector. Wild-type adenovirus can be produced from this process, since recombination between the two proviral vectors may occur. It is therefore important to isolate individual virus clones from individual plaques and examine their genomic structure.

The generation and propagation of current replication-defective adenovirus vectors relies on a unique helper cell line designated 293, which is transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses the E1 protein. Since the E3 region is optional in the adenovirus genome, current adenovirus vectors carry foreign DNA in the E1, D3 or both regions with the aid of 293 cells. In nature, adenoviruses can package about 105% of the wild-type genome, providing about 2kb of additional DNA capacity. In combination with the alternative DNA of about 5.5kb in the E1 and E3 regions, the current adenovirus vectors have a maximum capacity of less than 7.5kb, or about 15% of the total length of the vector. More than 80% of the adenovirus viral genome remains in the vector backbone and is the source of vector-borne cytotoxicity. Furthermore, replication defects of E1 deleted viruses are incomplete.

The helper cell line may be derived from a human cell, such as a human embryonic kidney cell, muscle cell, hematopoietic cell, or other human embryonic mesenchymal or epithelial cell. Or the helper cells may be derived from cells of other mammalian species that are permissive for human adenoviruses. Such cells include, for example, vero cells or other monkey embryo mesenchymal cells or epithelial cells. As described above, the preferred helper cell line is 293.

Adenoviruses of the disclosure are replication-defective, or at least conditionally replication-defective. Adenoviruses may have any of 42 different known serotypes or subgroups a-F. Type 5 adenovirus of subgroup C is an exemplary starting material that may be used to obtain the conditional replication defective adenovirus vectors for use in the present disclosure.

Other viral vectors may 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 be employed. They provide several attractive features for a variety of mammalian cells.

In embodiments, particular embodiments, the vector is an AAV vector. AAV is a small virus that infects humans and some other primate species. AAV is not known to cause disease. The virus elicits 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 adverse consequences. Gene therapy vectors using AAV can infect both dividing and resting cells and persist in an extrachromosomal state without integration into the host cell genome, although some viral-carried gene integration into the host genome does occur in natural viruses. These features make AAV a very attractive candidate for creating viral vectors for gene therapy and for creating isogenic human disease models. Recent human clinical trials of retinal gene therapy using AAV have shown promise. AAV belongs to the genus depended parvovirus, which in turn belongs to the parvoviridae family. The virus is a small (20 nm) replication-defective non-enveloped virus.

Wild-type AAV has attracted considerable interest to gene therapy researchers due to a number of features. Most of them, viruses are markedly devoid of pathogenicity. It also infects non-dividing cells and is capable of stable integration into the host cell genome at a specific site on the human chromosome 19 (designated AAVS 1). This feature makes it easier to predict than retroviruses, which present a threat of random insertion and mutation occurrence, sometimes leading to the development of cancer. AAV genomes integrate most often into the mentioned sites, whereas random incorporation into the genome is negligible in frequency. However, the development of AAV as a gene therapy vector eliminates this integration capability by removing rep and cap from the vector DNA. The desired gene is inserted between Inverted Terminal Repeats (ITRs) along with a promoter that drives gene transcription, helping to form concatamers 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 additional concatamers in the host cell nucleus. In non-dividing cells, these concatamers remain intact throughout the life cycle of the host cell. In dividing cells, AAV DNA is lost through cell division because the additional DNA is not replicated with the host cell DNA. Random integration of AAV DNA into the host genome is detectable, but occurs very infrequently. AAV also exhibits very low immunogenicity, and appears to be limited to the production of neutralizing antibodies, whereas they do not induce well-defined cytotoxic responses. This feature, together with the ability to infect resting cells, suggests that they are superior to adenoviruses in their use as vectors for human gene therapy.

AAV genomes are composed of single stranded deoxyribonucleic acid (ssDNA), sense or negative sense, about 4.7 kilobases long. The genome comprises 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 for the AAV lifecycle, while the latter contains overlapping capsid protein nucleotide sequences VP1, VP2 and VP3, which interact to form an icosahedral symmetrical capsid.

The Inverted Terminal Repeat (ITR) sequences each contain 145 bases. They are so named because of their symmetry, which has proved to be required for efficient proliferation of AAV genomes. The feature that these sequences confer to them this property is their ability to form hairpins, which contributes to the so-called self-priming (self-priming) which allows primer-independent synthesis of the second DNA strand. ITRs have also been shown to be required for both integration of AAV DNA into the host cell genome (human chromosome 19) and rescue therefrom, as well as for a combination of efficient encapsidation of AAV DNA and production of fully assembled deoxyribonuclease resistant AAV particles.

With respect to gene therapy, ITR appears to be the only sequence required for cis alongside the therapeutic gene-the structural (cap) and packaging (rep) proteins can be delivered in trans. With this assumption, many methods have been established for the efficient production of recombinant AAV (rAAV) vectors containing reporter or therapeutic genes. However, it is also disclosed that ITR is not the only element required for efficient replication and cladding cis. Some groups have identified a sequence within the coding sequence of the Rep gene, known as a cis-acting Rep-dependent element (CARE). When cis is present, CARE has been shown to enhance replication and encapsulation.

Each of the immunogenic compositions discussed herein can be used alone or in combination with one or more other antigens, either from the same viral pathogen or from another pathogenic source or sources. These compositions may be used for prophylactic (preventing infection) or therapeutic (treating disease after infection) purposes.

The term "pharmaceutically acceptable" may mean approved by a federal regulatory agency or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, excipient, or vehicle with which a therapeutic agent is administered. Such a pharmaceutical carrier may be a sterile liquid, such as water, and may preferably include an adjuvant. When the pharmaceutical composition is administered by injection, such as intramuscular injection, water is a specific carrier. Aqueous saline and aqueous dextrose and glycerol solutions can also be employed 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.

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

The immunogenic composition may comprise an adjuvant. Exemplary adjuvants that enhance the effectiveness of the composition include (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, and the like; (2) an oil-in-water emulsion formulation (with or without other specific adjuvants such as muramyl peptide (see below) or bacterial cell wall components) such as, for example, (a) an MF59 (PCT publication No. WO 90/14837), containing 5% squalene, 0.5% TWEEN 80 and 0.5% Span 85, formulated as submicron particles using a microfluidizer, (b) SAF, containing 10% squalane, 0.4% TWEEN 80, 5% Pluronic-blocked polymer L121 and thr-MDP, producing an emulsion of larger particle size by microfluidization to form a submicron emulsion or vortex, and (c) a RIBI ^TM adjuvant system (RAS) (RIBI Immunochem, hamilton, mont), containing 2% squalene, 0.2% TWEEN 80 and one or more bacterial cell wall components selected from the group consisting of monophosphoryl lipid a (MPL), trehalose Dimycolate (TDM), and Cell Wall Skeleton (CWS), preferably cwl+cwl), and (cwl), and (3, 35 f, 35 m, 3 f, 35 f (c) and 35 f (c) can be used to produce an emulsion of larger particle size by complete stimulation of cells such as cfm (cfm, 3, 35, 3 f, and 35 f-f (f) or the like, and (f) of a complex of particles such as those containing a complete stimulus such as cfm Macrophage colony-stimulating factor (M-CSF), tumor Necrosis Factor (TNF), and the like, (6) Toll-like receptor agonists, and (7) other substances that act as adjuvants to enhance the effectiveness of the composition. The composition may not contain an adjuvant. The composition may also comprise lipid nanoparticles. The composition may be formulated as nanoparticles. The composition may also comprise cationic or polycationic compounds, including protamine or other cationic peptides or proteins, such as poly-L-lysine (PLL).

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

The compositions described herein may comprise an immunologically effective amount of a polypeptide or polynucleotide, as well as any other component described above, as desired. By "immunologically effective amount" is meant that the amount is effective to elicit an immune response by administering to an individual a single dose or a fraction of a series of doses. The immune response elicited may be sufficient, for example, to treat and/or prevent and/or reduce the incidence of pain, infection, or disease. This amount will vary depending on the health and physical condition of the individual to be treated, the taxonomic group of the individual to be treated (e.g., non-human primate, etc.), the ability of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the assessment of the medical condition by the treating physician, and other relevant factors. The expected amounts will fall within a relatively broad range, which amounts can be determined by routine experimentation.

Any suitable route of administration may be used. For example, the engineered proteins of the present disclosure or nucleic acids encoding the engineered proteins can be formulated for parenteral administration, e.g., 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. Alternatively, the formulation may be administered directly to the mucosa by a topical route, for example by nasal drops, inhalation or by nebuliser.

The composition may be administered according to any suitable schedule. The dose treatment may be a single dose regimen or a multiple dose regimen. Multiple doses may be used for primary and/or booster immunization regimens. In a multiple dose regimen, the various doses may be administered by the same or different routes. Multiple doses will typically be administered at least 1 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 immunomodulators.

The compositions disclosed herein are useful for treating both children and adults. Thus, a human subject may be less than 1 year old, 1-5 years old, 5-16 years old, 16-55 years old, 55-65 years old, or at least 65 years old.

VI antibodies and diagnostic uses

The polypeptides described above can be used to produce polyclonal and monoclonal antibodies. If polyclonal antibodies are desired, selected mammals (e.g., mice, rabbits, goats, guinea pigs, horses, etc.) are immunized with an immunogenic polypeptide bearing a pre-fusion epitope for PIV F. Serum from immunized animals was collected and processed according to known procedures. If the serum containing polyclonal antibodies to the pre-fusion epitope of PIV F contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art.

Monoclonal antibodies directed against pre-fusion epitopes of PIV F can also be readily prepared by those skilled in the art. General methods for preparing monoclonal antibodies using hybridomas are known. Immortalized antibody producing cell lines may be created by cell fusion, but also by other techniques, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. The monoclonal antibody group generated against the PIV F pre-fusion epitope can be used for screening various characteristics, namely isotype, epitope affinity and the like.

Monoclonal and polyclonal antibodies directed against the pre-fusion epitope of PIV F are particularly useful in diagnostics, while neutralizing antibodies are useful in passive immunotherapy. In particular, monoclonal antibodies can be used to generate anti-idiotype antibodies.

Polypeptides that immunoreact with serum containing PIV F antibodies, as well as antibodies raised against these polypeptides, are useful in immunoassays to detect the presence of PIV F antibodies or the presence of viruses in biological samples, including, for example, blood or serum samples. There are great differences in the design of immunoassays, many of which are known in the art. For example, immunoassays can utilize polypeptides having the sequence shown in any one of SEQ ID NOs 1-7.

Alternatively, immunoassays can employ a combination of viral antigens derived from the polypeptides described herein. For example, monoclonal antibodies directed against at least one polypeptide described herein, combinations of monoclonal antibodies directed against polypeptides described herein, monoclonal antibodies directed against different viral antigens, polyclonal antibodies directed against polypeptides described herein, or polyclonal antibodies directed against different viral antigens may be used. For example, the protocol may be based on a competition or direct reaction, or may be a sandwich type assay. For example, the protocol may also use a solid support, or may be achieved by immunoprecipitation. Most assays involve the use of labeled antibodies or polypeptides, and the label may be, for example, a fluorescent, chemiluminescent, radioactive or dye molecule. Amplified probe signal assays are also known, examples of which are assays using biotin and avidin, and enzyme-labeled and mediated immunoassays, such as ELISA assays.

Kits suitable for immunodiagnosis and containing suitable labeling reagents are constructed by packaging suitable materials, including engineered PIV F proteins containing pre-fusion epitopes of PIV F or antibodies to epitopes, in a suitable container along with the remaining reagents and materials required to perform the assay and a set of suitable assay instructions.

The polynucleotide probes may also be packaged into a diagnostic kit. The diagnostic kit includes probe DNA, which may be labeled, or the probe DNA may not be labeled, and the labeled component may be contained in the kit. The kit may also contain other suitable packaged reagents and materials, such as standards, as required for a particular hybridization protocol, as well as instructions for performing the test.

VII immunoassay method

The present disclosure relates to immunoassay methods for binding, purifying, removing, quantifying, and otherwise generally detecting PIV F proteins. While such methods may be applied in a traditional sense, another use would be quality control and monitoring of vaccine reserves, where antibodies according to the present disclosure can be used to assess the amount or integrity (i.e., long term stability) of an antigen. Or the method may be used to screen various antibodies for appropriate/desired reactivity characteristics.

Some immunodetection methods include enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), immunoradiometric assays, fluoroimmunoassay, chemiluminescent assays, bioluminescent assays, western blots, and the like. In particular, competitive assays for detecting and quantifying PIV F proteins are also provided. The steps of various useful immunoassays have been described in the scientific literature, such as, for example, doolittle and Ben-Zeev (1999), gulbis and Galand (1993), de Jager et al (1993) and Nakamura et al (1987). In general, the immunological binding method comprises obtaining a sample suspected of containing PIV F protein, and contacting the sample with a first antibody according to the present disclosure (as the case may be) under conditions effective to allow formation of an immune complex.

These methods include methods for detecting or purifying PIV F protein from a sample. The antibodies will preferably be attached to a solid support, such as in the form of a column matrix, and a sample suspected of containing PIV F protein will be applied to the immobilized antibodies. Unwanted components will be washed off the column, leaving PIV F protein expressing cells immunocomplexed with immobilized antibodies, and the PIV F protein expressing cells are then collected by removing organisms or antigens from the column.

The immunological binding methods also include methods for detecting and quantifying the amount of PIV F protein or related components in a sample, and detecting and quantifying any immune complexes formed during the binding process. Here, one would obtain a sample suspected of containing PIV F protein and contact the sample with an antibody that binds PIV F protein or a component thereof, and then detect and quantify the amount of immune complex formed under the specific conditions. In terms of antigen detection, the biological sample analyzed may be any sample suspected of containing PIV F protein, such as a tissue slice or specimen, a homogenized tissue extract, a biological fluid (e.g., a nasal swab), including blood and serum or secretions, such as stool or urine.

Contacting the selected biological sample with the antibody under effective conditions and for a period of time sufficient to allow formation of an immune complex (primary immune complex) is typically about simply adding the antibody composition to the sample and incubating the mixture for a period of time sufficient for the antibody to form an immune complex with PIV F protein (i.e., bind to hMPV F protein-binding antibody). After this time, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot, or western blot, will typically be washed to remove any non-specifically bound antibody species, allowing only those antibodies that specifically bind within the primary immune complex to be detected.

In general, detection of immune complex formation is well known in the art and can be accomplished by applying a variety of methods. These methods are generally based on the detection of labels or markers, such as any of those radioactive, fluorescent, biological and enzymatic tags. Patents relating to the use of such labels include U.S. Pat. nos. 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, one may find additional advantages by using secondary binding ligands such as secondary antibodies and/or biotin/avidin ligand binding arrangements.

The antibody itself for detection may be linked to a detectable label, wherein one would then simply detect this label, allowing the amount of primary immune complex in the composition to be determined. Or primary antibodies bound within the primary immune complex may be detected by a secondary binding ligand having binding affinity for the antibody. In these cases, the secondary binding ligand may be linked to a detectable label. The secondary binding ligand itself is typically an antibody and may therefore be referred to as a "secondary" antibody. The primary immune complex is contacted with a labeled secondary binding ligand or antibody under effective conditions and for a period of time sufficient to allow formation of the secondary immune complex. The secondary immune complex is then typically washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complex is then detected.

Other methods include detection of the primary immune complex by a two-step method. Secondary binding ligands such as antibodies having binding affinity for the antibody are used to form secondary immune complexes, as described above. After washing, the secondary immune complex is again contacted with a tertiary binding ligand or antibody having binding affinity for the secondary antibody under effective conditions and for a period of time sufficient to allow formation of an immune complex (tertiary immune complex). The tertiary ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complex thus formed. The system may provide signal amplification if desired.

One immunoassay method uses two different antibodies. The first biotinylated antibody is used to detect the target antigen and then the second antibody is used to detect biotin attached to the complex biotin. In the method, the test sample is first incubated in a solution containing the first step antibody. If the target antigen is present, some antibodies will bind to the antigen to form biotinylated antibody/antigen complexes. The antibody/antigen complex is then amplified by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA and/or complementary biotinylated DNA, with each step adding additional biotin sites to the antibody/antigen complex. The amplification step is repeated until the appropriate level of amplification is reached, at which point the sample is incubated in a solution containing the second step antibody to biotin. This second step antibody is labeled, such as for example with an enzyme that can be used to detect the presence of the antibody/antigen complex by histology using a chromogenic substrate. By suitable amplification, macroscopically visible conjugates can be produced.

Another known immunoassay method utilizes an immuno-PCR (polymerase chain reaction) method. The PCR method is similar to the Cantor method until incubated with biotinylated DNA, but rather than using multiple rounds of streptavidin and biotinylated DNA, the DNA/biotin/streptavidin/antibody complex is washed away with a low pH or high salt buffer that releases the antibody. The resulting wash solution is then used to perform a PCR reaction with the appropriate primers and appropriate controls. At least in theory, the tremendous amplification capacity and specificity of PCR can be used to detect individual antigen molecules.

A.ELISA

In its simplest and direct sense, an immunoassay is a binding assay. Some preferred immunoassays are the various types of enzyme-linked immunosorbent assays (ELISA) and Radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and western blotting, dot blotting, FACS analysis, etc. may also be used.

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

In another exemplary ELISA, a sample suspected of containing PIV F protein (e.g., a potentially infected cell) is immobilized onto the well surface and then contacted with an anti-PIV F protein antibody of the present disclosure. After binding and washing to remove non-specifically bound immune complexes, bound anti-PIV F protein antibodies are detected. When the initial anti-PIV F protein antibody is linked to a detectable label, the immune complex can be detected directly. Likewise, a secondary antibody having binding affinity to a primary anti-PIV F protein antibody can be used to detect the immune complex, wherein the secondary antibody is linked to a detectable label.

Regardless of the format employed, ELISA has certain common features such as coating, incubation and binding, washing to remove non-specifically bound substances, and detection of bound immune complexes. These are described below.

When coating a plate with an antigen or antibody, one typically incubates the wells of the plate with a solution of the antigen or antibody overnight or for a specified number of hours. The wells of the plate will then be washed to remove the not fully adsorbed material. Any remaining available surface of the well is then "coated" with a non-specific protein that is antigen neutral with respect to the test antisera. These include Bovine Serum Albumin (BSA), casein or milk powder solutions. The coating allows blocking of non-specific adsorption sites on the immobilized surface, thereby reducing the background caused by non-specific binding of antisera to the surface.

In ELISA, it may be more customary to use secondary or tertiary detection means rather than direct procedures. Thus, after binding of the protein or antibody to the well, the background is reduced by coating with a non-reactive material and washing to remove unbound material, the immobilized surface is contacted with the biological sample to be tested under conditions effective to allow the formation of immune complexes (antigen/antibody). Detection of the immunocomplexes then requires a labeled secondary binding ligand or antibody, and a secondary binding ligand or antibody that binds to the labeled tertiary antibody or tertiary binding ligand.

By "under conditions effective to allow formation of immune complexes (antigen/antibody)" is meant that the conditions preferably include dilution of the antigen and/or antibody with a solution such as BSA, bovine Gamma Globulin (BGG) or Phosphate Buffered Saline (PBS)/Tween. These added additives also help reduce non-specific background.

By "suitable" conditions is also meant incubation at a temperature or for a period of time sufficient to allow effective binding. The incubation step is typically about 1 to 2 to 4 hours or so, with temperatures preferably of approximately 25 ℃ to 27 ℃, or may be about 4 ℃ or so overnight.

After all incubation steps in ELISA, the contacted surfaces were washed to remove uncomplexed material. Preferred washing procedures include washing with solutions such as PBS/Tween or borate buffers. After formation of specific immune complexes between the test sample and the initially bound material and subsequent washing, the presence of even minor amounts of immune complexes can be determined.

To provide a means of detection, the secondary or tertiary antibodies will have an associated label to allow detection. Preferably, this will be an enzyme that will develop color upon incubation with an appropriate chromogenic substrate. Thus, for example, one would wish to contact primary and secondary immune complexes with urease, glucose oxidase, alkaline phosphatase or catalase conjugated antibodies for a period of time or incubate both together for a period of time under conditions that favor the development of further immune complex formation (e.g., incubation in a PBS-containing solution such as PBS-Tween for 2 hours at room temperature).

After incubation with the labeled antibody and subsequent washing to remove unbound material, the amount of label is quantified, for example by incubation with chromogenic substrates such as urea, or bromocresol purple, or 2,2' -biazino-bis- (3-ethyl-benzothiazoline-6-sulfonic Acid (ABTS), or H ₂O₂ with peroxidase as enzyme label.

B. Western blot

Western blotting (or western immunoblotting) is an analytical technique used to detect specific proteins in a given tissue homogenate or extract sample. It uses gel electrophoresis to separate native or denatured proteins according to the length of the polypeptide (denaturing conditions) or the 3-D structure of the protein (native/non-denaturing conditions). The protein is then transferred to a membrane (typically nitrocellulose or PVDF) where it is probed (detected) using antibodies specific for the target protein.

The sample may be taken from whole tissue or cell culture. In most cases, the solid tissue is first mechanically disintegrated using a stirrer (for larger sample volumes), using a homogenizer (for smaller volumes) or by sonication. The cells may also be opened by one of the mechanical methods described above. Various detergents, salts and buffers may be employed to facilitate cell lysis and protein solubilization. Protease and phosphatase inhibitors are typically added to prevent the sample from being digested by its own enzymes. Tissue preparation is typically performed at low temperatures to avoid protein denaturation.

The proteins of the sample were separated using gel electrophoresis. Proteins may be isolated by isoelectric point (pI), molecular weight, charge, or a combination of these factors. The nature of the separation depends on the handling of the sample and the nature of the gel. This is a very useful way of determining proteins. Two-dimensional (2-D) gels may also be used to disperse proteins from a single sample into two dimensions. Proteins are separated according to isoelectric point (their pH with neutral net charge) in a first dimension and according to their molecular weight in a second dimension.

In order to make proteins available for antibody detection, they were moved from within the gel onto membranes made of nitrocellulose or polyvinylidene difluoride (PVDF). The membrane was placed over the gel and a stack of filter papers was placed over it. The entire stack is placed in a buffer solution which moves the paper upward by capillary action, thereby bringing about the proteins. Another method of transferring proteins is known as electroblotting, which uses an electric current to pull the proteins from a gel into a PVDF or nitrocellulose membrane. Proteins migrate from within the gel to the membrane while maintaining their tissue within the gel. As a result of this blotting procedure, the protein is exposed on a thin surface layer for detection (see below). Both membranes were chosen because they have non-specific protein binding properties (i.e., binding to all proteins is equivalent). Protein binding is based on hydrophobic interactions, as well as charged interactions between the membrane and the protein. Nitrocellulose membranes are cheaper than PVDF, but are much weaker and do not withstand repeated probing well. The uniformity and overall effectiveness of protein transfer from the gel to the membrane can be checked by staining the membrane with coomassie brilliant blue or ponceau dye. Once transferred, the protein is detected using either a primary antibody that is labeled or a primary antibody that is not labeled, and then indirectly detected using either a protein a that is labeled or a secondary antibody that is labeled that binds to the Fc region of the primary antibody.

C. immunohistochemistry

The antibodies of the present disclosure may also be used in combination with freshly frozen and/or formalin-fixed, paraffin-embedded tissue blocks prepared for investigation by Immunohistochemistry (IHC). Methods for preparing tissue blocks from these microparticle samples have been successfully used in previous IHC studies of various prognostic factors and are well known to those skilled in the art (Brown et al, 1990; abbond et al, 1990; allred et al, 1990).

Briefly, frozen sections can be prepared by rehydrating 50ng frozen "crushed" tissue in Phosphate Buffered Saline (PBS) in small plastic capsules at room temperature, granulating the particles by centrifugation, resuspending them in viscous embedding medium (OCT), inverting the capsules and/or granulating again by centrifugation, quick freezing in isopentane at-70 ℃, cutting the plastic capsules and/or removing the frozen tissue cylinders, fixing the tissue cylinders on a cryomicrotome chuck, and/or cutting 25-50 consecutive sections from the capsules. Or the entire frozen tissue sample may be used for serial sectioning.

Permanent sections can be prepared by a similar method involving the steps of rehydrating 50mg of the sample in a plastic microcentrifuge tube, pelleting, re-suspending in 10% formalin for 4 hours, washing/pelleting, re-suspending in warm 2.5% agar, pelleting, cooling in ice water to harden the agar, removing tissue/agar blocks from the tube, immersing and/or embedding the blocks in paraffin, and/or cutting up to 50 consecutive permanent sections. Also, the entire tissue sample may be replaced.

D. Immunoassay kit

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

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

Other suitable immunoassay reagents for use in the present kits include two-component reagents comprising a secondary antibody having binding affinity for a primary antibody, together with a tertiary antibody having binding affinity for the secondary antibody, wherein the tertiary antibody is linked to a detectable label. As noted above, many exemplary labels are known in the art and all such labels may be used in connection with the present disclosure.

The kit may further comprise suitable aliquots of PIV F protein composition, whether labeled or unlabeled, as may be used to prepare a standard curve for a detection assay. The kit may contain the antibody-label conjugate in the form of a complete conjugate, 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 in an aqueous medium or in lyophilized form.

The container means of the kit typically comprise at least one vial, test tube, flask, bottle, syringe or other container means in which the antibody may be placed or preferably suitably aliquoted. The kits of the present disclosure will also typically include means for hermetically containing antibodies, antigens, and any other reagent containers for commercial sale. Such containers may include injection molded or blow molded plastic containers with the desired vials held therein.

E. Flow cytometry and FACS

Antibodies of the present disclosure may also be used in flow cytometry or FACS. Flow cytometry is a laser or impedance-based technique used in many detection assays, including cell counting, cell sorting, biomarker detection, and protein engineering. The technique suspends cells in a fluid stream and passes them through an electronic detection device, which allows for multi-parameter analysis of physical and chemical characteristics of up to thousands of particles per second simultaneously. Flow cytometry is commonly used for diagnosis of disorders, particularly hematological cancers, but there are many other applications in basic research, clinical practice, and clinical trials.

Fluorescence Activated Cell Sorting (FACS) is a special type of cell counting. It provides a method for sorting heterogeneous mixtures of biological cells into two or more containers one cell at a time, based on specific light scattering and fluorescence characteristics of each cell. Generally, the technology involves a cell suspension entrained in a narrow, fast-flowing liquid flow center. The flow is arranged such that there is a great separation between the cells relative to their diameter. The vibration mechanism causes the cell stream to break up into individual droplets. Just prior to the cell stream breaking up into droplets, the cell stream passes through a fluorescence measurement station where the fluorescence of each cell is measured. A charging loop is placed at the point where the cell stream breaks up into droplets. Immediately prior to measuring fluorescence intensity, a charge is placed on the ring, while the opposite charge is trapped on the droplet as it breaks apart from the cell stream. The charged droplets then fall through an electrostatic deflection system that transfers the droplets into a container according to their charge.

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

VIII definition of

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 application, as claimed. In the present application, the use of the singular includes the plural unless specifically stated otherwise. In the present application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "include" and other forms such as "include" and "include (included)" are not limiting. In addition, unless specifically stated otherwise, terms such as "element" or "component" encompass both elements and components comprising one unit and elements and components comprising more than one subunit. Furthermore, the use of the term "portion" may include a portion of a portion or the entire portion.

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

The term "about" as used herein, when referring to a measurable value such as an amount, time interval, etc., is intended to encompass variations up to ±10% from the specified value. Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached 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. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

As used herein, "substantially free" with respect to a specified component is used herein to mean that none of the specified components are purposefully formulated into the composition and/or are present as contaminants or in trace amounts only. The total amount of the specified components resulting from any unintentional contamination of the composition is thus well below 0.05%, preferably below 0.01%. Most preferred are compositions wherein no amount of the specified component can be detected using standard analytical methods.

The term "antibody" refers to an intact immunoglobulin of any isotype or fragment thereof that can compete with intact antibodies for specific binding to a target antigen, and includes, for example, chimeric antibodies, humanized antibodies, fully human antibodies, and bispecific antibodies. An "antibody" is an antigen binding protein. An intact antibody will typically comprise at least two full length heavy chains and two full length light chains, but in some cases may comprise fewer chains, such as an antibody naturally occurring in a camelid that may comprise only heavy chains. Antibodies may be derived from only a single source or may be "chimeric", i.e., different portions of an antibody may be derived from two different antibodies, as described further below. Antigen binding proteins, antibodies or binding fragments may be produced in hybridomas by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. The term "antibody" includes, unless otherwise indicated, derivatives, variants, fragments and muteins thereof, examples of which are described below, in addition to antibodies comprising two full length heavy chains and two full length light chains. Further, unless expressly excluded, antibodies include monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimics"), 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 a peptibody (peptibody).

Naturally occurring antibody building blocks typically comprise tetramers. Each such tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one full length "light" chain (in certain embodiments, about 25 kDa) and one full length "heavy" chain (in certain embodiments, about 50-70 kDa). The amino-terminal portion of each chain typically includes a variable region of about 100 to 110 or more amino acids 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 generally classified into kappa light chains and lambda light chains. Heavy chains are generally classified as mu, delta, gamma, alpha or epsilon, and the isotypes of antibodies are defined as IgM, igD, igG, igA and IgE, respectively. IgG has several subclasses including, but not limited to, igG1, igG2, igG3, and IgG4. Subclasses of IgM include, but are not limited to, igM1 and IgM2.IgA is similarly subdivided into subclasses, including but not limited to IgA1 and IgA2. Within full length light and heavy chains, typically, the variable and constant regions are joined by a "J" region having about 12 or more amino acids, while heavy chains also include a "D" region having more than about 10 amino acids. See, e.g., fundamental Immunology, ch.7 (Paul, W.edit, 2 nd edition, RAVEN PRESS, N.Y. (1989)) (incorporated by reference in its entirety for all purposes). The variable regions of each light/heavy chain pair typically form an antigen binding site.

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

The variable regions typically exhibit the same general structure of relatively conserved Framework Regions (FR), also known as complementarity determining regions or CDRs, joined by three hypervariable regions. CDRs from each pair of two chains are typically aligned by a framework region, which allows for binding to a particular epitope. From the N-terminus to the C-terminus, the light and heavy chain variable regions typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is generally according to 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).

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

The explicit depiction of CDRs and identification of residues comprising the antibody binding site may be accomplished by solving the structure of the antibody and/or solving 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 employed to identify or coarsely estimate CDR regions. Examples of such methods include, but are not limited to, kabat definition, chothia definition, abM definition, and contact definition.

Kabat definition is a standard for numbering residues in antibodies and is commonly 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 the Chothia definition considers 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). AbM defines a computer program that uses an integrated suite of antibody constructs produced by Oxford Molecular Group to model. See, e.g., martin et al ,Proc Natl Acad Sci(USA),86:9268-9272(1989);"AbM^TM,A Computer Program for Modeling Variable Regions of Antibodies,"Oxford,UK;Oxford Molecular,Ltd..AbM, which define modeling the tertiary structure of antibodies from primary sequences using a combination of knowledge databases and de novo computing methods, such as those described by Samudrala et al ,"Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach,"PROTEINS,Structure,Function and Genetics Suppl.,3:194-198(1999). The contact definition is based on analysis of the complex crystal structure available. See, e.g., macCallum et al, J.mol.biol.,5:732-45 (1996).

Conventionally, CDR regions in the heavy chain are commonly referred to as H1, H2, and H3, and are numbered sequentially in the direction from the amino terminus to the carboxy terminus. CDR regions in the light chain are commonly referred to as L1, L2 and L3 and are numbered sequentially in the direction from the amino terminus to the carboxy terminus.

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

The term "heavy chain" includes full-length heavy chains and fragments thereof having sufficient variable region sequences to confer binding specificity. The full length heavy chain includes a variable region domain VH and three constant region domains CH1, CH2 and CH3. The VH domain is located at the amino terminus of the polypeptide, while the CH domain is located at the carboxy terminus, with CH3 closest to the carboxy terminus of the polypeptide. The heavy chain may be of any isotype including IgG (including IgG1, igG2, igG3 and IgG4 subtypes), igA (including IgA1 and IgA2 subtypes), igM and IgE.

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 ligation 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).

The term "antigen" refers to a substance capable of inducing an adaptive immune response. Specifically, an antigen is a substance that serves as a target of an adaptive immune response receptor. Typically, an antigen is a molecule that binds to an antigen-specific receptor but is not itself capable of inducing an immune response in vivo. Antigens are typically proteins and polysaccharides, and a few are also lipids. Antigens, as used herein, also include immunogens and haptens.

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

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

An antibody that "specifically binds to a particular polypeptide or an epitope on a particular polypeptide" or "has specificity for a particular polypeptide or an epitope on a particular polypeptide" is an antibody that binds to the particular polypeptide or an epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope. For example, PIV F protein-specific antibodies of the present disclosure are specific for PIV F proteins. Antibodies that bind to PIV F proteins may have dissociation constants (Kd) 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 to 10 ^-13 M, e.g., 10 ^-9 M to 10 ^-13 M).

The term "compete" when used in the context of antigen binding proteins (e.g., antibodies or antigen binding fragments thereof) that compete for the same epitope means that competition between antigen binding proteins, as determined by assays in which the antigen binding protein (e.g., antibody or antigen binding fragment thereof) being tested prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., ligand or reference antibody) to a cognate antigen (e.g., PIV F protein or fragment thereof). many types of competitive binding assays can be used to determine whether an antigen binding protein competes with another, such as a solid phase direct or indirect Radioimmunoassay (RIA), a solid phase direct or indirect Enzyme Immunoassay (EIA), a sandwich competition assay (see, e.g., stahli et al, 1983,Methods in Enzymology 9:242-253), a solid phase direct biotin-avidin EIA (see, e.g., kirkland et al, 1986, J. Immunol. 137:3614-3619), a solid phase direct labeling assay, Solid phase direct labeling sandwich assays (see, e.g., harlow and Lane,1988,Antibodies,A Laboratory Manual,Cold Spring Harbor Press), solid phase direct labeling RIA using 1-125 labels (see, e.g., morel et al, 1988, molecular. Immunol. 25:7-15), solid phase direct biotin-avidin EIA (see, e.g., cheung et al, 1990,Virology 176:546-552), and direct labeling RIA (Moldenhauer et al, 1990, scan. J. Immunol. 32:77-82). Typically, such assays involve the use of purified antigen bound to a solid surface or cell carrying either one of 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 a solid surface or cell in the presence of the test antigen binding protein. The antigen binding proteins are typically tested for their presence in excess. Antigen binding proteins identified by competition assays (competitive antigen binding proteins) include antigen binding proteins that bind to the same epitope as the reference antigen binding protein and antigen binding proteins that bind to an epitope that binds to the reference antigen binding protein in close enough proximity to allow steric hindrance. additional details regarding methods for determining competitive binding are provided in the embodiments herein. Typically, when the competing antigen binding protein is present in excess, it will inhibit (e.g., reduce) the specific binding of the reference antigen binding protein to the co-antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or more. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

As used herein, the term "epitope" refers to a specific group of atoms or amino acids on an antigen to which an antibody binds. The epitope may be a linear epitope or a conformational epitope. Linear epitopes are formed from contiguous amino acid sequences from an antigen and interact with antibodies according to their primary structure. Conformational epitopes, on the other hand, are made up of discrete parts of the amino acid sequence of the antigen and interact with antibodies according to the 3D structure of the antigen. Typically, an epitope is about five or six amino acids in length. If two antibodies exhibit competitive binding to an antigen, they may bind to the same epitope within the antigen.

A useful measure of antibody efficacy in the art is "50% neutralization titer". Another useful measure of antibody efficacy is any of the following "60% neutralization titer", 70% neutralization titer ", 80% neutralization titer", and 90% neutralization titer. For example, to determine 50% neutralization titers, serum from immunized animals was diluted to assess how the diluted serum would still retain the ability to block 50% of infectious virus from entering cells. For example, a titer of 700 means that the serum retains the ability to neutralize 50% of the infectious virus after 700-fold dilution. Thus, higher titers indicate a stronger neutralizing antibody response. The titer can be in 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 upper limit of the 50%, 60%, 70%, 80%, or 90% neutralization titer range may be about 400, about 600, about 800, about 1000, about 1500, about 2000, about 2500, about 3000, about 3500, about 4000, about 4500, about 5000, about 5500, about 6000, about 6500, about 7000, about 8000, about 9000, about 10000, about 11000, about 12000, about 13000, about 14000, about 15000, about 16000, about 17000, about 18000, about 19000, about 20000, about 21000, about 22000, about 23000, about 24000, about 25000, about 26000, about 27000, about 28000, about 29000, or about 30000. For example, 50% neutralization titers can be about 3000 to about 6500. "about" means plus or minus 10% of the value.

The term "host cell" means a cell that has been transformed with a nucleic acid sequence or is capable of being transformed with a nucleic acid sequence and thereby expressing a gene of interest. The term includes progeny of a parent cell, whether or not the progeny are identical in morphology or genetic composition to the original parent cell, as long as the gene of interest is present.

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

In calculating the percent identity, the sequences being compared are typically aligned in a manner that gives the greatest match between the sequences. One example of a computer program that may be used to determine the percent identity is the GCG package, which includes GAPs (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 two polypeptides or polynucleotides for which the percentage of sequence identity is to be determined. The sequences are aligned to obtain the best match of their respective amino acids or nucleotides ("match span", as determined by the algorithm). The gap opening penalty (calculated as 3 x average diagonal, where "average diagonal" is the average of the diagonals of the comparison matrix used; the "diagonal" is the fraction or number assigned to each perfect amino acid match by a particular comparison matrix) and the gap expansion penalty (typically 1/10 times the gap opening penalty) are used in conjunction with the algorithm, as are comparison matrices such as PAM 250 or BLOSUM 62. Standard comparison matrices (see, dayhoff et al, 1978,Atlas of Protein Sequence and Structure5:345-352 for PAM 250 comparison matrices; henikoff et al, 1992, proc. Natl. Acad. Sci. U.S. A.89:10915-10919 for BLOSUM 62 comparison matrices) can also be used by the algorithm.

Examples of parameters that can be used to determine the percent identity of a polypeptide or nucleotide sequence using the GAP program are found in Needleman et al, 1970, J.mol.biol.48:443-453.

Some alignment schemes for aligning two amino acid sequences may result in only one short region matching the two sequences, and this small alignment region may have very high sequence identity even though there is no significant relationship between the two full length sequences. Thus, if desired, the selected alignment method (GAP program) may be adjusted to produce an alignment of at least 50 or other numbers of consecutive amino acids across the target polypeptide.

As used herein, the term "linked" refers to association via an intramolecular interaction such as a covalent bond, a metallic bond, and/or an ionic bond or an intermolecular interaction such as a hydrogen bond or a noncovalent bond.

The term "operably connected" refers to an arrangement of elements wherein the components so described are configured to perform their usual functions. 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 will direct the expression of the coding sequence. Promoters or other control elements need not be adjacent to a coding sequence so long as they function to direct their expression. For example, there may be an intervening untranslated but transcribed sequence between the promoter sequence and the coding sequence, and the promoter sequence may still be considered "operably linked" to the coding sequence.

The term "or" is used in the claims to mean "and/or" unless explicitly indicated to mean only alternatives or alternatives are mutually exclusive, although the disclosure supports definitions of only alternatives and "and/or". As used herein, "another" may mean at least a second or more.

The term "polynucleotide" or "nucleic acid" includes both single-and double-stranded nucleotide polymers. The nucleotides making up the polynucleotide may 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, phosphoroselenate, phosphorodiselenate, phosphoroanilino-phosphorothioate, phosphoroanilino-phosphate, and phosphoramidate.

The term "polypeptide" or "protein" means a macromolecule having the amino acid sequence of a native protein, i.e., a protein produced by a naturally occurring and non-recombinant cell, or it is produced by a genetically engineered or recombinant cell and comprises a molecule having the amino acid sequence of a native protein, or a molecule having the deletion, addition, and/or substitution of one or more amino acids of a native sequence. The term also includes amino acid polymers in which one or more amino acids are chemical analogs of the corresponding naturally occurring amino acid and polymer. The terms "polypeptide" and "protein" specifically encompass PIV F protein binding proteins, antibodies or sequences having deletions, additions and/or substitutions of one or more amino acids of an antigen binding protein. The term "polypeptide fragment" refers to a polypeptide having an amino terminal deletion, a carboxy terminal deletion, and/or an internal deletion as compared to the full-length native protein. Such fragments may also contain modified amino acids as compared to the native protein. Fragments may be about 5 to 500 amino acids in length. For example, a fragment may 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, including binding domains. In the case of PIV F protein binding antibodies, useful fragments include, but are not limited to, CDR regions, variable domains of heavy and/or light chains, a portion of an antibody chain or variable regions thereof comprising only two CDRs, and the like.

Pharmaceutically acceptable carriers are conventional. Remington's Pharmaceutical Sciences, e.w. martin, mack Publishing co., easton, PA, 15 th edition (1975) describes compositions and formulations suitable for drug delivery of the fusion proteins disclosed herein. In general, the nature of the carrier will depend on the particular mode of administration employed. For example, parenteral formulations typically contain an injectable fluid as a vehicle, which includes pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, and the like. For solid compositions (e.g., in the form of powders, pills, tablets, or capsules), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to the biologically neutral carrier, the pharmaceutical composition to be administered may contain small amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives and pH buffering agents and the like, for example sodium acetate or sorbitan laurate.

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

As used herein, the term "therapeutically effective amount" or "effective dose" refers to the dose or concentration of a drug effective to treat a disease or disorder. For example, in connection with the treatment of viral infections using monoclonal antibodies or antigen binding fragments thereof as disclosed herein.

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

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

IX. example

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1

The sequence used for the design of the extracellular domain of hPIV 3F contains residues 19-481 of SEQ ID NO. 1. To screen for single substitution designs, the L168Q substitution was included to increase solubility and provide more consistent purification. Including the GCN4 CC tri2 trimerization domain. The construct also comprises an HRV3C protease recognition site cloned into the mammalian expression plasmid pαh, an octahistidine tag and a tandem Twin-Strep tag. The sequence of the base construct with the L168Q substitution is provided in SEQ ID NO. 8, wherein lysine is at position 141.

Using a structure-based design, substitutions aimed at facilitating pre-fusion structural stability were introduced into the base construct (PIV 3F L Q ectodomain with GCN4 trimerization tag, which adopts post-fusion conformation, FIG. 3; SEQ ID NO:8, where lysine is at position 141). Less than a distance apartThe core-facing residues of (c) are substituted with aromatic side chains or pairs of aromatic and positively charged side chains, respectively, to facilitate pi-pi or pi-cationic interactions. Or substitution of residues with extended or larger hydrophobic side chains to fill pre-existing internal cavities. Disulfide bonds are designed to increase overall stability or prevent formation of a post-fusion conformation. The purpose of the charge or polarity substitution is to predictNatural residues within the scope establish hydrogen bonds or salt bridges.

The single substitution design is brought into a combinatorial screening round. In this round, the L168Q substitution in the first round was converted back to leucine. For some combinatorial variants, the L168Q substitution was later reintroduced. Furthermore, the foldon trimerization motif of T4 fibritin is comprised between the GCN4 CC tri2 trimerization domain and the HRV3C protease recognition site (SEQ ID NO:11, wherein lysine is at position 141).

Table 1 provides an exemplary single substitution design. Table 2 provides exemplary substitution combinations.

Plasmids encoding hPIV 3F variants were transiently transfected into FreeStyle293F cells (Thermo Fisher) using polyethylenimine, and 5. Mu. Mkifunensine was added 3 hours after transfection. The cultures were grown for 4-6 days, and the culture supernatants were isolated by centrifugation and passage through a 0.22 μm filter. Proteins were purified from the supernatant using STREPTACTIN resin (IBA), followed by Size Exclusion Chromatography (SEC) using Superose 6 10/300 column (GE HEALTHCARE) in a buffer consisting of 2mM Tris pH 8.0, 200mM NaCl and 0.02% NaN ₃ to further purify the hPIV 3F variants. For initial purification and characterization, single substitution and combination variants were purified from 40mL cultures. Protein purity, monodispersity and expression level were determined by SDS-PAGE (e.g., fig. 4, 8, 10, 11, 17, 18 and 21) and SEC (e.g., fig. 9, 12, 13A and 22). The first peak corresponds to the multimer of hPIV 3F after trimer fusion, and the second peak corresponds to the monomeric hPIV 3F trimer.

Negative staining electron microscope (nsEM) analysis was performed on some of the hPIV 3F variants. The purified hPIV 3F variant was diluted to a concentration of 0.06mg/mL in 2mM Tris pH 8.0, 200mM NaCl and 0.02% NaN ₃. Each protein was deposited on a CF-400-CU grid (Electron Microscopy Sciences) that had been plasma cleaned in a Solarus 950 plasma cleaner (Gatan) at a 4:1O ₂/H₂ ratio for 30 seconds and stained with 2% (w/v) uranyl acetate. In a 2010f TEM (Japan electron optical laboratory) running at 200kV and equipped with a OneView camera (Gatan), at a magnification of 60,000X (corresponding toA calibrated pixel size) of the grid. Exemplary data are shown in fig. 6 and 17.

To confirm that stable substitutions did not result in any unexpected conformational changes, various designs of low temperature EM structures were determined (fig. 3, 13B, 14-16, 19 and 20). Purified hPIV 3F variants were diluted to a concentration range of 1-3mg/mL in 2mM Tris pH 8.0, 200mM NaCl, 0.02% NaN ₃ and applied to a plasma-washed CF-400 1.2/1.3 grid or UltrAuFoil 1.2.2/1.3 grid, then blotted dry in a Vitrobot Mark IV (thermo Fisher) for 3-6 seconds and frozen in liquid ethane. Photomicrographs were collected from individual grids using either (i) Titan Krios (thermo fisher) equipped with a K3 direct electron detector (Gatan) or (ii) Glacios (Thermofisher) equipped with Falcon IV. The data is the calibrated magnification at KriosAnd Falcon 4 calibration magnificationAnd (5) collecting the waste water.

***

All methods disclosed and claimed herein can be performed 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 of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. Rather, it will be apparent that certain agents that are both 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.

Reference to the literature

The following references are specifically incorporated herein by reference to the extent that they provide exemplary procedures or other details supplementary to those set forth herein.

International patent publication WO 2018/081289

International patent publication WO 2022/207839

U.S. patent publication 2022/0024987

Claims (53)

1. An engineered protein comprising a respiratory virus fusion protein (PIV F) extracellular domain having at least 90% sequence identity to amino acids 19-481 of SEQ ID NO: 1 or 2, wherein the engineered protein comprises at least one substitution or a group of substitutions selected from the group consisting of: H27C/F437C; H27C/T439C; H27C/P440C; H27C/I443C; H27Y; V28M; V30I; N33C/K295C; G37C/S337C; S41C/P283C; Y48C/I169C; Y48C/I169C/A 140Y; L49C/L278C; L49F; L49W; I50C/A171C; I50C/T277C; I50W; S52C/K173C; S52C/S275C; S52 L;L53C/S174C;P55C/V175C;P55C/Q176C;K56C/N155C;I57F;E58C/I183C;G64C/G196C;G64C/ G200C; Q67C/L199C; Q67C/G200C; Y71C/L203C; L86C/V266C; Q89C/A131C; K90Y; I93C/G116C; V 94C/G116C; T95C/G116C; T117P; I118C/G381C; A119C/G381C; L120C/G381C; A123P; T124P; S12 5C/P374C; S125P; S125W; A126P; I128F; I128W; L134C/I267C; A137C/I267C; I144W; L147C/A17 1C; I151C/A171C; A157C/Q176C; A157C/D177C; A157F; V158L; Q159C/A171C; L168Q; V170I; V17 0M; A171V; K173Q; V175L; V175P; V179L; E182F; E182W; P185C/A195C; G191P; G200E; I201F; I2 01W; A202T; E209W; I213C/I226C; I213C/G230C; G219C/E333C; L228C/V264C; L228F; L228W; R2 36W; R236Y; S246V; L256Y; V264F; V264W; V266F; V266W; S275F; S275M; T277F; T277L; T277W; L2 78F; L278W; V280F; V280W; R281Y; L282F; L282W; D327C/P344C; A334S; G345M; F346C/T369C; F3 46C/S370C; N349P; L356F; S361C/T444C; Q362C/N447C; P364C/N447C; T366C/V449C; T367R; N3 80C/G433C; G381C/K431C; G382C/G433C; V384I; T413C/A436C; G433F; I443W; I443Y; V449C/I4 1 or 2. 2. The engineered protein of claim 1 , comprising at least one paired cysteine substitution selected from the group consisting of: H27C/F437C; H27C/T439C; H27C/P440C; H27C/I443C; N33C/K295C; G37C/S337C; S41C/P283C; Y48C/I169C; Y48C/I169C/A140Y; L49C/L278C; I50C/A171C; I50C/T277C; S52C/K173C; S52C /S275C; L53C/S174C; P55C/V175C; P55C/Q176C; K56C/N155C; E58C/I183C; G64C/G196C; G64C/G200C; Q67C/L199C; Q67C /G200C; Y71C/L203C; L86C/V266C; Q89C/A131C; I93C/G116C; V94C/G116C; T95C/G116C; 20C/G381C; S125C/P374C; L134C/I267C; A137C/I267C; L147C/A171C; I151C/A171C; A157C/Q176C; A157C/D177C; Q159 C/A171C; P185C/A195C; I213C/I226C; I213C/G230C; G219C/E333C; L228C/V264C; D327C/P344C; F346C/T369C; F346C/S 370C;S361C/T444C;Q362C/N447C;P364C/N447C;T366C/V449C;N380C/G433C;G381C/K431C;G382C/G433C;T413C/A436C;V449C/I454C;V449C/D455C;V449C/I456C;V449C/S457C;S457C/V449C;S457C/I456C;K464C/V449C; and S470C/K471C. 3. The engineered protein of claim 1 or 2, further comprising at least one pair of cysteine substitutions selected from the group consisting of: S186C/A195C. 4. The engineered protein of claim 2 or 3, wherein the paired cysteine substitutions form disulfide bonds. 5. The engineered protein of any one of claims 1-4, comprising at least one cavity filling substitution or a group of cavity filling substitutions selected from the group consisting of: H27Y; V28M; V30I; L49F; L49W; I50W; S52L; I57F; K90Y; A140Y; I144W; A157F; V158L; V170I; V170M; A171V; V175L; V179L; E182F; E182W; G200E; I201F; I20 1W; L228F; L228W; R236Y; S246V; L256Y; V264F; V264W; V266F; V266W; S275F; S275M; T277F; T277L; T277W; L278F; L278W; V280F; V280W; R281Y; L282F; L282W; G345M; L356F; V384I; G433F; I443W; I443Y; A450F; I454F; and I456W. 6. The engineered protein of claim 5, wherein the substitution forms a salt bridge within a substitution pair or between a single substitution and a native amino acid in the protein. 7. The engineered protein of any one of claims 1-6, comprising at least one substitution or a group of substitutions selected from the group consisting of: T117P; A123P; T124P; S125P; A126P; V175P; G191P; N349P; L451P; and D452P. 8. The engineered protein of any one of claims 1-7, comprising at least one substitution or a group of substitutions selected from the group consisting of: S125W; I128F; I128W; E209W; and R236W. 9. The engineered protein of any one of claims 1-8, comprising at least one substitution or a group of substitutions selected from the group consisting of: K173Q; A202T; A334S; T367R; D455K; K471A; K471L; and W473A. 10. The engineered protein of any one of claims 1-9, comprising an E at position 108. 11. The engineered protein of any one of claims 1-10, comprising a combination of at least one engineered disulfide bond and at least one cavity-filling substitution. 12. The engineered protein of any one of claims 1-10, comprising a combination of at least one engineered disulfide bond and at least one proline substitution. 13. The engineered protein of any one of claims 1-10, comprising a set of substitutions selected from the group consisting of: G64C/G196C/V28M; G64C/G196C/V175L; G64C/G196C/V158L; G64C/G196C/A123P; G64C/G196C/S125P; G64C/G196C/I201W; G64C/G196C/L 282F; 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/V15 8L; A137C/I267C/A123P; A137C/I267C/S125P; A137C/I267C/I201W; A137C/I267C/L282F; A13 7C/I267C/L228W; A137C/I267C/R281Y; A137C/I267C/L282W; A137C/I267C/N349P; A137C/I267 C/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/A 171C/L228W; L147C/A171C/R281Y; L147C/A171C/L282W; L147C/A171C/N349P; L147C/A171C/T 367R; L147C/A171C/K471A; G64C/G196C/A137C/I267C/K471A; G64C/G196C/A137C/I267C/V175 L; G64C/G196C/A137C/I267C/S125P; G64C/G196C/A137C/I267C/S125P/V175L; G64C/G196C/A 137C/I267C/T367R; G64C/G196C/A137C/I267C/K471A/S125P; G64C/G196C/A137C/I267C/K471 A/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/A17 1C/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/V175 L/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/I15 1C/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/V4 49C/S457C; L168Q/G64C/G196C/A137C/I267C/K471A; L168Q/G64C/G196C/A137C/I267C/V175 L; L168Q/G64C/G196C/A137C/I267C/S125P; L168Q/G64C/G196C/A137C/I267C/S125P/V175L; L 168Q/G64C/G196C/A137C/I267C/T367R; L168Q/G64C/G196C/A137C/I267C/K471A/S125P; L168 Q/G64C/G196C/A137C/I267C/K471A/S125P/T367R/V175L; L168Q/G64C/G196C/A137C/I267C; L 168Q/G64C/G196C/A137C/I267C/K471A/S125P/L282F/V175L; L168Q/G64C/G196C/I151C/A17 1C/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/V2 8M/V175L/I201W/L282F/L228W/R281Y; L168Q/I151C/A171C/V449C/S457C; L168Q/I151C/A171 C/V449C/S457C/V28M/V175L/R281Y; I151C/A171C/S186C/A195C/V28M/V175L/R281Y; L168Q/ I151C/A171C/L228W/L282F/R821Y/V175L; I151C/A171C/L228W/L282F/R821Y/V175L; I151C/A 171C/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. The engineered protein of any one of claims 1-13, wherein the substitution or substitution group is selected from any one of the substitutions and substitution groups of Tables 1 and 2. 15. The engineered protein of any one of claims 1-14, comprising an L168Q substitution. 16. The engineered protein of any one of claims 1-15, wherein the PIV F extracellular domain is a human PIV (hPIV) F extracellular domain. 17. The engineered protein of any one of claims 1-16, wherein the human PIV F extracellular domain is the hPIV3 F extracellular domain. 18. The engineered protein of any one of claims 1-17, wherein the protein does not comprise the cytoplasmic tail of PIV F. 19. The engineered protein of any one of claims 1-18, wherein the protein is fused to or conjugated to a trimerization domain. 20. The engineered protein of claim 19, wherein the protein is fused to a trimerization domain. 21. The engineered protein of claim 19 or 20, wherein the trimerization domain comprises a T4 fibrin trimerization domain, a GCN4 domain, a 4J4A domain, or a combination thereof. 22. The engineered protein of any one of claims 19-21, wherein the trimerization domain comprises a sequence selected from the group consisting of: LKQIVLRIMEIEARIAKI AKIEGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG,LKQIVLRIMEI EARIAKIEGSEFNSLKQIVLRIMEIEARIAKIE,LKQIVLRIMEIEARIAKIEGSLKQIVLRIMEIEARIAKIE,LKQIVLRIMEIEARIAKIEGSLE LIKLRIMEIEARIAKIEKDRAIL. 23. The engineered protein of any one of claims 1-22, wherein the protein is fused to or conjugated to a transmembrane domain. 24. The engineered protein of claim 23, wherein the protein is fused to a transmembrane domain. 25. The engineered protein of claim 23 or 24, wherein the transmembrane domain comprises a PIV F protein transmembrane domain. 26. The engineered protein of claim 25, wherein the PIV F protein transmembrane domain comprises the sequence IIIILIMMIILFIINITIITI. 27. The engineered protein of claim 23 or 24, wherein the transmembrane domain does not comprise a PIV F protein transmembrane domain. 28. The engineered protein of any one of claims 1-27, comprising an N-terminal signal sequence. 29. The engineered protein of any one of claims 1-28, wherein the protein exhibits improved solubility or stability compared to native PIV F in a post-fusion conformation. 30. The engineered protein of any one of claims 1-29, wherein the protein is immunogenic. 31. An engineered parainfluenza virus fusion protein (PIV F) trimer comprising three engineered proteins according to any one of claims 1 to 30. 32. The engineered trimer of claim 31 , wherein the trimer is stabilized in a prefusion conformation relative to a trimer of native PIV F protein subunits. 33. The engineered trimer of claim 31 or 32, wherein the trimer comprises at least one engineered disulfide bond between subunits. 34. The engineered trimer of claim 33, wherein the trimer comprises at least one engineered disulfide bond between subunits selected from the group consisting of: I118C/G381C; A119C/G381C; L120C/G381C; S125C/P374C; G219C/E333C; F346C/T369C; F346C/S370C; and V449C/S457C. 35. A nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of the engineered protein of any one of claims 1-30. 36. The nucleic acid molecule of claim 35, wherein the nucleic acid molecule further comprises a DNA expression vector. 37. The nucleic acid molecule of claim 35, wherein the nucleic acid molecule is mRNA. 38. The nucleic acid molecule of claim 35, wherein the nucleic acid molecule is a self-replicating RNA molecule. 39. The nucleic acid molecule of any one of claims 35-38, further comprising at least one chemical modification. 40. The nucleic acid molecule of claim 39, wherein the at least one chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thiol-1-methyl-1-deaza-pseudouridine, 2-thiol-1-methyl-pseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxy-pseudouridine, 4-thiol-1-methyl-pseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and 2'-0-methyluridine. 41. A pharmaceutical composition comprising (i) the engineered protein of any one of claims 1-30, (ii) the engineered trimer of any one of claims 31-34, or (iii) the nucleic acid molecule of any one of claims 35-40; and a pharmaceutically acceptable carrier. 42. The pharmaceutical composition of claim 41, further comprising an adjuvant. 43. The pharmaceutical composition of claim 41 or 42, comprising another PIV antigen. 44. The pharmaceutical composition of any one of claims 41-43, wherein the composition is formulated within cationic lipid nanoparticles. 45. A method for preventing parainfluenza virus (PIV) infection or a disease associated with PIV infection in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of any one of claims 41-44. 46. A method of eliciting an immune response in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition of any one of claims 41-44. 47. A method for reducing parainfluenza virus (PIV) viral shedding in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition of any one of claims 41-44. 48. The pharmaceutical composition of any one of claims 41-44, for use in treating or preventing parainfluenza virus (PIV) infection or a disease associated with PIV infection in a subject. 49. The method of any one of claims 45-47 or the pharmaceutical composition of claim 48, wherein the subject is a mammal. 50. The pharmaceutical composition of any one of claims 41-44, for use in eliciting an immune response against parainfluenza virus (PIV). 51. Use of the pharmaceutical composition of any one of claims 41 to 44 in the manufacture of a medicament for treating or preventing parainfluenza virus (PIV) infection or a disease associated with PIV infection. 52. A composition comprising (i) the engineered protein of any one of claims 1-30 or (ii) the engineered trimer of any one of claims 31-34 bound to an antibody. 53. The composition of claim 52, wherein the antibody specifically binds to the PIV F extracellular domain in the prefusion conformation.