Stabilized prefusion PIV3 F protein

Abstract

The present invention relates to stabilized pre-fusion human parainfluenza virus 3 (HPIV3) F proteins and fragments thereof. The present invention also relates to nucleic acid molecules encoding such proteins and fragments, and uses of the proteins, fragments, and nucleic acid molecules.

Description

[Technical Field]

[0001] The present invention relates to the field of medicine. In particular, the present invention relates to recombinant PIV3 pre-fusion F proteins, nucleic acid molecules encoding the PIV3 F proteins, and their use, for example, in vaccines. [Background technology]

[0002] Background of the Invention Human parainfluenza type III (HPIV3) primarily causes respiratory complications in children and immunocompromised individuals, but it has recently been recognized as a concern in adult populations as well. In the United States, HPIV3 causes over 11,000 pediatric hospitalizations annually (Weinberg et al., J Pediatr. 154:694-699, 2009), and HPIV3 is also a significant cause of mortality, morbidity, and healthcare burden in other vulnerable populations (Ison et al., Clin. Microbiol Rev 32, 2019). Most children aged 5 years and older have antibodies to HPIV-3, indicating that most children have experienced HPIV3 infection by that age.

[0003] Currently, there is no vaccine or specific antiviral treatment to prevent HPIV disease. Treatment is supportive, although the use of corticosteroids and nebulized epinephrine has been found to be beneficial in croup.

[0004] Four HPIV serotypes (HPIV-1 to -4) are known, which are associated with different clinical symptoms and seasonal occurrences, with HPIV-3 being the most prevalent and generally manifesting as bronchiolitis / pneumonia. Seasonal variations and spontaneous outbreaks in the different serotypes result in overall variable occurrence and complex epidemiology.

[0005] HPIV3 is an enveloped RNA virus in the Paramyxoviridae family of the Mononegavirales. Its genome is approximately 15,000 nucleotides long and encodes six major proteins in the following gene sequence: 3'-NPMF-HN-L-5. Virus-cell fusion occurs through the cooperative action of two envelope glycoproteins that constitute the viral entry machinery: the receptor-binding protein hemagglutinin neuraminidase (HN) and the fusion protein (F). HN, a molecule with both receptor-binding and cleavage activities, stimulates and activates the F protein upon binding to a sialic acid-containing target receptor. The F protein fuses the viral membrane with the host cell membrane through irreversible protein refolding from an unstable pre-fusion conformation to a stable post-fusion conformation. The structures of both conformations have been determined for several paramyxoviruses, providing insight into the complex mechanism of this fusion protein. As a type I membrane protein, the F protein is translated in the endoplasmic reticulum and transported to the plasma membrane via the Golgi apparatus and trans-Golgi network. Similar to other class I fusion proteins, the inactive precursor PIV3 F0 requires cleavage by an appropriate host endoprotease (presumably TMPRSS2) at a monobasic cleavage site into disulfide-linked subunits, F1 and F2. After this cleavage, F1 contains a hydrophobic fusion peptide (FP) at its N-terminus. To refold from the pre-fusion conformation to the post-fusion conformation, refolding region 1 (RR1) between residues 110 and 213 (where the numbering is based on the numbering of amino acid residues in SEQ ID NO: 1), which includes FP and heptad repeat A (HRA), must be converted from a collection of helices, loops, and strands to a long, continuous helix. This allows the FP located in the N-terminal segment of RR1 to extend outward from the viral membrane and insert into the proximal membrane of the target cell.Next, refolding region 2 (RR2), which forms the C-terminal stem of the prefusion F spike and contains the heptad repeat B (HRB), moves to the opposite side of the PIV3 F head and binds to the HRB domain along with the HRA coiled-coil trimer to form a six-helix bundle. The formation of the RR1 coiled-coil and the repositioning of RR2 to complete the six-helix bundle are the most dramatic structural changes that occur during the refolding process. Class I fusion proteins have been shown to be intrinsically unstable, and although structure-based stabilization of viral fusion proteins in the prefusion conformation has been shown to induce superior neutralization and protection in animal models and clinical trials (Krarup et al., Nat Commun. 6:8143, 2015; De Taeye, Cell 163(7):1702-1715, 2015; McLellan et al., Science. 342(6158):592-598, 2013; Stewart-Jones et al., PNAS 48:12265-12270, 2018; Crank et al., Science 365(6452):505-509, 2019; Sadoff et al., JID doi:10.1093 / infdis / jiab003 2021; Sadoff et al., NEJM, doi:10.1056 / NEJMoa2034201 2021), currently, no vaccine is still available and no treatment exists for the prevention or treatment of hPIV3.

[0006] Therefore, there remains a need for an effective vaccine against PIV3, particularly a vaccine that includes or is based on the PIV3 F protein in a pre-fusion conformation. Indeed, a vaccine, preferably indicated for children and high-risk patients (e.g., the elderly and COPD patients), could provide broad-impact intervention from a fairly upstream stage of serious disease, thereby reducing the overall frequency of HPIV3 and associated morbidity and mortality. The present invention aims to provide a means for obtaining such a stable pre-fusion PIV3 F protein for use in vaccination against PIV3. Summary of the Invention

[0007] Overview of the invention The present invention provides stable recombinant pre-fusion human parainfluenza type III (HPIV3) fusion (F) proteins, i.e., recombinant HPIV3 F proteins and fragments thereof stabilized in the pre-fusion conformation. The pre-fusion HPIV3 F protein or fragments thereof contain at least one epitope specific to the pre-fusion conformation F protein, as determined, for example, by specific binding of an antibody specific for the pre-fusion conformation to the protein. In certain preferred embodiments, the pre-fusion HPIV3 F protein is a soluble multimeric (preferably trimeric) protein. The present invention also provides nucleic acid molecules encoding the pre-fusion HPIV3 F protein or fragments thereof, and vectors, e.g., adenovectors, containing such nucleic acid molecules.

[0008] The present invention also relates to a method for stabilizing HPIV3 F protein in the pre-fusion conformation and to the pre-fusion PIV3 F protein obtainable by said method.

[0009] The present invention further relates to compositions, preferably pharmaceutical compositions, comprising the PIV3 F protein, nucleic acid molecule, and / or vector described herein, and their use in inducing an immune response to the PIV3 F protein, in particular their use as a vaccine against PIV3. The present invention also relates to a method for inducing an anti-parainfluenza virus type III (PIV3) immune response in a subject, comprising administering to the subject an effective amount of a pre-fusion HPIV3 F protein, a nucleic acid molecule encoding the HPIV3 F protein, and / or a vector comprising the nucleic acid molecule described herein. Preferably, the induced immune response is characterized by the induction of neutralizing antibodies against PIV3 and / or protective immunity against PIV3. In a specific aspect, the present invention relates to a method for inducing anti-parainfluenza virus type III (PIV3) F antibodies in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising the pre-fusion HPIV3 F protein, a nucleic acid molecule encoding the PIV3 F protein, and / or a vector comprising the nucleic acid molecule described herein. [Brief explanation of the drawings]

[0010] [Figure 1] Schematic representation of conserved elements of the PIV3 F protein in both the full-length membrane-bound protein ("Full Length," top panel) and the mature soluble ectodomain ("Ectodomain," bottom panel). The N-terminal F2 domain is preceded by a signal peptide sequence (SP), which is cleaved off during protein maturation. The fusion peptide (FP) is located at the N-terminus of F1. Heptad repeats A, B, and C are indicated (HRA, HRB, and HRC, respectively). Additionally, the transmembrane region (TM) and cytoplasmic tail (CT) are also indicated. The soluble ectodomain may be accompanied by a C-terminal GCN4 trimerization motif. The cleavage sites between SP and F2 and between F2 and F1 are indicated by arrows. [Figure 2]Binding of PIA174 to PIV3 preF in cell supernatants measured by biolayer interferometry. Quantitative octet measurements were performed using PIV3 F in crude cell supernatants by immobilizing the antibody PIA174 on an anti-human IgG sensor. Initial binding rates are plotted. Cell culture medium from PIV3 F ("wild-type," SEQ ID NO: 2) without stabilizing mutations and without GCN4 and mock-transfected cells ("mock") served as negative controls. Measurements were performed on the day of harvest ("day 0") and repeated after 20 days of storage at 4°C ("day 20"). Single and double mutations were tested in the GCN4 trimerization domain and backbone with the D452N mutation, as indicated. [Figure 3-1] Analytical SEC profiles of various PIV3 F proteins with stabilizing mutations in crude cell supernatants. The indicated protein variants (black, solid lines) were compared with mock-transfected supernatants (dashed lines). The peak at 4.4–4.5 min corresponds to the PIV3 preF trimer. [Figure 3-2] Analytical SEC profiles of various PIV3 F proteins with stabilizing mutations in crude cell supernatants. The indicated protein variants (black, solid lines) were compared with mock-transfected supernatants (dashed lines). The peak at 4.4–4.5 min corresponds to the PIV3 preF trimer. [Figure 3-3] Analytical SEC profiles of various PIV3 F proteins with stabilizing mutations in crude cell supernatants. The indicated protein variants (black, solid lines) were compared with mock-transfected supernatants (dashed lines). The peak at 4.4–4.5 min corresponds to the PIV3 preF trimer. [Figure 4-1]Binding of PIA174 to PIV3 preF in cell supernatants measured by biolayer interferometry for single and all possible double combinations of stabilizing mutations. Quantitative octet measurements were performed using PIV3 F in crude cell supernatants by immobilizing the antibody PIA174 on an anti-human IgG sensor. Initial binding rates are plotted. Cell culture medium from PIV3 F ("wild-type," SEQ ID NO: 2) without stabilizing mutations and without GCN4, as well as mock-transfected cells ("mock"), served as negative controls. Measurements were performed on the day of harvest. Single and double mutations were tested in the GCN4 trimerization domain and backbone with the D452N mutation, as indicated. [Figure 4-2] Binding of PIA174 to PIV3 preF in cell supernatants measured by biolayer interferometry for single and all possible double combinations of stabilizing mutations. Quantitative octet measurements were performed using PIV3 F in crude cell supernatants by immobilizing the antibody PIA174 on an anti-human IgG sensor. Initial binding rates are plotted. Cell culture medium from PIV3 F ("wild-type," SEQ ID NO: 2) without stabilizing mutations and without GCN4, as well as mock-transfected cells ("mock"), served as negative controls. Measurements were performed on the day of harvest. Single and double mutations were tested in the GCN4 trimerization domain and backbone with the D452N mutation, as indicated. [Figure 5-1] Figure 1 shows binding of PIA174 to PIV3 preF in 5-fold diluted cell supernatants, measured by biolayer interferometry, for all possible combinations of selected stabilizing mutations. Quantitative octet measurements were performed using PIV3 F in 5-fold diluted crude cell supernatants and immobilizing the antibody PIA174 on an anti-human IgG sensor. Initial binding rates are plotted. Mutation combinations were tested in the GCN4 trimerization domain and backbone with D452N, Q89M, Q222I, and L168P mutations, as indicated. [Figure 5-2]Figure 1 shows binding of PIA174 to PIV3 preF in 5-fold diluted cell supernatants, measured by biolayer interferometry, for all possible combinations of selected stabilizing mutations. Quantitative octet measurements were performed using PIV3 F in 5-fold diluted crude cell supernatants and immobilizing the antibody PIA174 on an anti-human IgG sensor. Initial binding rates are plotted. Mutation combinations were tested in the GCN4 trimerization domain and backbone with D452N, Q89M, Q222I, and L168P mutations, as indicated. [Figure 6-1] Stabilized PIV3 preF in the absence of the GCN4 trimerization domain. (A) Binding of PIA174 to PIV3 preF PIV200941 (which does not contain the GCN4 trimerization domain but contains the D452N+Q89M+Q222I+L168P mutations) in cell supernatants as measured by biolayer interferometry. S470V and / or S477V were introduced into the stem of the PIV3 F protein. The initial binding rate is plotted. (B) Sample A tested in analytical SEC. The peak at approximately 4.8 minutes corresponds to PIV3 preF trimers. (C) Binding of PIA174 to unstabilized PIV3 preF (PIV190058, SEQ ID NO: 2) without the GCN4 trimerization domain in cell supernatants as measured by biolayer interferometry. S470V or S477V (PIV200960 and PIV200962, respectively) were introduced into the stem of the PIV3 F protein, and the initial binding rates are plotted. [Figure 6-2]Stabilized PIV3 preF in the absence of the GCN4 trimerization domain. (A) Binding of PIA174 to PIV3 preF PIV200941 (which does not contain the GCN4 trimerization domain but contains the D452N+Q89M+Q222I+L168P mutations) in cell supernatants as measured by biolayer interferometry. S470V and / or S477V were introduced into the stem of the PIV3 F protein. The initial binding rate is plotted. (B) Sample A tested in analytical SEC. The peak at approximately 4.8 minutes corresponds to PIV3 preF trimers. (C) Binding of PIA174 to unstabilized PIV3 preF (PIV190058, SEQ ID NO: 2) without the GCN4 trimerization domain in cell supernatants as measured by biolayer interferometry. S470V or S477V (PIV200960 and PIV200962, respectively) were introduced into the stem of the PIV3 F protein, and the initial binding rates are plotted. [Figure 7-1] Further stabilization of PIV3 preF in the absence of the GCN4 trimerization domain. (A) Binding of PIA174 to a matrix of PIV3 preF designed without the GCN4 trimerization domain in cell supernatants as measured by biolayer interferometry. The initial binding rate is plotted. (B) Samples from (A) tested by analytical SEC. The peak at approximately 4.8 minutes corresponds to PIV3 preF trimers. [Figure 7-2] Further stabilization of PIV3 preF in the absence of the GCN4 trimerization domain. (A) Binding of PIA174 to a matrix of PIV3 preF designed without the GCN4 trimerization domain in cell supernatants as measured by biolayer interferometry. The initial binding rate is plotted. (B) Samples from (A) tested by analytical SEC. The peak at approximately 4.8 minutes corresponds to PIV3 preF trimers. [Figure 7-3]Further stabilization of PIV3 preF in the absence of the GCN4 trimerization domain. (A) Binding of PIA174 to a matrix of PIV3 preF designed without the GCN4 trimerization domain in cell supernatants as measured by biolayer interferometry. The initial binding rate is plotted. (B) Samples from (A) tested by analytical SEC. The peak at approximately 4.8 minutes corresponds to PIV3 preF trimers. [Figure 7-4] Further stabilization of PIV3 preF in the absence of the GCN4 trimerization domain. (A) Binding of PIA174 to a matrix of PIV3 preF designed without the GCN4 trimerization domain in cell supernatants as measured by biolayer interferometry. The initial binding rate is plotted. (B) Samples from (A) tested by analytical SEC. The peak at approximately 4.8 minutes corresponds to PIV3 preF trimers. [Figure 8] Removal of S470V and S477V opens the PIV3 preF trimer. (A) Crude cell supernatants of PIV201105 and PIV201103 from Figure 7B were tested by analytical SEC-MALS. The hydrodynamic radius and molecular weight (MW) of the major peaks (indicated by arrows) were measured and are shown in (B). The MALS signal corresponding to the molecular weight is indicated by a dotted line for each peak. [Figure 9-1] Analytical SEC after heat stress. The indicated proteins in crude cell supernatant were incubated at 4°C (dashed line), 50°C (black line), or 60°C (gray line) for 30 minutes. Samples were then analyzed by analytical SEC to measure the loss of PIV3 preF trimers. The backbone used contained the S41P+Q89M+Q222I+N167P+L168P+D452N+S470V+S477V stabilizing mutations and did not have a heterologous trimerization domain. [Figure 9-2]Analytical SEC after heat stress. The indicated proteins in crude cell supernatant were incubated at 4°C (dashed line), 50°C (black line), or 60°C (gray line) for 30 minutes. Samples were then analyzed by analytical SEC to measure the loss of PIV3 preF trimers. The backbone used contained the S41P+Q89M+Q222I+N167P+L168P+D452N+S470V+S477V stabilizing mutations and did not have a heterologous trimerization domain. [Figure 10-1] Stability of PIV3 preF mutants. Crude cell supernatants of the indicated proteins were analyzed by differential scanning fluorometry (DSF) to determine melting temperatures. [Figure 10-2] Stability of PIV3 preF mutants. Crude cell supernatants of the indicated proteins were analyzed by differential scanning fluorometry (DSF) to determine melting temperatures. [Figure 11-1] Characterization of purified PIV3 preF protein. Protein was prepared from expiHEK supernatant using C-tag purification followed by size-exclusion chromatography. (A) Overview of different protein designs and their yields after purification. (B) SDS-PAGE under reducing and non-reducing conditions. Unprocessed PIV3 F protein migrates at approximately 50 kD. (C) SEC-MALS and (D) DSF analysis of purified protein. [Figure 11-2] Characterization of purified PIV3 preF protein. Protein was prepared from expiHEK supernatant using C-tag purification followed by size-exclusion chromatography. (A) Overview of different protein designs and their yields after purification. (B) SDS-PAGE under reducing and non-reducing conditions. Unprocessed PIV3 F protein migrates at approximately 50 kD. (C) SEC-MALS and (D) DSF analysis of purified protein. [Figure 11-3]Characterization of purified PIV3 preF protein. Protein was prepared from expiHEK supernatant using C-tag purification followed by size-exclusion chromatography. (A) Overview of different protein designs and their yields after purification. (B) SDS-PAGE under reducing and non-reducing conditions. Unprocessed PIV3 F protein migrates at approximately 50 kD. (C) SEC-MALS and (D) DSF analysis of purified protein. [Figure 12-1] A: Table of constructs used. The absence (-) or presence (-) of the HR2 stem mutation S470V+S477V in the design and the absence (-) or introduction (-) of various amino acid substitutions in the head domain of the PIV3 F protein are shown. Single head mutations were evaluated, except for the combination Q889M+Q221I (the side chains interact in the prefusion structure). B. Detection of prefusion PIV3 F trimers in the supernatants of cells transfected with the mutants in A), as measured by binding of the prefusion-specific PIA174 antibody with biolayer interferometry (qOctet). Quantitative Octet measurements were performed using PIV3 F in crude cell supernatants and immobilizing the antibody PIA174 on an anti-human IgG sensor. Initial binding rates are plotted. C. Detection of PIV3 F trimers in the supernatants of cells transfected with the mutants shown in A), as measured by analytical SEC. PIV3 F trimers (indicated by "T") eluted at 4.6-4.8 min. Each panel compares the absence (dotted line) or presence (solid line) of the S470V+S477V mutations combined with an amino acid substitution in the head domain of PIV3 F (specific mutations indicated above each graph). [Figure 12-2]A: Table of constructs used. The absence (-) or presence (-) of the HR2 stem mutation S470V+S477V in the design and the absence (-) or introduction (-) of various amino acid substitutions in the head domain of the PIV3 F protein are shown. Single head mutations were evaluated, except for the combination Q889M+Q221I (the side chains interact in the prefusion structure). B. Detection of prefusion PIV3 F trimers in the supernatants of cells transfected with the mutants in A), as measured by binding of the prefusion-specific PIA174 antibody with biolayer interferometry (qOctet). Quantitative Octet measurements were performed using PIV3 F in crude cell supernatants and immobilizing the antibody PIA174 on an anti-human IgG sensor. Initial binding rates are plotted. C. Detection of PIV3 F trimers in the supernatants of cells transfected with the mutants shown in A), as measured by analytical SEC. PIV3 F trimers (indicated by "T") eluted at 4.6-4.8 min. Each panel compares the absence (dotted line) or presence (solid line) of the S470V+S477V mutations combined with an amino acid substitution in the head domain of PIV3 F (specific mutations indicated above each graph). [Figure 12-3]A: Table of constructs used. The absence (-) or presence (-) of the HR2 stem mutation S470V+S477V in the design and the absence (-) or introduction (-) of various amino acid substitutions in the head domain of the PIV3 F protein are shown. Single head mutations were evaluated, except for the combination Q889M+Q221I (the side chains interact in the prefusion structure). B. Detection of prefusion PIV3 F trimers in the supernatants of cells transfected with the mutants in A), as measured by binding of the prefusion-specific PIA174 antibody with biolayer interferometry (qOctet). Quantitative Octet measurements were performed using PIV3 F in crude cell supernatants and immobilizing the antibody PIA174 on an anti-human IgG sensor. Initial binding rates are plotted. C. Detection of PIV3 F trimers in the supernatants of cells transfected with the mutants shown in A), as measured by analytical SEC. PIV3 F trimers (indicated by "T") eluted at 4.6-4.8 min. Each panel compares the absence (dotted line) or presence (solid line) of the S470V+S477V mutations combined with an amino acid substitution in the head domain of PIV3 F (specific mutations indicated above each graph). [Figure 13] A. Description of the constructs used and the melting temperatures of PIV3 F trimers in the supernatants of transfected Expi293 cells, as measured by differential scanning fluorometry (DSF). Removal of single or double mutations from PIV211368 (by reverting them to wild-type amino acids) is indicated in bold. B. Yields of PIV3 F trimers in the supernatants of cells transfected with the mutants shown in A), as measured by analytical SEC. PIV3 F trimers elute with a retention time of 4.6–4.8 min. [Figure 14]A. Analytical SEC of purified PIV3 F trimer PIV211368. Tag-free PIV3 F was purified from cell-free supernatant of transfected Expi293 cells by ion-exchange purification and polishing by size-exclusion chromatography. B. Table of purified PIV211368 PIV3 F protein properties, including yield, trimer size, hydrodynamic radius, and melting temperature (DSF). C. Slow-freeze stability of purified PIV3 F trimer PIV211368 in various buffers. Recovery of PIV3 F trimer after slow freezing of the protein from 20°C to -70°C over 24 hours is compared to trimer recovery after storage at 4°C (histograms are the average of n=5 individual measurements (open circles)). Buffer composition: FB12: 20 mM histidine, 75 mM NaCl, 5% sucrose, 0.02% PS80, 0.4% (w / w) EtOH, 0.1 mM EDTA, pH 6.5. PS4P4: 20 mM KHPO4, 75 mM NaCl, 4% sucrose, 0.01% PS20, pH 6.5. TS5P2: 20 mM Tris, 75 mM NaCl, 5% sucrose, 0.02% PS20, 0.4% EtOH, pH 7.5. [Figure 15] A. Analytical SEC of purified PIV3 F trimer PIV210235. PIV3 F was purified from cell-free supernatant of transfected GnT1 cells by C-tag purification and polishing with size-exclusion chromatography. B. Table of purified PIV210235 PIV3 F protein properties, including yield, trimer size, hydrodynamic radius, and melting temperature (DSF). [Figure 16] SDS-PAGE under reducing or non-reducing conditions followed by Coomassie staining. The dotted circle indicates partial processing of PIV211368. [Figure 17] Melting temperatures of purified HPIV3 F trimers measured by differential scanning fluorometry (DSF). N=3 replicates. Individual and average values ​​are shown as gray and black solid lines, respectively. [Figure 18]Binding of PIA174 to purified PIV3 preF protein measured by biolayer interferometry. PIA174 antibody was immobilized on an anti-human IgG sensor. The initial binding rate is plotted. A negative control (NC) protein that is not PIV3 preF protein and 1x kinetic buffer were used as negative controls.

[0011] Detailed Description of the Invention As previously described, the fusion protein (F) of parainfluenza virus (PIV3) is involved in the fusion of the viral membrane with the host cell membrane, which is necessary for infection. PIV3 F mRNA is translated into a 539-amino acid precursor protein designated F0, which contains an N-terminal signal peptide sequence (e.g., amino acid residues 1-18 of SEQ ID NO: 1) that is removed by a signal peptidase in the endoplasmic reticulum. F0 is presumably cleaved in the plasma membrane between amino acid residues 109 and 110 by a cellular protease (likely TMPRSS2 or a TMPRSS2-like enzyme) to generate two domains or subunits designated F1 and F2. The F1 domain (amino acid residues 110-539) contains a hydrophobic fusion peptide at its N-terminus and a transmembrane (TM) region (amino acid residues 494-516) and a cytoplasmic region (amino acid residues 517-539) at its C-terminus. The F2 domain (amino acid residues 19–109) is covalently linked to F1 by a single disulfide bond (Figure 1). The F1-F2 heterodimer assembles as a homotrimer on the virion surface. The mature ectodomain of the PIV3 F protein (comprising amino acid residues 19–493) can be structurally divided into a globular head domain (amino acid residues 19–451) and a fibrous stem region (amino acid residues 452–484).

[0012] A vaccine against PIV3 infection is not currently available. One potential approach for producing a vaccine is a subunit vaccine based on purified PIV3 F protein. However, for this approach, it is desirable that the purified PIV3 F protein be stable over time, i.e., remain in a pre-fusion conformation (e.g., as determined by specific binding of the PIV3 F protein to an antibody specific for the pre-fusion conformation of the PIV3 F protein), be in a conformation similar to the pre-fusion conformation of the PIV3 F protein, and be produced in sufficient quantities. Furthermore, for a soluble subunit-based vaccine, the PIV3 F protein must be truncated by deleting the transmembrane (TM) and cytoplasmic regions to generate a soluble secreted F protein ectodomain (sF). Because the TM region provides membrane anchoring and enhances stability, the F protein ectodomain is significantly less stable than the full-length protein and more readily refolds to its final post-fusion state. Therefore, to obtain a soluble F protein in the pre-fusion conformation that exhibits high expression levels and high stability, the pre-fusion conformation needs to be stabilized.

[0013] Stabilization of the prefusion conformation is also desirable for full-length PIV3 F protein (i.e., including the TM and cytoplasmic regions), e.g., for any live-attenuated or vector-based vaccine approach, since full-length (membrane-bound) PIV3 F protein is also metastable.

[0014] Recently, a HPIV-3 protein mutant containing several stabilizing amino acid substitutions that stabilize the prefusion conformation has been described (Stewart-Jones et al., PNAS 115(48)12265-12270, 2018). However, this mutant has several drawbacks: i) the expression and stability of this PIV3 preF protein were insufficient for the full development of a successful vaccine; ii) some of these mutations are located on the surface of the protein, which may affect antigenicity and immunogenicity; and / or iii) this mutant is fused to the GCN4 trimerization domain at the C-terminus, which may affect immunogenicity and may induce irrelevant antibodies against this trimerization domain. Such irrelevant antibodies do not cross-react with the virus and may impair immunogenicity if this domain is used in other (future) vaccines to enhance its immunodominance.

[0015] Described herein is a stabilized pre-fusion human parainfluenza virus 3 (HPIV3) F protein comprising an F1 domain and an F2 domain, the F protein comprising the amino acid sequence of the F1 and F2 domains of the HPIV3 strain, comprising hydrophobic amino acids at positions 470 and 477, where the numbering of amino acid positions is according to the numbering of amino acid residues in SEQ ID NO: 1. Preferably, the protein is a trimer. The hydrophobic amino acid at positions 470 and / or 477 can be any hydrophobic amino acid, including, but not limited to, valine, leucine, isoleucine, methionine, and phenylalanine. The amino acid residues at positions 470 and 477 can be the same hydrophobic amino acid or can be different hydrophobic amino acids. In certain preferred embodiments, the hydrophobic amino acid at positions 470 and / or 477 is valine (V), and preferably, both amino acids at positions 470 and 477 are valine (V).

[0016] The protein may comprise one or more additional mutations. Thus, in certain embodiments, the amino acid residue at position 452 is N, and / or the amino acid residue at position 41 is P, and / or the amino acid residue at position 167 is P, and / or the amino acid residue at position 168 is P, and / or the amino acid residue at position 335 is P, and / or the amino acid residue at position 89 is M, and the amino acid residue at position 222 is I, and / or the amino acid residue at position 165 is P, and / or the amino acid residue at position 198 is L, and / or comprises a disulfide bridge between amino acid residues 85 and 221 and / or between amino acid residues 186 and 195, wherein the numbering of the amino acid positions is according to the numbering of the amino acid residues in SEQ ID NO: 1.

[0017] Further described is a protein, wherein the amino acid residue at position 204 is D, and / or the amino acid residue at position 367 is L, and / or the amino acid residue at position 436 is P, and / or the protein comprises a disulfide bridge between amino acid residues 38 and 291.

[0018] The present invention provides a protein comprising an F1 domain and an F2 domain, comprising the amino acid sequence of the F1 domain and the F2 domain of the F protein of HPIV3 strain, wherein the amino acid residue at position 41 is P, the amino acid residue at position 89 is M, the amino acid residue at position 222 is I, the amino acid residue at position 168 is P, the amino acid residue at position 470 is V, the amino acid residue at position 477 is V, and the amino acid residue at position 109 is Q, wherein the numbering of the amino acid positions is according to the numbering of the amino acid residues in SEQ ID NO:1.

[0019] The present invention provides stabilized trimeric pre-fusion HPIV-3 proteins that exhibit high expression levels and enhanced stability. Additionally, the proteins of the present invention are single-chain proteins, i.e., the proteins are not processed (cleaved). The proteins are resistant to proteolysis, thereby improving the manufacturability of the proteins.

[0020] According to the present invention, it has been demonstrated that the presence of one or more specific amino acid residues at the indicated positions enhances the stability of the HPIV3 F protein and / or the HPIV3 F protein ectodomain in the prefusion conformation, for example, compared to an HPIV3 F protein that does not contain these amino acid residues at these positions. According to the present invention, the specific amino acids can already be present in the amino acid sequence or can be introduced by substitution (mutation) of an amino acid at that position with a specific amino acid according to the present invention.

[0021] Please note that throughout this application the terms HPIV-3 and PIV-3 are used interchangeably.

[0022] In certain embodiments, the protein has enhanced stability (thermostability) upon storage at 4°C and / or 50°C and / or 60°C compared to HPIV3 F protein without the presence of these amino acid residues at these positions. "Storage stable" means that when the protein is stored in solution (e.g., culture medium) at 4°C, 50°C and / or 60°C for a period of time, the protein still displays at least one epitope specific to a pre-fusion-specific antibody.

[0023] Additionally or alternatively, the protein may have enhanced thermal stability, as indicated by, for example, an increased melting temperature (eg, as measured by differential scanning fluorimetry).

[0024] The present invention also provides fragments of the HPIV-3 F protein. As used herein, the term "fragment" refers to an HPIV3 polypeptide having an amino-terminal deletion (e.g., by cleavage of the signal sequence) and / or a carboxy-terminal deletion (e.g., by deletion of the transmembrane region and / or cytoplasmic tail) and / or an internal deletion, but in which the remaining amino acid sequence is identical to the corresponding positions in the sequence of the HPIV3 F protein (e.g., the full-length sequence of the HPIV3 F protein). It will be understood that, to induce an immune response, and generally for vaccination purposes, the protein need not be full-length or possess all of its wild-type functions; fragments of the protein will be equally useful. Fragments according to the invention are immunologically active fragments and typically contain at least 15 or at least 30 amino acids of the HPIV3 F protein. In specific embodiments, the fragment comprises at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 460, 470, 480, 490, 500, or 510 amino acids of the HPIV3 F protein. In a preferred embodiment, the fragment is the HPIV3 F protein ectodomain, consisting of amino acid residues 19 to 484 of the HPIV3 F protein.

[0025] In certain embodiments, the proteins or fragments thereof of the present invention do not contain a signal sequence. Those skilled in the art will understand that a signal sequence (also sometimes referred to as a signal peptide, targeting signal, localization signal, localization sequence, transit peptide, leader sequence, or leader peptide) functions to prompt the cell to translocate the protein, usually to the cell membrane. The signal peptidase can be cleaved during or after translocation to generate a free signal peptide and a mature protein.

[0026] In certain embodiments, the PIV3 F protein ectodomain comprises a truncated F1 domain, preferably the truncated F1 domain does not comprise the transmembrane and cytoplasmic regions of the HPIV3 F protein. According to the present invention, the truncated F1 domain can comprise amino acids 110 to 484, preferably amino acids 110 to 485. In certain embodiments, the truncated F1 domain consists of amino acids 110 to 484, preferably amino acids 110 to 485 of the HPIV3 F protein.

[0027] To promote stable trimerization of the HPIV3 F ectodomain, a heterologous trimerization domain can be linked to the truncated F1 domain.

[0028] As mentioned above, because the TM region provides membrane anchoring and enhanced stability, the ectodomain of the F protein is significantly less stable than the full-length protein and will more readily refold to its final post-fusion state. In certain embodiments, to obtain a stable, soluble F protein in the pre-fusion conformation that exhibits high expression levels and high stability, a heterologous trimerization domain can be linked to a truncated F1 domain. The heterologous trimerization domain can be a GCN4 leucine-zipper domain. According to the present invention, the heterologous trimerization domain preferably comprises or consists of the amino acid sequence of SEQ ID NO: 3. Alternative forms of the GCN4 domain or other heterologous trimerization domains are also suitable for the present invention.

[0029] The amino acid positions used throughout this application are indicated with reference to the wild-type sequence of the HPIV3 F protein of SEQ ID NO: 1. Thus, the expression "amino acid residue at position 'x' of the F protein" used herein means the amino acid residue corresponding to the amino acid residue at position 'x' in the HPIV3 F protein of SEQ ID NO: 1. In the numbering system used throughout this application, 1 refers to the N-terminal amino acid of the immature F0 protein (SEQ ID NO: 1). When an F protein of another HPIV-3 strain is used, the amino acid positions of the F protein are numbered with reference to the numbering of the F protein of SEQ ID NO: 1 by aligning the sequence of said other HPIV3 F protein with the F protein of SEQ ID NO: 1, inserting gaps as necessary. Sequence alignment can be performed using methods well known in the art, for example, by CLUSTALW, Bioedit, or CLC Workbench.

[0030] The stabilized pre-fusion human parainfluenza virus 3 (HPIV3) F protein ectodomain comprises truncated F1 and F2 domains that contain the amino acid sequences of the F1 and F2 domains of an HPIV3 strain F protein, wherein the amino acid residues at positions 470 and / or 477 are hydrophobic amino acids, and the protein does not contain a heterotrimerization domain, wherein the numbering of the amino acid positions is according to the numbering of the amino acid residues in SEQ ID NO:1.

[0031] According to the present invention, it has been demonstrated that a stable soluble trimeric pre-fusion PIV-3 ectodomain (i.e., a soluble trimeric pre-fusion PIV-3 protein) can be obtained without the presence of a heterologous trimerization domain when the amino acid residue at positions 470 and / or 477 is a hydrophobic amino acid, preferably when both amino acid residues at positions 470 and 477 are hydrophobic.

[0032] The hydrophobic amino acid at positions 470 and / or 477 can be any hydrophobic amino acid, including, but not limited to, valine, leucine, isoleucine, methionine, and phenylalanine. The amino acid residues at positions 470 and 477 can be the same hydrophobic amino acid or different hydrophobic amino acids. In certain preferred embodiments, the hydrophobic amino acid at positions 470 and / or 477 is valine (V), and preferably, both amino acids at positions 470 and 477 are valine (V).

[0033] In certain embodiments, the truncated F1 domain does not include the transmembrane and cytoplasmic regions. Preferably, the truncated F1 domain comprises amino acids 110 to 484, preferably amino acids 110 to 485. In certain embodiments, the truncated F1 domain consists of amino acids 110 to 484, preferably amino acids 110 to 485 of the HPIV3 F protein.

[0034] In particular embodiments, furthermore, the amino acid residue at position 95 is A, and / or the amino acid residue at position 441 is A, and / or the amino acid residue at position 58 is D.

[0035] In certain embodiments, the protein comprises an amino acid sequence or a fragment thereof selected from the group consisting of SEQ ID NOs: 243 to 250. Preferably, the protein comprises the amino acid sequence of SEQ ID NO: 243.

[0036] In certain embodiments, the protein does not include a signal sequence (ie, the sequence corresponding to amino acids 1-18 of SEQ ID NO:1).

[0037] In certain embodiments, the protein does not include a C-terminal tag (C-tag).

[0038] Nucleotide sequences used throughout this application are presented in 5' to 3' orientation, and amino acid sequences are presented in N-terminal to C-terminal orientation, as is conventional in the art.

[0039] The amino acids according to the present invention can be any of the 20 naturally occurring amino acids (or "standard" amino acids). Standard amino acids can be classified into several groups based on their properties. Important factors are charge, hydrophilicity or hydrophobicity, size, and functional group. These properties are important for protein structure and protein-protein interactions. Some amino acids have special properties. For example, cysteine ​​can form covalent disulfide bonds (or disulfide bridges) with other cysteine ​​residues, proline induces rotation of the protein backbone, and glycine is more flexible than other amino acids. Table 1 shows the abbreviations and properties of the standard amino acids.

[0040] It will be understood by those skilled in the art that mutations can be introduced into proteins using routine molecular biology techniques. The mutations according to the present invention preferably result in increased expression levels and / or increased stabilization of the pre-fusion PIV3 F protein compared to a PIV3 F protein that does not contain these mutations.

[0041] The present invention further provides a nucleic acid molecule encoding the PIV3 F protein of the present invention. The nucleic acid molecule can be DNA or RNA. According to the present invention, the RNA can be mRNA, modified mRNA, self-replicating RNA, or circular mRNA.

[0042] In a preferred embodiment, the nucleic acid molecule encoding the protein according to the present invention is codon-optimized for expression in mammalian cells, preferably human cells. Codon optimization methods are known and have been described (e.g., WO 96 / 09378). A sequence is considered codon-optimized if, compared to the wild-type sequence, at least one non-preferred codon is replaced with a more preferred codon. Here, a non-preferred codon is a codon that is used less frequently in an organism than another codon encoding the same amino acid, and a more preferred codon is a codon that is used more frequently in an organism than the non-preferred codon. The codon usage frequency in a particular organism can be found, for example, in a codon frequency table at http: / / www.kazusa.or.jp / codon. Preferably, multiple non-preferred codons, preferably most or all non-preferred codons, are replaced with more preferred codons. Preferably, the codon most frequently used in the organism is used in the codon-optimized sequence. Replacement with a preferred codon generally results in higher expression.

[0043] Those skilled in the art will understand that, as a result of the degeneracy of the genetic code, many different polynucleotides and nucleic acid molecules can encode the same protein. Those skilled in the art will also understand that, using conventional techniques, nucleotide substitutions that do not affect the protein sequence encoded by a nucleic acid molecule can be made to reflect the codon usage of any particular host organism in which the protein will be expressed. Thus, unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNAs may or may not contain introns.

[0044] Nucleic acid sequences can be cloned using conventional molecular biology techniques or can be generated de novo by DNA synthesis, which can be performed using conventional methods by service companies operating in the field of DNA synthesis and / or molecular cloning (e.g., GeneArt, GenScripts, Invitrogen, Eurofins).

[0045] The present invention also provides a vector comprising said nucleic acid molecule. Thus, in a particular embodiment, the nucleic acid molecule according to the invention is part of a vector.

[0046] In a specific embodiment of the present invention, the vector is an adenovirus vector. The adenovirus according to the present invention belongs to the Adenoviridae family, preferably the Mastadenovirus genus. It can be a human adenovirus, but it can also be an adenovirus that infects other species, including, but not limited to, bovine adenovirus (e.g., bovine adenovirus 3, BAdV3), canine adenovirus (e.g., CAdV2), porcine adenovirus (e.g., PAdV3 or 5), or simian adenovirus (including simian adenovirus and ape adenovirus, such as chimpanzee adenovirus or gorilla adenovirus). Preferably, the adenovirus is a human adenovirus (HAdV or AdHu) or a simian adenovirus, such as chimpanzee or gorilla adenovirus (ChAd, AdCh, or SAdV), or rhesus adenovirus (RhAd). In the present invention, when Ad is referred to without any species designation, it means human adenovirus, for example, the abbreviation "Ad26" means the same as HAdV26, which is human adenovirus serotype 26. Also, the designation "rAd" used herein means recombinant adenovirus, for example, "rAd26" means recombinant human adenovirus 26.

[0047] Most advanced research has been conducted using human adenoviruses, and human adenoviruses are preferred in certain aspects of the present invention. In certain preferred embodiments, the recombinant adenoviruses according to the present invention are based on human adenoviruses. In preferred embodiments, the recombinant adenoviruses are based on human adenovirus serotypes 5, 11, 26, 34, 35, 48, 49, 50, 52, etc. According to particularly preferred embodiments of the present invention, the adenovirus is human adenovirus serotype 26. Advantages of these serotypes include low seroprevalence in the human population and / or low pre-existing neutralizing antibody titers, as well as experience with human subjects in clinical trials.

[0048] Simian adenoviruses also generally have low seroprevalence and / or low pre-existing neutralizing antibody titers in human populations, and a substantial amount of research has been reported using chimpanzee adenovirus vectors (e.g., US6083716; WO 2005 / 071093; WO 2010 / 086189; WO 2010 / 085984; Farina et al., 2001, J Virol 75:11603-13; Cohen et al., 2002, J Gen Virol 83:151-55; Kobinger et al., 2006, Virology 346:394-401; Tatsis et al., 2007, Molecular Therapy 15:608-17; also, Bangari and Mittal, 2006, Vaccine 24:849-62; and the review by Lasaro and Ertl, 2009, Mol Ther 17:1333-39). Thus, in other embodiments, the recombinant adenovirus according to the invention is based on a simian adenovirus, such as a chimpanzee adenovirus. In certain embodiments, the recombinant adenovirus is based on simian adenovirus type 1, 7, 8, 21, 22, 23, 24, 25, 26, 27.1, 28.1, 29, 30, 31.1, 32, 33, 34, 35.1, 36, 37.2, 39, 40.1, 41.1, 42.1, 43, 44, 45, 46, 48, 49, 50, or SA7P. In certain embodiments, the recombinant adenovirus is based on a chimpanzee adenovirus, such as ChAdOx 1 (see, e.g., WO 2012 / 172277) or ChAdOx 2 (see, e.g., WO 2018 / 215766). In certain embodiments, the recombinant adenovirus is based on a chimpanzee adenovirus, such as BZ28 (see, e.g., WO 2019 / 086466). In certain embodiments, the recombinant adenovirus is based on a gorilla adenovirus, such as BLY6 (see, e.g., WO 2019 / 086456) or BZ1 (see, e.g., WO 2019 / 086466).

[0049] In a preferred embodiment of the present invention, the adenoviral vector comprises a capsid protein from a rare serotype, including, for example, Ad26. In a typical embodiment, the vector is a rAd26 virus. "Adenoviral capsid protein" refers to a protein on the capsid of an adenovirus (e.g., an Ad26, Ad35, rAd48, or rAd5HVR48 vector) that is involved in determining the serotype and / or tropism of a particular adenovirus. Adenoviral capsid proteins typically include fiber, penton, and / or hexon proteins. As used herein, a "capsid protein" of a particular adenovirus, e.g., an "Ad26 capsid protein," can be, for example, a chimeric capsid protein comprising at least a portion of an Ad26 capsid protein. In a specific embodiment, the capsid protein is the entire capsid protein of Ad26. In a specific embodiment, the hexon, penton, and fiber are those of Ad26.

[0050] Those skilled in the art will recognize that elements from multiple serotypes can be combined in a single recombinant adenoviral vector. Thus, chimeric adenoviruses can be produced that combine desirable properties from different serotypes. Thus, in some embodiments, the chimeric adenoviruses of the present invention can combine the absence of pre-existing immunity of a first serotype with properties such as temperature stability, assembly, anchoring, production yield, targeted or improved infection, DNA stability in target cells, etc. See, for example, WO 2006 / 040330 for the chimeric adenovirus Ad5HVR48, which comprises an Ad5 backbone with a partial capsid derived from Ad48. See also WO 2019 / 086461 regarding chimeric adenoviruses Ad26HVRPtr1, Ad26HVRPtr12, and Ad26HVRPtr13, which comprise an Ad26 viral backbone with partial capsid proteins of Ptr1, Ptr12, and Ptr13, respectively.

[0051] In certain preferred embodiments, recombinant adenovirus vectors useful in the present invention are derived in large or in entirety from Ad26 (i.e., the vector is rAd26). In some embodiments, the adenovirus is replication-deficient because, for example, it contains a deletion in the E1 region of its genome. In the case of adenoviruses derived from non-group C adenoviruses, such as Ad26 or Ad35, the adenovirus E4-orf6 coding sequence is typically replaced with the E4-orf6 of a human subgroup C adenovirus, such as Ad5. This allows the growth of such adenoviruses in well-known complementation cell lines that express the E1 gene of Ad5, such as 293 cells, PER.C6 cells, etc. (See, e.g., Havenga et al., 2006, J Gen Virol 87:2135-43; WO 03 / 104467). However, such adenoviruses would not be able to replicate in non-complementing cells that do not express the E1 gene of Ad5.

[0052] The production of recombinant adenovirus vectors is well known in the art. The production of rAd26 vectors is described, for example, in WO 2007 / 104792 and Abbink et al. (2007) Virol 81(9):4654-63. The exemplary genome sequence of Ad26 can be found in GenBank accession EF 153474 and SEQ ID NO: 1 of WO 2007 / 104792. Examples of vectors useful in the present invention include, for example, those described in WO 2012 / 082918 (the disclosure of which is incorporated herein by reference in its entirety).

[0053] Typically, vectors useful in the present invention are produced using nucleic acids (e.g., plasmids, cosmids, or baculovirus vectors) containing the entire recombinant adenovirus genome. Thus, the present invention also provides isolated nucleic acid molecules encoding the adenovirus vectors of the present invention. The nucleic acid molecules of the present invention can be in the form of DNA or RNA, obtained by cloning or produced synthetically. The DNA can be double-stranded or single-stranded.

[0054] Adenoviral vectors useful in the present invention are typically replication-deficient. In these embodiments, the virus is made replication-deficient by deleting or inactivating a region critical for viral replication, such as the E1 region. This region can be substantially deleted or inactivated, for example, by inserting a gene of interest (e.g., a gene encoding a stabilized pre-fusion PIV3 F protein (usually linked to a promoter) or a gene encoding a pre-fusion PIV3 F protein fragment (usually linked to a promoter)) into this region. In some embodiments, the vector of the present invention can contain deletions in other regions, such as the E2, E3, or E4 regions, or can contain the insertion of a heterologous gene linked to a promoter in one or more of these regions. In the case of E2 and / or E4 mutant adenoviruses, cell lines that complement E2 and / or E4 are generally used to produce recombinant adenoviruses. Mutations in the E3 region of adenovirus do not need to be complemented by cell lines because E3 is not required for replication.

[0055] To produce sufficient quantities of adenoviral vectors for use in the present invention, packaging cell lines are typically used. Packaging cells are cells that contain deleted or inactivated genes in replication-deficient vectors, allowing the virus to replicate within the cells. Suitable packaging cell lines for adenoviruses with deletions in the E1 region include, for example, PER.C6, 911, 293, and E1A549.

[0056] In a preferred embodiment of the present invention, the vector is an adenovirus vector, more preferably an rAd26 vector, and most preferably an rAd26 vector having at least one deletion in the E1 region of the adenovirus genome, such as the vector described in Abbink, J Virol, 2007, 81(9):4654-63, which is incorporated herein by reference. Typically, a nucleic acid sequence encoding a pre-fusion PIV3 F protein is cloned into the E1 and / or E3 region of the adenovirus genome.

[0057] Host cells containing nucleic acid molecules encoding pre-fusion PIV3 F proteins also form part of the present invention. Pre-fusion PIV3 F proteins can be produced by recombinant DNA technology, including expression of the molecules in host cells, such as Chinese hamster ovary (CHO) cells, tumor cell lines, BHK cells, human cell lines such as HEK293 cells and PER.C6 cells, or yeast, fungi, or insect cells, or in transgenic animals or plants. In certain embodiments, the cells are derived from multicellular organisms, and in certain embodiments, the cells are derived from vertebrates or invertebrates. In certain embodiments, the cells are mammalian cells. In certain embodiments, the cells are human cells. Generally, production of a recombinant protein, such as a pre-fusion PIV F protein of the present invention, in a host cell involves introducing a heterologous nucleic acid molecule encoding the protein in an expressible form into the host cell, culturing the cell under conditions suitable for expression of the nucleic acid molecule, and expressing the protein in the cell. A nucleic acid molecule encoding a protein in an expressible form can be in the form of an expression cassette and usually requires sequences that can effect expression of the nucleic acid, such as enhancers, promoters, polyadenylation signals, etc. Those skilled in the art will recognize that a variety of promoters can be used to express genes in host cells. Promoters can be constitutive or regulatable and can be available from a variety of sources, including viral, prokaryotic, or eukaryotic, or can be artificially designed.

[0058] Cell culture media are available from various commercial suppliers, and an appropriate medium can be routinely selected for host cells expressing the protein of interest (here, the pre-fusion PIV3 F protein). The appropriate medium may or may not contain serum.

[0059] A "heterologous nucleic acid molecule" (also referred to herein as a "transgene") is a nucleic acid molecule that does not naturally occur in a host cell. It is introduced, for example, into a vector, by standard molecular biology techniques. The transgene is generally operably linked to an expression control sequence. This can be done, for example, by placing the nucleic acid encoding the transgene under the control of a promoter. Further regulatory sequences can be added. Numerous promoters are available for transgene expression and are known to those skilled in the art; for example, these can include viral, mammalian, and synthetic promoters. Non-limiting examples of suitable promoters for achieving expression in eukaryotic cells include the CMV promoter (US Pat. No. 5,385,839), such as the CMV immediate early promoter, which includes, for example, nt. -735 to +95 from the CMV immediate early gene enhancer / promoter. A polyadenylation signal, for example, the bovine growth hormone polyA signal (US Pat. No. 5,122,458), can be present after the transgene. Alternatively, several widely used expression vectors are available in the art and from commercial sources, such as Invitrogen's pcDNA and pEF vector series, BD Sciences' pMSCV and pTK-Hyg, Stratagene's pCMV-Script, etc., which can be used to recombinantly express proteins of interest or to provide appropriate promoter and / or transcription terminator sequences, polyA sequences, etc.

[0060] Cell culture can be any type of cell culture, including adherent cell culture (e.g., cells attached to the surface of a culture vessel or to microcarriers) and suspension culture. Most large-scale suspension cultures are performed as batch or fed-batch processes because they are the easiest to implement and scale up. Nowadays, continuous processes based on the perfusion principle are becoming more common and are similarly suitable. Suitable culture media are also well known to those skilled in the art and are generally available in large quantities from commercial sources or can be specially prepared according to standard protocols. Culturing can be carried out using batch, fed-batch, continuous systems, etc., in dishes, roller bottles, or bioreactors. Suitable conditions for culturing cells are known (see, e.g., Tissue Culture, Academic Press, eds. Kruse and Paterson (1973), and R.I. Freshney, Culture of Animal Cells: A Manual of Basic Technique, fourth edition (Wiley-Liss Inc., 2000, ISBN 0-471-34889-9)).

[0061] The present invention further provides compositions comprising the pre-fusion PIV3 F protein and / or fragments thereof and / or nucleic acid molecules and / or vectors described herein. Accordingly, the present invention provides compositions comprising a pre-fusion PIV3 F protein or fragment thereof that displays an epitope that is present in the pre-fusion conformation of the PIV3 F protein but not in the post-fusion conformation. The present invention also provides compositions comprising nucleic acid molecules and / or vectors encoding such pre-fusion PIV3 F proteins or fragments. The present invention further provides pharmaceutical compositions, e.g., vaccine compositions, comprising the pre-fusion PIV3 F protein, PIV3 F protein fragment, and / or nucleic acid molecule and / or vector and one or more pharmaceutically acceptable excipients.

[0062] The present invention also provides use of a stabilized pre-fusion PIV3 F protein (fragment), nucleic acid molecule, and / or vector according to the present invention for inducing an immune response against PIV3 F protein in a subject. Furthermore, a method for inducing an immune response against PIV3 F protein in a subject, comprising administering to the subject a pre-fusion PIV3 F protein (fragment) and / or nucleic acid molecule and / or vector according to the present invention. Also provided are pre-fusion PIV3 F proteins (fragment) and / or nucleic acid molecules and / or vectors according to the present invention for use in inducing an immune response against PIV3 F protein in a subject. Furthermore, the present invention provides use of a pre-fusion PIV3 F protein (fragment) and / or nucleic acid molecule and / or vector according to the present invention for the manufacture of a medicament for use in inducing an immune response against PIV3 F protein in a subject. In particular, the present invention provides a pre-fusion PIV3 F protein (fragment) and / or nucleic acid molecule and / or vector according to the present invention for use as a vaccine.

[0063] The prefusion PIV3 F protein (fragment), nucleic acid molecule, or vector of the present invention can be used for the prevention (protection) and / or treatment of PIV3 infection. In a specific embodiment, the prevention and / or treatment can be targeted to patient groups susceptible to PIV3 infection. Such patient groups include, but are not limited to, elderly people (e.g., 50 years or older, 60 years or older, preferably 65 years or older), young people (e.g., 5 years or younger, 1 year or younger), pregnant women (due to maternal immunization), hospitalized patients, and patients who have been treated with antiviral compounds but have shown an inadequate antiviral response.

[0064] The pre-fusion PIV3 F protein, fragments, nucleic acid molecules and / or vectors according to the invention may be used in the sole treatment and / or prevention of diseases or conditions caused by PIV3, or in combination with other prophylactic and / or therapeutic treatments, such as (existing or future) vaccines, antiviral agents and / or monoclonal antibodies.

[0065] The present invention further provides a method for preventing and / or treating PIV3 infection in a subject using a pre-fusion PIV3 F protein or a fragment thereof, a nucleic acid molecule, and / or a vector according to the present invention. In a specific embodiment, the method for preventing and / or treating PIV3 infection in a subject comprises administering an effective amount of the pre-fusion PIV3 F protein (fragment), nucleic acid molecule, and / or vector to a subject in need thereof. A therapeutically effective amount refers to an amount of protein, nucleic acid molecule, or vector effective to prevent, ameliorate, and / or treat a disease or condition caused by infection with PIV3. Prevention includes inhibiting or reducing the spread of PIV3, or inhibiting or reducing the onset, development, or progression of one or more symptoms associated with PIV3 infection. As used herein, amelioration can refer to a reduction in visible or detectable disease symptoms, viremia, or any other measurable sign of PIV3 infection.

[0066] For administration to a subject, such as a human, the present invention may use pharmaceutical compositions comprising the pre-fusion PIV3 F protein (fragment), nucleic acid molecule, and / or vector described herein and a pharmaceutically acceptable carrier or excipient. In this context, the term "pharmaceutically acceptable" means that, at the dosages and concentrations used, the carrier or excipient does not cause any undesirable or harmful effects in the subject to which it is administered. Such pharmaceutically acceptable carriers and excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A.R. Gennaro, ed., Mack Publishing Company

[1990] ; Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, eds., Taylor & Francis

[2000] ; and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, ed., Pharmaceutical Press

[2000] ). The PIV3 F protein or nucleic acid molecule is preferably formulated and administered as a sterile solution, although lyophilized formulations may also be utilized. Sterile solutions are prepared by sterile filtration or other methods known in the art. The solution is then lyophilized or filled into pharmaceutical administration containers. The pH of the solution generally ranges from 3.0 to 9.5, e.g., 5.0 to 7.5. The PIV3 F protein is typically present in a solution containing a suitable pharmaceutically acceptable buffer, and the composition may also contain a salt. Optionally, a stabilizer, such as albumin, may be present. In certain embodiments, a surfactant is added. In certain embodiments, the PIV3 F protein may be formulated into an injectable preparation.

[0067] In certain embodiments, the composition according to the present invention further comprises one or more adjuvants. Adjuvants are known in the art to further enhance the immune response to an applied antigenic determinant. The terms "adjuvant" and "immunostimulatory substance" are used interchangeably herein and are defined as one or more substances that cause stimulation of the immune system. In this context, an adjuvant is used to enhance the immune response to the PIV3 F protein of the present invention. Examples of suitable adjuvants include: aluminum salts, such as aluminum hydroxide and / or aluminum phosphate; oil emulsion compositions (or oil-in-water compositions), such as squalene-water emulsions, e.g., MF59 (see, e.g., WO 90 / 14837); saponin preparations, such as QS21 and immunostimulating complexes (ISCOMS) (see, e.g., US Pat. No. 5,057,540; WO 90 / 03184, WO 96 / 11711, WO 2004 / 004762, WO 2005 / 002620); bacterial or microbial derivatives, e.g., Examples of adjuvants include monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), CpG motif-containing oligonucleotides, ADP-ribosylating bacterial toxins or mutants thereof, such as Escherichia coli (E. coli) heat-labile enterotoxin LT and cholera toxin CT; eukaryotic proteins, such as antibodies or fragments thereof (e.g., antibodies against antigens themselves or CD1a, CD3, CD7, CD80), and ligands for receptors (e.g., CD40L, GMCSF, GCSF, etc.), which stimulate immune responses upon interaction with recipient cells. In a specific embodiment, the compositions of the present invention contain aluminum as an adjuvant, e.g., in the form of aluminum hydroxide, aluminum phosphate, potassium aluminum phosphate, or a combination thereof, at a concentration of 0.05 to 5 mg, e.g., 0.075 to 1.0 mg, of aluminum per dose.

[0068] In other embodiments, the composition does not include an adjuvant.

[0069] In a specific embodiment, the present invention provides a method for producing a vaccine against respiratory syncytial virus (PIV3), comprising preparing a PIV3 F protein (fragment), nucleic acid, or vector according to the present invention and incorporating it into a pharmaceutically acceptable composition. The term "vaccine" refers to a substance or composition containing an active ingredient effective to induce a degree of immunity in a subject against a particular pathogen or disease, resulting in at least a reduction (up to complete elimination) of the severity, duration, or other signs associated with infection or disease by the pathogen. In the present invention, a vaccine comprises an effective amount of a pre-fusion PIV3 F protein (fragment) and / or a nucleic acid molecule encoding the pre-fusion PIV3 F protein and / or a vector containing the nucleic acid molecule, which induces an effective immune response against PIV3. This provides a method for preventing serious lower respiratory tract diseases that lead to hospitalization and reduces the frequency of complications, such as pneumonia and bronchiolitis, caused by PIV3 infection and replication in subjects. The term "vaccine" according to the present invention refers to a pharmaceutical composition and, therefore, typically includes a pharmaceutically acceptable diluent, carrier, or excipient. It may or may not contain additional active ingredients. In certain embodiments, it may be a combination vaccine (combination vaccine) further comprising other components that induce an immune response, for example, against other proteins of PIV3 and / or against other infectious agents, such as RSV, HMPV, and / or influenza. The administration of the additional active ingredients may be carried out, for example, by separate administration or by administration of a combination product of the vaccine of the present invention and the additional active ingredients.

[0070] The composition according to the present invention can be administered by any standard route of administration. Non-limiting examples include parenteral administration, such as intradermal, intramuscular, subcutaneous, or transdermal administration, or mucosal administration, such as intranasal or oral administration. In one embodiment, the composition is administered by intramuscular injection. Various possibilities for administering a composition, such as a vaccine, to induce an immune response against the antigen in the vaccine are known to those skilled in the art.

[0071] As used herein, a subject is preferably a mammal, such as a rodent, such as a mouse, cotton rat, or non-human primate, or a human. Preferably, the subject is a human subject.

[0072] The proteins, fragments, nucleic acid molecules, vectors, and / or compositions can be administered as a prime or boost in an allogeneic or heterologous prime-boost regimen. If a boost vaccination is administered, such a boost vaccination is typically administered to the same subject one week to one year, preferably two weeks to four months, after the subject's initial administration of the composition (which in such cases is referred to as a "prime vaccination"). In certain embodiments, the administration includes a prime administration and at least one boost administration.

[0073] The present invention further provides a method for producing a vaccine against PIV3, comprising providing a serotype 26 recombinant human adenovirus containing a nucleic acid encoding a pre-fusion PIV3 F protein or a fragment thereof described herein, propagating the recombinant adenovirus in a host cell culture, isolating and purifying the recombinant adenovirus, and formulating the recombinant adenovirus into a pharmaceutically acceptable composition. In a specific embodiment, a method for producing adenoviral particles containing a nucleic acid molecule (transgene) encoding a PIV3 F protein or a fragment thereof is provided herein. The method comprises (a) contacting a host cell of the present invention with an adenoviral vector of the present invention, and (b) growing the host cell under conditions in which adenovirions containing the transgene are produced. Recombinant adenovirus can be produced and propagated in host cells according to well-known methods, including cell culture of host cells infected with adenovirus. The cell culture can be any type of cell culture, including adherent cell culture (e.g., cells attached to the surface of a culture vessel or microcarriers) and suspension culture.

[0074] Most large-scale suspension cultures are performed as batch or fed-batch processes because they are the easiest to implement and scale up. Nowadays, continuous processes based on the perfusion principle are becoming more common and are similarly suitable (see, e.g., WO 2010 / 060719 and WO 2011 / 098592, both of which are incorporated herein by reference, which describe suitable methods for obtaining and purifying large amounts of recombinant adenovirus).

[0075] The present invention further provides an isolated recombinant nucleic acid forming the genome of a recombinant human adenovirus of serotype 26, comprising a nucleic acid encoding a PIV3 F protein or fragment thereof described herein.

[0076] The proteins of the invention can also be used as diagnostic tools for testing the immune status of an individual, for example, by determining whether antibodies capable of binding to the proteins of the invention are present in the individual's serum. Accordingly, the present invention also relates to an in vitro diagnostic method for detecting the presence of PIV3 infection in a patient, comprising the steps of: a) contacting a biological sample obtained from the patient with a protein of the invention; and b) detecting the presence of an antibody-protein complex.

[0077] The present invention will be further illustrated in the following examples, which are not intended to limit the invention in any way and are merely intended to clarify the invention.

[0078] Example Example 1: Instability of soluble PIV3 F ectodomain protein A plasmid encoding the wild-type PIV3 F protein ectodomain (SEQ ID NO: 2), in which the transmembrane and cytoplasmic tails were replaced with C-tags, was synthesized in Genscript and codon-optimized. The construct was cloned into pCDNA2004 by standard methods well known in the art, including site-directed mutagenesis and PCR, and sequenced. The protein was expressed in the expi293F cell line. Expi293F cells were transiently transfected using ExpiFectamine (Life Technologies) according to the manufacturer's instructions and cultured at 37°C and 10% CO2 for 3 days. The culture supernatant was harvested, and cells and cell debris were removed by centrifugation at 300g for 5 minutes. The clarified supernatant was then sterile-filtered using a 0.22 μm vacuum filter and stored at 4°C until use.

[0079] Biolayer interferometry (BLI) assays using quantitative octet measurements with the prefusion-specific monoclonal antibody PIA 174 (Stewart-Jones et al., PNAS 115(48)12265-12270, 2018) immobilized on an anti-human IgG sensor were used to detect the PIV3 F protein ectodomain in crude supernatants. On the day of harvest (day 0), a low but clear signal for wild-type (i.e., unstabilized) PIV3 preF protein was present, but after 20 days of storage at 4°C, it was undetectable (Figure 2; wild-type).

[0080] Example 2: Stabilizing mutations analyzed by biolayer interferometry and analytical SEC To stabilize the unstable prefusion conformation of the PIV3 F protein, the PIV3 F protein ectodomain was fused to the GCN4 trimerization motif (SEQ ID NO: 3) at the C-terminus, and the amino acid residue Asp at position 452 was mutated to Asn (D452N). Additional mutations were then introduced in this background, as shown in Figure 2. A plasmid encoding a recombinant PIV3 F protein ectodomain with a C tag was expressed in Expi293F cells, and 3 days after transfection, the supernatant was tested for binding to PIA174 using a quantitative Octet assay (Figure 2). The mutant showed binding to the prefusion trimer-specific Mab PIA174 on the day of harvest, and binding was maintained after 20 days of storage at 4°C. The D452N mutation and the addition of GCN4 stabilized the prefusion conformation. The additional stabilizing mutations S41P, (Q89M+Q222I), V165P, N167P, L168P, Q198L, F335P, (S186C+A195C), and (G85C+L221C) increased the amount of pre-fusion PIV3 F protein in crude cell supernatants compared to constructs containing only the D452N mutation, and also maintained the pre-fusion conformation for 20 days in supernatants stored at 4°C.

[0081] Cell culture supernatants of various PIV3 F constructs containing stabilizing mutations were analyzed on the day of harvest using analytical size-exclusion chromatography (SEC) (Figure 3). Analytical SEC experiments were performed using a μDAWN TREOS instrument (Wyatt) connected to an Optilab μT-rEX refractive index detector (Wyatt) coupled with an in-line Nanostar DLS reader (Wyatt) and an ultra-high-performance liquid chromatography system (Vanquish, Thermo Scientific). Clarified crude cell culture supernatant was applied at 0.35 mL / min to a 300 Å column (Sepax Cat# 231300-4615) with a corresponding guard column (Sepax) equilibrated in running buffer (150 mM sodium phosphate, 50 mM NaCl, pH 7.0). When analyzing supernatant samples, the μMALS detector was offline, and analytical SEC data were analyzed using the Chromeleon 7.2.8.0 software package. As shown in the antibody binding studies above, SEC analysis also demonstrated an increase in trimer content due to the introduction of stabilizing mutations. Compared with the wild-type (SEQ ID NO: 2) or a mutant containing only the GCN4 trimerization domain and the D452N substitution (PIV171432; SEQ ID NO: 4), mutants with additional stabilizing substitutions exhibited higher trimer content by analytical SEC of culture supernatants (Figure 3). Compared with the soluble F mutant containing D452N and the C-terminal GCN4 domain, mutants containing the additional stabilizing substitutions S41P, (Q89M + Q222I), V165P, N167P, L168P, Q198L, F335P, (S186C + A195C), and (G85C + L221C) exhibited higher trimer content by analytical SEC of culture supernatants (Figure 3).

[0082] Example 3: Analysis of additive and synergistic stabilizing mutations by biolayer interferometry To further stabilize the unstable prefusion conformation of the PIV3 F protein ectodomain, constructs containing additional mutations at amino acid residues 41, 89, 165, 167, 168, 198, 204, 222, 335, 367, and / or 436 in the D452N background were generated (thus, all constructs contained the D452N mutation). Plasmids encoding these recombinant PIV3 F protein ectodomains fused to GCN4 (SEQ ID NO: 3) at the C-terminus and equipped with a C-tag were expressed in Expi293F cells, and 3 days after transfection, supernatants were tested for binding to PIA174 using a quantitative Octet assay (Figure 4). The mutants showed binding to the prefusion trimer-specific Mab PIA174 on the day of harvest. Furthermore, many of the double mutations showed higher binding than the individual single mutations at positions 41, 165, 167, 168, 198, 204, 335, 367 and 436 or the double mutation at positions 89+222 in the D452N background, indicating an additive or even synergistic stabilizing effect.

[0083] To further stabilize the PIV3 F protein ectodomain containing the D452N + (Q89M + Q222I) + L168P mutation (i.e., PIV200309, SEQ ID NO: 76), the above stabilizing mutations were added in various combinations. Plasmids encoding these recombinant PIV3 F protein ectodomains fused to GCN4 at the C-terminus and equipped with a C-tag were expressed in Expi293F cells. Three days after transfection, the supernatants were diluted 5-fold in mock transfection medium and tested for binding to PIA174 using a quantitative Octet assay (Figure 5). The mutants showed binding to the prefusion trimer-specific Mab PIA174 on the day of harvest, with the highest binding observed for PIV200884 (SEQ ID NO: 108) (Q89M / Q222I + L168P + S41P + N167P + D452N).

[0084] Example 4:Stabilizing mutations in the stem allow removal of the GCN4 trimerization domain while maintaining trimeric organization when analyzed by biolayer interferometry and analytical SEC To stabilize the unstable trimeric prefusion conformation of the PIV3 F protein ectodomain in the absence of GCN4, amino acid residues 470 and 477 were mutated in the stem region of the PIV3 protein (residues 452-481). A plasmid encoding a recombinant PIV3 F protein ectodomain with a C tag was expressed in Expi293F cells, and 3 days after transfection, the supernatant was tested for binding to PIA174 using the quantitative Octet assay (Figure 6A). The PIV3 backbone used was stabilized by D452N+Q89M+Q222I+L168P mutations in the absence of the GCN4 trimerization domain (PIV200941). Mutants of PIV200941 containing 470V and / or 477V showed binding to the trimer-specific Mab PIA174, whereas constructs containing wild-type amino acids at positions 470 and 477 showed little prefusion trimers in the supernatant. Thus, the addition of the 470V and 477V mutations stabilized the native trimeric quaternary structure of the prefusion F protein in the absence of the GCN4 trimerization domain.

[0085] Cell culture supernatants of various PIV3 F constructs containing the 470V and / or 477V stabilizing mutations were analyzed using analytical size-exclusion chromatography (SEC) on the day of harvest (Fig. 7B). Compared with a variant that does not contain stabilizing mutations in the stem and does not contain the GCN4 trimerization domain (PIV200941; containing D452N+Q89M+Q222I+L168P stabilizing mutations in the head domain), variants containing the 470V and / or 477V stabilizing mutations exhibited higher trimer content by analytical SEC of the culture supernatants (Fig. 6B).

[0086] We also studied S470V and S477V in the wild-type backbone (PIV190058), which does not contain the GCN4 trimerization domain or stabilizing mutations (Figure 6C). Introduction of S470V (PIV200960) or S477V (PIV200962) improved binding to PIA174, indicating that these mutations stabilize the prefusion conformation without any additional mutations or heterologous trimerization domains.

[0087] Example 5: Combinations of stabilizing mutations in the head (residues 19-451) and stem (residues 452-481) enhance the expression and stability of the prefusion PIV3 F protein as measured by biolayer interferometry, analytical SEC, and differential scanning fluorimetry. To stabilize the unstable trimeric prefusion conformation of the PIV3 F protein ectodomain in the absence of GCN4, the amino acid residues at positions 41, 89, 167, 168, 222, 335, 452, 470, and / or 477 were mutated. A plasmid encoding a recombinant PIV3 F protein ectodomain with a C tag was expressed in Expi293F cells, and 3 days after transfection, the supernatant was tested for binding to PIA174 using quantitative Octet (Figure 7A) and analytical SEC (Figure 7B). In the absence of 470V and 477V, the protein migrated with a shorter retention time, indicating that the protein was larger. MALS analysis of PIV201113 and PIV201105 (Figure 8) showed no significant difference in molecular weight (165 kDa and 156 kDa, respectively). The shorter retention time of PIV201113 is likely due to the increased apparent size of the protein due to the opening of the stem region; PIV201113 partially trimerizes at the apex, but not as small as PIV201105, which also trimerizes at the base due to optimized stem mutations. Thus, amino acids 470V and / or 477V are required to keep the protein in the native trimeric conformation in the absence of a heterologous trimerization domain.

[0088] The stability of various proteins in the supernatant was also measured by incubating samples in a heat block at 4°C, 50°C, or 60°C for 30 minutes. The samples were then centrifuged at 15,000 rpm for 10 minutes to remove larger aggregates, and the supernatant was run on an analytical SEC (Figure 9). In the absence of 470V, the protein loses its trimeric conformation already at 50°C. In the absence of 477V, the protein loses its trimeric conformation at 60°C. The absence of 41P also reduces the stability of the protein, as the trimeric peak completely disappears after 30 minutes of incubation at 60°C.

[0089] The stability of various proteins in the supernatant was also determined by measuring the melting temperature (Tm) using differential scanning fluorometry (DSF). For this purpose, SYPRO Orange 5000x (S6650, Invitrogen) was diluted (1:250) in PBS to prepare a 20x working solution. For each reaction, 15 μL of supernatant was mixed with 5 μL of SYPRO 20x in a MicroAmp Fast Optical 96-well plate (4346906, ThermoFisher). PBS was used as a negative control. The plate was covered with MicroAmp Optical Adhesive Film (4311971, ThermoFisher) and then read in a ViiA7 real-time PCR instrument. The construct containing all stabilizing mutations (S41P + Q89M + Q222I + N167P + L168P + D452N + S470V + S477V + F335P) but without GCN4 had a Tm50 of 70.7°C ("backbone + F335P" Figure 10). Removal of 335P reduced the Tm50 to 66.4°C (Figure 10). Further removal of 470V and / or 477V dramatically reduced the Tm50 to below 59°C. Further removal of 41P or 89M + 222I reduced the Tm to 61.1°C and 64.7°C, respectively. This indicates that S470V and S477V in particular are required for stable soluble trimers in the absence of the GCN4 trimerization domain. Additionally, S41P, F335P and Q89M+Q222I increase the melting temperature and therefore further stabilize the protein.

[0090] Example 6: Characterization of purified PIV3 F protein by SDS-PAGE, analytical SEC, and differential scanning fluorometry A set of PIV3 F designs (outlined in Figure 11A) was transiently transfected into expi293 cells using ExpiFectamine (Life Technologies) according to the manufacturer's instructions and cultured at 37°C, 10% CO2 for 5 days. The culture supernatant was harvested and centrifuged at 600g for 10 minutes to remove cells and cell debris. The centrifuged supernatant was then sterile filtered using a 0.22 μm vacuum filter and stored at 4°C until use. PIV3 F protein was purified using a two-step purification protocol involving a CaptureSelect™ C-tag affinity column followed by size-exclusion chromatography using a HiLoad Superdex200pg 16 / 600 column (GE Healthcare).

[0091] The yield (mg / L) for each protein design after purification is shown in Figure 11A. The purified proteins were analyzed by SDS-PAGE under reducing and non-reducing conditions and developed with Coomassie gel (Figure 11B). The proteins migrated as a single unprocessed band, indicating that cleavage at the F2 / F1 boundary did not occur in expiHEK cells. SEC-MALS showed a clean trace with a sharp peak at the size of the PIV3 F trimer (Figure 11C). Differential scanning fluorometry (DSF) showed a 3.6-3.8°C increase in melting temperature for the F335P-containing designs (PIV201255 and PIV201256) compared to their counterparts (PIV201254 and PIV201110, respectively) that did not contain F335P (Figure 11D).

[0092] Example 7: Analysis by biolayer interferometry and analytical SEC showed that the stabilizing mutations S470V+S477V in the HR2 stem are sufficient for trimer formation. To stabilize the unstable trimeric prefusion conformation of the PIV3 F protein ectodomain in the absence of GCN4, amino acid residues 470 and 477 were mutated in the stem region of the PIV3 protein (residues 452-481). A plasmid encoding a recombinant PIV3 F protein ectodomain with a C tag was expressed in Expi293F cells, and 3 days after transfection, the supernatant was tested for binding to PIA174 using the quantitative Octet method described in Example 1 (Figure 12B) and analyzed for trimer content using analytical size-exclusion chromatography (SEC) as described in Example 2 (Figure 12C). The wild-type PIV3 backbone (containing neither the stabilizing mutations nor the GCN4 trimerization domain; PIV190058) did not bind to the trimer-specific Mab PIA174 (Figure 12B), nor did it show a detectable trimer peak in the supernatant (Figure 12C). In contrast, when the S470V and S477V HR2 stem mutations were introduced (PIV210294), PIA174 MAb binding and a detectable trimer peak were observed, demonstrating that the addition of the 470V and 477V mutations stabilized the native trimeric quaternary structure of the pre-fusion F protein, which does not contain the GCN4 trimerization domain. Subsequent introduction of various head domain mutations (Figure 12A) enhanced PIA174 binding in quantitative Octet assays and improved trimer yield in analytical SEC compared with S470V + S477V alone. However, the head domain mutations alone did not result in detectable trimer binding or expression in the supernatant (Figure 12C, dotted line). This highlights the importance of HR2 stabilization for the native trimeric quaternary structure of soluble PIV3 pre-fusion F (Figure 12C, solid line).

[0093] Example 8: Contribution of individual mutations to the stability and yield of PIV3 prefusion F construct PIV211368 Expression of PIV3 F proteins containing head-stabilizing mutations S41P, Q89M+Q222I, and L168P and stem-stabilizing mutations S470V+S477V (PIV211368) was compared with PIV3 F mutants in which single or double mutations were systematically removed by reverting their amino acids to wild type (shown in bold in Figure 13A). Plasmids encoding recombinant PIV3 F protein ectodomains without purification tags were expressed in Expi293F cells, and 3 days after transfection, the stability of the various proteins in the supernatant was measured by measuring the melting temperature (Tm50) using differential scanning fluorometry (DSF) as described in Example 5 (Figure 13A). Trimeric content was assessed by analytical SEC as described in Example 2 (Figure 13B). Head-domain mutations that contribute to stability are S41P and Q98M+Q122I. This is because, when reverted to wild-type, they exhibit lower melting temperatures (60.8°C and 63.6°C, respectively) than that of PIV211368 (65.8°C). HR2 mutations S470V and S477V also contribute to the thermal stability of PIV3 F, exhibiting lower melting temperatures of 50.3°C and 58.0°C, respectively. In contrast, the head domain L168P substitution reduces thermal stability, as indicated by the increase in melting temperature of 67.1°C when reverted to wild-type (Figure 13A).

[0094] Head domain mutations either have little or no effect on PIV3 F trimer content (P41S; PIV211886) or have a positive effect (M89Q+I222Q; PIV211887 and P168L; PIV211890), as demonstrated by the reduced trimer peak of the wild-type revertant (Figure 13B).

[0095] In conclusion, in this particular stabilized protein design, the HR2 substitutions S470V and S477V contribute significantly to the stability of the PIV3 F protein, whereas the head domain mutation L168P contributes significantly to trimer expression but not to protein stability. The head domain mutations S41P and Q89M+Q222I contribute to thermal stability, and the latter combination also increases trimer yield.

[0096] Example 9: Characterization of purified tag-free PIV3 F protein as measured by analytical SEC, differential scanning fluorimetry, and slow freeze stability PIV3 F design PIV211368, which does not contain a purification tag and contains the stabilizing mutations S41P, Q89M / Q222I, L168P, S470V, and S477V, was transiently transfected into Expi293F cells using ExpiFectamine (Life Technologies) according to the manufacturer's instructions and cultured at 37°C and 10% CO2 for 5 days. The culture supernatant was collected and centrifuged at 600g for 10 minutes to remove cells and cell debris. The supernatant was then sterile filtered using a 0.22µm vacuum filter and stored at 4°C until use. PIV3 F protein was purified using a two-step purification protocol, including ion exchange purification at pH 4.0 and polishing by size exclusion chromatography using a Superdex 200 increase 16 / 40 column. Trimeric fractions were pooled and further characterized by SEC-MALS (Figure 14A). The trimer yield, molecular weight, and hydrodynamic radius are shown in Figure 14B. Differential scanning fluorometry (DSF) showed that purified PIV211368 had a melting temperature of 66.5°C (Figure 14B), slightly higher than that measured for the protein in crude cell culture supernatant (Figure 13A). The stability of purified PIV211368 was further tested by slow-freezing the protein from 20°C to -70°C for 24 hours in various buffer compositions (FB12, PS4P4, and TS5P2). The recovery of PIV3 F trimer after slow freezing was measured by analytical SEC and compared to the trimer recovery after storage at 4°C. The recovery ranged from 92 to 98%, indicating minimal trimer loss in any of the buffers tested.

[0097] Example 10: Characterization of purified C-tagged PIV3 F protein by analytical SEC and differential scanning fluorometry PIV3 F design PIV210235, equipped with a C-tag and containing stabilizing mutations S41P, Q89M / Q222I, S470V, and S477V, was transiently transfected into Expi293 GnT1 cells using ExpiFectamine (Life Technologies) according to the manufacturer's instructions and cultured at 37°C and 10% CO2 for 5 days. The culture supernatant was collected and centrifuged at 600g for 10 minutes to remove cells and cell debris. The supernatant was then sterile filtered using a 0.22μm vacuum filter and stored at 4°C until use. PIV3 F protein was purified using a two-step purification protocol involving a CaptureSelect™ C-tag affinity column followed by size exclusion chromatography using a Superdex200 10 / 300 column (GE Healthcare). Trimeric fractions were pooled and further characterized by SEC-MALS (Figure 15A). The trimer yield, molecular weight, and hydrodynamic radius are shown in Figure 15B. Differential scanning fluorometry (DSF) showed that purified PIV210235 had a melting temperature of 67.5°C (Figure 15B).

[0098] Example 11: Completely single-chain proteins according to the present invention Purification (IEX followed by SEC) of PIV211368 (SEQ ID NO: 237) yielded an unexpectedly partially processed protein to F2 and F1 as detected by reducing SDS-PAGE followed by Coomassie staining [even though the cleavage site ("RTER") is not recognized by furin-like proteases and the native protease TMPRSS2 is not expressed in expiHEK cells] (Figure 16). Introduction of R109Q (and T95A) yielded a protein that was entirely single-chain after IEX / SEC purification. Introduction of the E58D mutation did not affect processing (compare PIV220923 with PIV220922). All three proteins exhibited similar thermostability as measured by DSF (Figure 17). Purified PIV220922 and PIV220923 proteins exhibited slightly higher binding to the PIV3 prefusion-specific antibody PIA174 (Figure 18). [Table 1] [ka] TIFF2025533104000003.tif62162TIFF2025533104000004.tif236163TIFF2025533104000005.tif244163TIFF2025533104000006.tif244163TIFF2025533104000007.tif233163TIFF2025533104000008.tif93163TIFF2025533104000009.tif234163TIFF2025533104000010.tif243164TIFF2025533104000011.tif195163TIFF2025533104000012.tif236164TIFF2025533104000013.tif243162TIFF2025533104000014.tif244163TIFF2025533104000015.tif235161TIFF2025533104000016.tif245163TIFF2025533104000017.tif235163TIFF2025533104000018.tif243164TIFF2025533104000019.tif243163TIFF2025533104000020.tif235164TIFF2025533104000021.tif242163TIFF2025533104000022.tif235163TIFF2025533104000023.tif243163TIFF2025533104000024.tif245163TIFF2025533104000025.tif234162TIFF2025533104000026.tif242163TIFF2025533104000027.tif235163TIFF2025533104000028.tif242163TIFF2025533104000029.tif242163TIFF2025533104000030.tif234162TIFF2025533104000031.tif243162TIFF2025533104000032.tif234163TIFF2025533104000033.tif244163TIFF2025533104000034.tif243162TIFF2025533104000035.tif232163TIFF2025533104000036.tif242162TIFF2025533104000037.tif236164TIFF2025533104000038.tif243163TIFF2025533104000039.tif243161TIFF2025533104000040.tif233163TIFF2025533104000041.tif242163TIFF2025533104000042.tif234163TIFF2025533104000043.tif243162TIFF2025533104000044.tif243163TIFF2025533104000045.tif234162TIFF2025533104000046.tif243162TIFF2025533104000047.tif234162TIFF2025533104000048.tif243162TIFF2025533104000049.tif243163TIFF2025533104000050.tif234163TIFF2025533104000051.tif244162TIFF2025533104000052.tif234163TIFF2025533104000053.tif242163TIFF2025533104000054.tif244162TIFF2025533104000055.tif235163TIFF2025533104000056.tif242163TIFF2025533104000057.tif233163TIFF2025533104000058.tif243162TIFF2025533104000059.tif246163TIFF2025533104000060.tif242162TIFF2025533104000061.tif232163. TIFF2025533104000062.tif241162TIFF2025533104000063.tif234162TIFF2025533104000064.tif244162TIFF2025533104000065.tif242162TIFF2025533104000066.tif234163TIFF2025533104000067.tif245162TIFF2025533104000068.tif233163TIFF2025533104000069.tif242163TIFF2025533104000070.tif243163TIFF2025533104000071.tif248164TIFF2025533104000072.tif236162TIFF2025533104000073.tif241163TIFF2025533104000074.tif234162TIFF2025533104000075.tif243164TIFF2025533104000076.tif243162TIFF2025533104000077.tif240163TIFF2025533104000078.tif235162TIFF2025533104000079.tif232162TIFF2025533104000080.tif239162TIFF2025533104000081.tif246163TIFF2025533104000082.tif61163

Claims

1. 1. A stabilized pre-fusion human parainfluenza virus 3 (HPIV3) F protein comprising an F1 domain and an F2 domain that comprise the amino acid sequences of the F1 domain and F2 domain of an F protein of an HPIV3 strain, wherein the amino acid residue at position 41 is P, the amino acid residue at position 89 is M, the amino acid residue at position 222 is I, the amino acid residue at position 168 is P, the amino acid residue at position 470 is V, the amino acid residue at position 477 is V, and the amino acid residue at position 109 is Q, and the numbering of the amino acid positions is according to the numbering of the amino acid residues in SEQ ID NO:

1.

2. The protein of claim 1, further comprising: an amino acid residue at position 95 that is A; an amino acid residue at position 441 that is A; and an amino acid residue at position 58 that is D.

3. 10. A protein according to any one of the preceding claims, comprising a truncated F1 domain.

4. The protein of claim 3, wherein the truncated F1 domain does not include the transmembrane and cytoplasmic regions.

5. The protein of claim 3, wherein the truncated F1 domain comprises amino acids 110 to 484, preferably amino acids 110 to 485 of the HPIV3 F protein.

6. The protein of any one of claims 1 to 5, wherein the heterotrimerization domain is linked to a truncated F1 domain.

7. 10. The protein according to any one of the preceding claims, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 243 to 250 or a fragment thereof, preferably comprising the amino acid sequence of SEQ ID NO: 243 or a fragment thereof.

8. A nucleic acid molecule encoding the protein according to any one of claims 1 to 7.

9. The nucleic acid of claim 8, wherein the nucleic acid molecule is DNA or RNA.

10. 10. The nucleic acid of claim 9, wherein the RNA is mRNA, modified mRNA, self-replicating RNA or circular mRNA.

11. 11. The nucleic acid of claim 8, 9 or 10, which encodes a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 243 to 250 or a fragment thereof, preferably comprising the amino acid sequence of SEQ ID NO: 243 or a fragment thereof.

12. A vector comprising the nucleic acid according to any one of claims 8 to 11.

13. The vector of claim 12, wherein the vector is a human recombinant adenovirus vector.

14. 14. The vector of claim 13, wherein the adenoviral vector is a replication-incompetent Ad26 adenoviral vector having deletions of the E1 and E3 regions.

15. A composition comprising a protein according to any one of claims 1 to 7, a nucleic acid according to any one of claims 8 to 11, and / or a vector according to claim 12, 13 or 14.

16. 16. A method for vaccinating a subject against PIV3, comprising administering to the subject the composition of claim 15.

17. A method for preventing PIV3 infection and / or replication in a subject, comprising administering the vaccine of claim 15 to the subject.

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