Stable pre-fusion PIV3F proteins

Abstract

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

Description

[0001] The present invention relates to the field of medicine. The present invention particularly relates to a recombinant prefusion PIV3 F protein, a nucleic acid molecule encoding the PIV3 F protein, and its use, for example, in vaccines. Background Art

[0002] Human parainfluenza virus type 3 (HPIV3) mainly induces respiratory complications in children and immunocompromised populations; however, in recent years, it has also been identified as a concern in the adult population. More than 11,000 children are hospitalized annually in the United States due to HPIV3 (Weinberg et al., J Pediatr. 154:694 - 699, 2009), and HPIV3 is also an important cause of mortality, morbidity, and healthcare costs in other vulnerable populations (Ison et al., Clin. Microbiol Rev 32, 2019). Most children 5 years of age and older have antibodies against HPIV - 3, indicating that most children have experienced HPIV3 infection before this age.

[0003] Currently, there are no vaccines and specific antiviral therapies for preventing HPIV disease. Except for croup where the use of corticosteroids and nebulized epinephrine has been found to be beneficial, medical care is supportive.

[0004] Four serotypes of HPIV are known (HPIV - 1 through HPIV - 4), which are associated with different clinical manifestations and seasonal incidences, where HPIV3 is the most prevalent and typically presents as bronchiolitis / pneumonia. Seasonal variations in different serotypes and spontaneous outbreaks drive overall variable incidences and complex epidemiologies.

[0005] HPIV3 is an enveloped RNA virus of the Paramyxoviridae family within the order Mononegavirales. It has a genome of ~15,000 nucleotides in length that encodes six key proteins in the following gene sequence: 3'-N-P-M-F-HN-L-5'. Viral-cell fusion results from the concerted action of two envelope glycoproteins that make up the viral entry machinery—the receptor-binding protein, hemagglutinin-neuraminidase (HN), and the fusion protein (F). After binding to a target receptor containing sialic acid, HN, a molecule with both receptor-binding and cleavage activities, triggers and activates the F protein. The F protein fuses the viral membrane and the host cell membrane through an irreversible protein refolding from an unstable prefusion conformation to a stable postfusion conformation. The structures of both conformations have been determined for several paramyxoviruses, providing insights into the complex mechanism of this fusion protein. As a type I membrane protein, the F protein is translated at the endoplasmic reticulum and transported to the plasma membrane through the Golgi apparatus and the trans-Golgi network. Like other class I fusion proteins, the inactive precursor PIV3 F0 needs to be cleaved by an appropriate host endoprotease (possibly 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. For refolding from the prefusion to the postfusion conformation, the refolding region 1 (RR1) between residues 110 and 213, which includes the FP and heptad repeat A (HRA) (where the numbering is based on the amino acid residues in SEQ ID NO:1), must be transformed from an assembly of helices, loops, and strands into a long continuous helix. The FP, located at the N-terminal segment of RR1, can then extend from the viral membrane and insert into the proximal membrane of the target cell. Next, the refolding region 2 (RR2), which forms the C-terminal stem of the prefusion F spike and includes heptad repeat B (HRB), relocates to the other side of the PIV3 F head and binds the HRA coiled-coil trimer to the HRB domain to form a six-helix bundle. The formation of the RR1 coiled coil and the relocation of RR2 to complete the six-helix bundle are the most significant structural changes that occur during the refolding process.Class I fusion proteins have been shown to be inherently unstable, while structure-based stabilization of viral fusion proteins in the pre-fusion conformation has been shown to induce excellent 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), but to date, no vaccine is available and there is also no therapy for the prevention or treatment of hPIV3.

[0006] Accordingly, there remains a need for an effective vaccine against PIV3, particularly a vaccine that comprises or is based on the PIV3 F protein in the pre-fusion conformation. Indeed, a vaccine preferably indicated for children and high-risk patients (e.g., the elderly and patients with COPD) could provide an intervention with broad impact early in the course of severe illness, thereby reducing the overall incidence of HPIV3 as well as the associated morbidity and mortality. The present invention aims to provide means for obtaining such a stabilized pre-fusion PIV3 F protein for use in vaccination against PIV3. Summary of the Invention

[0007] The present invention provides a stabilized recombinant pre-fusion human parainfluenza virus type III (HPIV3) fusion (F) protein, i.e., a recombinant HPIV3 F protein stabilized in the pre-fusion conformation and fragments thereof. The pre-fusion HPIV3 F protein or a fragment thereof comprises at least one epitope specific for the pre-fusion conformation F protein, e.g., as determined 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 protein, preferably a trimeric protein. The present invention also provides a nucleic acid molecule encoding the pre-fusion HPIV3 F protein or a fragment thereof, and a vector comprising such a nucleic acid molecule, e.g., an adenovector.

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

[0009] The present invention further relates to compositions, preferably pharmaceutical compositions, comprising a PIV3 F protein, a nucleic acid molecule, and / or a vector as described herein, and to their use in inducing an immune response against the PIV3 F protein, in particular their use as a vaccine against PIV3. The present invention also relates to methods for inducing an immune response against parainfluenza virus type III (PIV3) in a subject, which comprise administering to the subject an effective amount of a prefusion HPIV3 F protein as described herein, a nucleic acid molecule encoding said HPIV3 F protein, and / or a vector comprising said nucleic acid molecule. Preferably, the induced immune response is characterized by the induction of neutralizing antibodies against PIV3 and / or protective immunity from PIV3. In a particular aspect, the present invention relates to a method for inducing PIV3 F antibodies in a subject, which comprises administering to the subject an effective amount of a pharmaceutical composition comprising a prefusion HPIV3 F protein as described herein, a nucleic acid molecule encoding said PIV3 F protein, and / or a vector comprising said nucleic acid molecule. BRIEF DESCRIPTION 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’, upper panel) and the mature soluble extracellular domain (‘Extracellular domain’, lower panel). The signal peptide sequence (SP), which is cleaved off during protein maturation, precedes the N-terminal F2 domain. The fusion peptide (FP) is located at the N-terminus of F1. The heptad repeats A, B, and C (HRA, HRB, and HRC, respectively) are indicated. The transmembrane region (TM) and cytoplasmic tail (CT) are further indicated. The soluble extracellular domain may be equipped with a C-terminal GCN4 trimerization motif. The cleavage sites between the SP and F2 and between F2 and F1 are indicated by arrows.

[0011] Figure 2 : Binding of PIA174 to PIV3 preF in cell supernatant determined by biolayer interferometry. Quantitative octet measurements were performed by immobilizing the antibody PIA174 to an anti-human IgG sensor and using PIV3 F in crude cell supernatant. The initial binding rate was plotted. Cell culture medium from mock-transfected cells (‘Mock’) and PIV3 F without stable mutations and without GCN4 (‘Wild-type’, SEQ ID NO:2) was obtained as a negative control. Measurements were performed on the day of harvest (‘Day 0’) and repeated after 20-day storage at 4°C (‘Day 20’). Single and double mutations were tested in the backbone with the GCN4 trimerization domain and the D452N mutation, as indicated.

[0012] Figure 3: Analytical SEC profiles of different PIV3 F proteins with stabilizing mutations in the crude cell supernatant. The indicated protein variants (black, solid line) were compared to mock-transfected supernatant (dashed line). The peak between 4.4 and 4.5 minutes corresponds to the PIV3 preF trimer.

[0013] Figure 4 : Binding of PIA174 to PIV3 preF in cell supernatant determined by biolayer interferometry of single and all possible double combinations of stabilizing mutations. Quantitative octet measurements were performed by immobilizing the antibody PIA174 to an anti-human IgG sensor and using PIV3 F in the crude cell supernatant. The initial binding rates were plotted. Cell media from mock-transfected cells (‘mock’) and PIV3 F without stabilizing mutations and without GCN4 (‘wild type’, SEQ ID NO:2) were obtained as controls. Measurements were performed on the day of harvest. Single and double mutations were tested in the backbone with the GCN4 trimerization domain and the D452N mutation as indicated.

[0014] Figure 5 : Binding of PIA174 to PIV3 preF in 5-fold diluted cell supernatant determined by biolayer interferometry of all possible combinations of selected stabilizing mutations. Quantitative octet measurements were performed by immobilizing the antibody PIA174 to an anti-human IgG sensor and using PIV3 F in 5-fold diluted crude cell supernatant. The initial binding rates were plotted. Combinations of mutations were tested in the backbone with the GCN4 trimerization domain and the D452N, Q89M, Q222I, and L168P mutations as indicated.

[0015] Figure 6 : PIV3 preF stabilized in the absence of the GCN4 trimerization domain. (A) Binding of PIA174 to PIV3 preF PIV200941 (without GCN4 trimerization domain but containing the D452N+Q89M+Q222I+L168P mutations) in cell supernatant as determined by biolayer interferometry. S470V and / or S477V were introduced into the stalk of the PIV3 F protein. The initial binding rates were plotted. (B) Samples of A tested in analytical SEC. The peak at ~4.8 minutes corresponds to the PIV3 preF trimer. (C) Binding of PIA174 to unstabilized PIV3 preF (PIV190058, SEQ ID NO:2) without GCN4 trimerization domain in cell supernatant as determined by biolayer interferometry. S470V or S477V (PIV200960 and PIV200962, respectively) were introduced into the stalk of the PIV3 F protein. The initial binding rates were plotted.

[0016] Figure 7 : Further stabilization of PIV3 preF in the absence of the GCN4 trimerization domain. (A) Binding of PIA174 to a matrix designed for PIV3 preF without the GCN4 trimerization domain in cell supernatants, as determined by biolayer interferometry. The initial binding rate was plotted. (B) Samples from (A) were tested in analytical SEC. The peak at ~4.8 min corresponds to the PIV3 preF trimer.

[0017] Figure 8 : Removal of S470V and S477V opens the PIV3 preF trimer. (A) Crude cell supernatants of PIV201105 and PIV201103 in Figure 7 B were tested in analytical SEC-MALS. The hydrodynamic radius and molecular weight (MW) of the main peak (indicated by an arrow) were determined and reported in (B). The MALS signal corresponding to the molecular weight is shown as a dashed line for the respective peaks.

[0018] Figure 9 : Analytical SEC after heat stress. The indicated proteins in the crude cell supernatants were incubated at 4 °C (dashed line), 50 °C (black line), or 60 °C (grey line) for 30 min. Samples were then analyzed by analytical SEC to determine the loss of the PIV3 preF trimer. The backbone used contained the S41P+Q89M+Q222I+N167P+L168P+D452N+S470V+S477V stabilizing mutations and did not have a heterologous trimerization domain.

[0019] Figure 10 : Stability of PIV3 preF variants. Crude cell supernatants of the indicated proteins were analyzed by differential scanning fluorimetry (DSF) to determine the melting temperature.

[0020] Figure 11 : Characterization of purified PIV3 preF protein. Proteins were purified from expiHEK supernatants 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. Untreated PIV3 F protein runs at ~50 kD. (C) SEC-MALS and (D) DSF analysis of the purified protein.

[0021] Figure 12: A: Table of constructs used, indicating the absence (-) or presence of the HR2 stem mutations S470V + S477V in the design, and the absence (-) or introduction of various amino acid substitutions in the head domain of the PIV3 F protein. Single head mutations were evaluated, except for the combination of Q889M + Q221I whose side chains interact in the prefusion structure. B. Detection of prefusion PIV3 F trimers in the supernatants of cells transfected with the variants indicated in A), as determined by binding of the prefusion-specific PIA174 antibody using biolayer interferometry (qOctet). Quantitative octet measurements were performed by immobilizing the antibody PIA174 to an anti-human IgG sensor and using PIV3 F in the crude cell supernatant. The initial binding rate was plotted. C. Detection of PIV3 F trimers in the supernatants of cells transfected with the variants indicated in A), as determined by analytical SEC. The PIV3 F trimer (indicated by 'T') eluted between 4.6 and 4.8 minutes. Each panel compares the absence (dashed line) or presence (solid line) of the S470V + S477V mutation in combination with amino acid substitutions in the head domain of PIV3 F (the specific mutations are indicated above each graph).

[0022] Figure 13 : A. Description of the corresponding melting temperatures of the PIV3 F trimer in the constructs used and in the supernatants of transfected Expi293 cells, as determined by differential scanning fluorimetry (DSF). Single or double mutations from PIV211368 were removed by restoring them to wild-type amino acids, indicated in bold. B. Yield of PIV3 F trimers in the supernatants of cells transfected with the variants indicated in A), as determined by analytical SEC. The PIV3 F trimer eluted with a retention time between 4.6 and 4.8 minutes.

[0023] Figure 14 : A. Analytical SEC of purified PIV3 F trimer PIV211368. The tag-free PIV3 F was purified from the cell-free supernatant of transfected Expi293 cells by ion-exchange purification and refinement via size-exclusion chromatography. B. Table of the properties of the purified PIV211368 PIV3 F protein, including yield, trimer size, hydrodynamic radius, and melting temperature (DSF).

[0024] C. Slow-freezing stability of purified PIV3 F trimer PIV211368 in different buffers. The recovery of PIV3 F trimer after slow-freezing the protein from 20 °C to -70 °C over a 24-hour period was compared to the trimer recovery after storage at 4 °C (histogram is the mean of n = 5 individual measurements (open circles)). Buffer compositions: 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% PS200, pH 6.5. TS5P2; 20 mM Tris, 75 mM NaCl, 5% sucrose, 0.02% PS20, 0.4% EtOH, pH 7.5.

[0025] Figure 15 : A. Analytical SEC of purified PIV3 F trimer PIV210235. PIV3 F was purified from the cell-free supernatant of transfected GnT1-cells by C-tag purification and refinement via size exclusion chromatography. B. Table of the properties of purified PIV210235 PIV3 F protein, including yield, trimer size, hydrodynamic radius, and melting temperature (DSF).

[0026] Figure 16 : SDS-PAGE under reducing or non-reducing conditions, followed by Coomassie staining. The dashed circle indicates partial processing of PIV211368.

[0027] Figure 17 : Melting temperature of purified HPIV3 F trimer as determined by differential scanning fluorimetry (DSF). N = 3 replicate measurements, and individual values and the mean are reported as grey and black solid lines, respectively.

[0028] Figure 18 : Binding of PIA174 to purified PIV3 preF protein as determined by biolayer interferometry. The PIA174 antibody was immobilized to an anti-human IgG sensor. The initial binding rate is plotted in the figure. A negative control (NC) protein that is not PIV3 preF and 1x kinetic buffer were obtained as negative controls. DETAILED DESCRIPTION

[0029] As described above, the fusion protein (F) of parainfluenza virus (PIV3) is involved in the fusion of the viral membrane with the host cell membrane, which is essential for infection. PIV3 F mRNA is translated into a 539-amino acid precursor protein designated F0, which contains a signal peptide sequence at the N-terminus (e.g., amino acid residues 1-18 of SEQ ID NO:1), which is removed by signal peptidase in the endoplasmic reticulum. F0 is likely cleaved at the cell membrane between amino acid residues 109 and 110 by a cellular protease (most likely TMPRSS2 or a TMPRSS2-like enzyme), generating 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 the C-terminus contains a transmembrane region (TM) (amino acid residues 494-516) and a cytoplasmic region (amino acid residues 517-539). The F2 domain (amino acid residues 19-109) is covalently linked to F1 by a disulfide bridge( Figure 1 ). The F1-F2 heterodimer assembles into a homotrimer on the surface of the virion. The mature extracellular domain 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 stalk region (amino acid residues 452-484).

[0030] Vaccines against PIV3 infection are currently not available. One potential method for producing a vaccine is a subunit vaccine based on the purified PIV3 F protein. However, for this method, it is desirable for the purified PIV3 F protein to be in a conformation similar to that of the pre-fusion state of the PIV3 F protein, which is stable over time, i.e., remains in the 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, and can be produced in sufficient quantities. Additionally, for a soluble subunit-based vaccine, the PIV3 F protein needs to be truncated by deletion of the transmembrane region (TM) and cytoplasmic region to produce a soluble secreted extracellular domain of the F protein (sF). Since the TM region is responsible for membrane anchoring and increasing stability, the extracellular domain of the F protein is more unstable than the full-length protein and is even more prone to refolding into the post-fusion end state. To obtain a soluble F protein in the pre-fusion conformation that exhibits high expression levels and high stability, the pre-fusion conformation thus needs to be stabilized.

[0031] Since the full-length (membrane-bound) PIV3 F protein is also metastable, stabilization of the pre-fusion conformation is also desirable for the full-length PIV3 F protein (i.e., including the TM and cytoplasmic regions), which is used, for example, in any live attenuated vaccine or vector-based vaccine approach.

[0032] Recently, HPIV-3 protein variants have been described that contain several stabilizing amino acid substitutions that stabilize the prefusion conformation (Stewart-Jones et al., PNAS 115(48) 12265-12270, 2018). However, such variants have several limitations; namely, i) the expression and stability of this PIV3 preF protein are not sufficient to fully develop a successful vaccine; ii) several of these mutations are located at the surface of the protein, which may affect antigenicity and immunogenicity; and / or iii) the C-terminus of this variant is fused to the GCN4 trimerization domain, which may affect immunogenicity and induce antibodies that are not related to this trimerization domain and do not cross-react with the virus, and may hinder immunogenicity when this domain is used in other (future) vaccines, which would increase its immunodominance.

[0033] Described herein is a stable prefusion human parainfluenza virus 3 (HPIV3) F protein comprising F1 and F2 domains, said F1 and F2 domains comprising the amino acid sequences of the F1 and F2 domains of the F protein of an HPIV3 strain, which comprises hydrophobic amino acids at position 470 and at position 477, wherein the numbering of said amino acid positions is according to the numbering of the amino acid residues in SEQ ID NO:1. Preferably, the protein is a trimer. The hydrophobic amino acid at position 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 may be the same hydrophobic amino acid, or different hydrophobic amino acids. In certain preferred embodiments, the hydrophobic amino acid at position 470 and / or 477 is valine (V), preferably both amino acids at positions 470 and 477 are valine (V).

[0034] The protein may contain 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 sequence 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 186 and 195, wherein the numbering of said amino acid positions is according to the numbering of the amino acid residues in SEQ ID NO:1.

[0035] In addition, proteins are described 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 wherein the protein comprises a disulfide bridge between amino acid residues 38 and 291.

[0036] The present invention provides a protein comprising F1 and F2 domains, said F1 and F2 domains comprising the amino acid sequences of the F1 and F2 domains of the F protein of an HPIV3 strain, wherein the amino acid residue at position 41 is P, and the amino acid residue at position 89 is M, and the amino acid residue at position 222 is I, and the amino acid residue at position 168 is P, and the amino acid residue at position 470 is V, and the amino acid residue at position 477 is V, and the amino acid residue at position 109 is Q, wherein the numbering of said amino acid positions is according to the numbering of the amino acid residues in SEQ ID NO:1.

[0037] The present invention provides a stable prefusion HPIV-3 protein trimer that exhibits high expression levels and increased stability. Additionally, the protein according to the present invention is a single-chain protein, i.e., the protein is untreated (uncut). Since the protein is protease-resistant, the manufacturability of the protein is increased.

[0038] According to the present invention, it has been demonstrated that the presence of one or more specific amino acid residues at the indicated positions increases the stability of, for example, the HPIV3 F protein in the prefusion conformation and / or the extracellular domain of the HPIV3 F protein, as compared to the HPIV3 F protein that does not contain these amino acid residues at these positions. According to the present invention, the specific amino acids may already be present in the amino acid sequence, or may be introduced into the specific amino acids of the present invention by amino acid substitution (mutation) at that position.

[0039] It should be noted that the terms HPIV-3 and PIV-3 are used interchangeably throughout the present application.

[0040] In certain embodiments, the protein has increased stability (thermal stability) after storage at 4 °C and / or 50 °C and / or 60 °C, as compared to the HPIV3 F protein that does not contain these amino acid residues at these positions. "Stability after storage" means that after the protein is stored in a solution (e.g., medium) at 4 °C, 50 °C, and / or 60 °C for a predetermined period of time, the protein still exhibits at least one epitope specific for a prefusion-specific antibody.

[0041] Additionally or alternatively, the protein may have increased thermal stability, as indicated, for example, by an increased melting temperature (measured by, for example, differential scanning fluorimetry).

[0042] The present invention also provides fragments of the HPIV-3F protein. As used herein, the term "fragment" refers to an HPIV3 polypeptide having an amino terminus (e.g., by removing the signal sequence) and / or a carboxyl terminus (e.g., by deleting the transmembrane region and / or cytoplasmic tail) and / or internal deletions, provided that 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 should be understood that for inducing an immune response and generally for vaccination purposes, the protein need not be full-length nor need it have all of its wild-type functions, and fragments of the protein are equally useful. Fragments according to the present invention are immunologically active fragments and typically contain at least 15 amino acids or at least 30 amino acids of the HPIV3 F protein. In certain embodiments, the fragment contains 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 extracellular domain of the HPIV3 F protein consisting of amino acid residues 19-484 of the HPIV3 F protein.

[0043] In certain embodiments, the protein or fragment thereof according to the present invention does not contain a signal sequence. Those skilled in the art will understand that a signal sequence (sometimes referred to as a signal peptide, targeting signal, localization signal, localization sequence, transit peptide, leader sequence or leader peptide) functions to cause a cell to translocate a protein, typically to the cell membrane. The signal peptidase may cleave during or after translocation to generate a free signal peptide and a mature protein.

[0044] In certain embodiments, the extracellular domain of the PIV3 F protein contains a truncated F1 domain, preferably, the truncated F1 domain does not contain the transmembrane region and cytoplasmic region of the HPIV3 F protein. According to the present invention, the truncated F1 domain may contain amino acids 110-484, preferably amino acids 110-485. In certain embodiments, the truncated F1 domain consists of amino acids 110-484 of the HPIV3F protein, preferably amino acids 110-485.

[0045] To facilitate stable trimerization of the HPIV3 F extracellular domain, a heterotrimerization domain may be linked to the truncated F1 domain.

[0046] As described above, because the TM region is responsible for membrane anchoring and increased stability, the extracellular domain of the F protein is less stable than the full-length protein and is even more prone to refolding into the final post-fusion state. To obtain a stable soluble F protein in the pre-fusion conformation that exhibits high expression levels and high stability, in certain embodiments, a heterotrimerization domain may be linked to the truncated F1 domain. The heterotrimerization domain can be the GCN4 leucine zipper domain. According to the present invention, the heterotrimerization domain preferably comprises or consists of the amino acid sequence of SEQ ID NO:3. Alternative forms of the GCN4 domain or other heterotrimerization domains are also suitable according to the present invention.

[0047] As used throughout this application, amino acid positions are given with reference to the wild-type sequence of the HPIV3 F protein of SEQ ID NO:1. As used in the present invention, the term "amino acid residue at position 'x' of the F protein" thus refers to the amino acid residue corresponding to the amino acid residue at position 'x' in the HPIV3 F protein of SEQ ID NO:1. It should be noted that the numbering system 1 used throughout this application refers to the N-terminal amino acid of the immature F0 protein (SEQ ID NO:1). When using the F protein of another HPIV-3 strain, 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 the other HPIV3 F protein with the F protein of SEQ ID NO:1 (inserting gaps as needed). The sequence alignment can be done using methods well known in the art, such as by CLUSTALW, Bioedit or CLC Workbench.

[0048] A stable pre-fusion human parainfluenza virus 3 (HPIV3) F protein extracellular domain comprises a truncated F1 domain and an F2 domain, which comprises the amino acid sequences of the F1 and F2 domains of the F protein of an HPIV3 strain, wherein the amino acid residue at position 470 and / or 477 is a hydrophobic amino acid, wherein the protein does not comprise a heterotrimerization domain, and wherein the numbering of the amino acid positions is according to the numbering of the amino acid residues in SEQ ID NO:1.

[0049] According to the present invention, it has been demonstrated that when the amino acid residue at position 470 and / or the amino acid residue at position 477 is a hydrophobic amino acid, preferably when the amino acid residues at both positions 470 and 477 are hydrophobic, a stable soluble trimeric pre-fusion PIV-3 extracellular domain (i.e., a soluble trimeric pre-fusion PIV-3 protein) without a heterotrimerization domain can be obtained.

[0050] The hydrophobic amino acid(s) at position 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 may be the same hydrophobic amino acid or different hydrophobic amino acids. In certain preferred embodiments, the hydrophobic amino acid at position 470 and / or 477 is valine (V), preferably both amino acids at positions 470 and 477 are valine (V).

[0051] In certain embodiments, the truncated F1 domain does not contain a transmembrane region and a cytoplasmic region. Preferably, the truncated F1 domain comprises amino acids 110 - 484, preferably 110 - 485. In certain embodiments, the truncated F1 domain consists of amino acids 110 - 484 of the HPIV3 F protein, preferably amino acids 110 - 485.

[0052] In certain embodiments, in addition, 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.

[0053] In certain embodiments, the protein comprises an amino acid selected from SEQ ID NO: 243 - 250 or a fragment thereof. Preferably, the protein comprises the amino acid sequence of SEQ ID NO: 243.

[0054] In certain embodiments, the protein does not contain a signal sequence (i.e., amino acids 1 - 18 corresponding to SEQ ID NO: 1).

[0055] In certain embodiments, the protein does not contain a C - terminal tag (C - tag).

[0056] As used throughout this application, nucleotide sequences are provided in the 5' to 3' direction and amino acid sequences are provided from the N - terminus to the C - terminus, as is customary in the art.

[0057] The amino acids according to the invention can be any of the twenty naturally occurring amino acids (or'standard' amino acids). The standard amino acids can be grouped based on their properties. Important factors are charge, hydrophilicity or hydrophobicity, size, and functional groups. These properties are important for protein structure and protein - protein interactions. Some amino acids have special properties, such as cysteine which can form covalent bonds (or disulfide bridges) with other cysteine residues, proline which induces a turn in the protein backbone, and glycine which is more flexible than other amino acids. Table 1 shows the abbreviations and properties of the standard amino acids.

[0058] Those skilled in the art should understand that proteins can be mutated by conventional molecular biology procedures. Compared with the PIV3 F protein that does not contain these mutations, the mutations according to the present invention preferably result in an increased expression level and / or increased stability of the pre-fusion PIV3 F protein.

[0059] The present invention further provides a nucleic acid molecule encoding the PIV3 F protein according to the present invention. The nucleic acid molecule may be DNA or RNA. According to the present invention, the RNA may be mRNA, modified mRNA, self-replicating RNA or circular mRNA.

[0060] 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. Methods for codon optimization are known and have been previously described (e.g., WO 96 / 09378). A sequence is considered codon-optimized if at least one non-preferred codon is replaced with a more preferred codon compared to the wild-type sequence. In the present context, a non-preferred codon is a codon that is less frequently used in an organism than another codon encoding the same amino acid, while a more preferred codon is a codon that is more frequently used in an organism than the non-preferred codon. The codon usage frequencies for a particular organism can be found in codon frequency tables, such as http: / / www.kazusa.or.jp / codon. Preferably, more than one non-preferred codon, preferably most or all non-preferred codons, are replaced with more preferred codons. Preferably, the most frequently used codons in the organism are used in the codon-optimized sequence. Replacement of preferred codons generally results in higher expression.

[0061] Those skilled in the art should understand that due to the degeneracy of the genetic code, numerous different polynucleotides and nucleic acid molecules can encode the same protein. It should also be understood that those skilled in the art may use conventional techniques to prepare nucleotide substitutions that do not affect the protein sequence encoded by the nucleic acid molecule to reflect the codon usage of any particular host organism in which the protein is to be expressed. Thus, unless otherwise specified, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate forms of one another and encode the same amino acid sequence. The nucleotide sequences encoding proteins and RNAs may or may not include introns.

[0062] The nucleic acid sequence can be cloned using conventional molecular biology techniques or generated de novo by DNA synthesis, which can be performed by service companies with operations in the field of DNA synthesis and / or molecular cloning (e.g., GeneArt, GenScripts, Invitrogen, Eurofins) using conventional procedures.

[0063] The present invention also provides a vector comprising the nucleic acid molecule as described above. In certain embodiments, the nucleic acid molecule according to the present invention is thus part of a vector.

[0064] In certain embodiments of the present invention, the vector is an adenovirus vector. The adenovirus according to the present invention belongs to the family Adenoviridae, and is preferably a virus belonging to the genus Mastadenovirus of mammals. It can be a human adenovirus or an adenovirus infecting 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 (which includes monkey 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 monkey adenovirus (RhAd). In the present invention, if referred to as Ad without indicating the species, it means a human adenovirus. For example, the brief notation "Ad26" means the same as HAdV26, which is human adenovirus serotype 26. Also as used herein, the notation "rAd" means a recombinant adenovirus. For example, "rAd26" refers to recombinant human adenovirus 26.

[0065] The most advanced research has been carried out using human adenoviruses, and human adenoviruses are preferred according to certain aspects of the present invention. In certain preferred embodiments, the recombinant adenovirus according to the present invention is based on a human adenovirus. In a preferred embodiment, the recombinant adenovirus is based on human adenovirus serotypes 5, 11, 26, 34, 35, 48, 49, 50, 52, etc. According to a particularly preferred embodiment of the present invention, the adenovirus is a human adenovirus of serotype 26. The advantages of these serotypes include a low serum prevalence and / or low pre-existing neutralizing antibody titers in the population, as well as having experience in clinical trials for human subjects.

[0066] Simian adenoviruses generally also have a low seroprevalence and / or low pre-existing neutralizing antibody titers in the human population, and a large amount of work using chimpanzee adenovirus vectors has been reported (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 see reviews by Bangari and Mittal, 2006, Vaccine 24:849-62; and by Lasaro and Ertl, 2009, Mol Ther 17:1333-39). Thus, in other embodiments, the recombinant adenoviruses according to the invention are based on simian adenoviruses, such as chimpanzee adenoviruses. In certain embodiments, the recombinant adenoviruses are based on simian adenovirus types 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 adenoviruses are based on chimpanzee adenoviruses such as ChAdOx 1 (see, e.g., WO 2012 / 172277) or ChAdOx 2 (see, e.g., WO 2018 / 215766). In certain embodiments, the recombinant adenoviruses are based on chimpanzee adenoviruses such as BZ28 (see, e.g., WO 2019 / 086466). In certain embodiments, the recombinant adenoviruses are based on gorilla adenoviruses such as BLY6 (see, e.g., WO 2019 / 086456) or BZ1 (see, e.g., WO 2019 / 086466).

[0067] In a preferred embodiment of the present invention, the adenovirus vector comprises a capsid protein from a rare serotype, such as including Ad26. In a typical embodiment, the vector is an rAd26 virus. "Adenovirus capsid protein" refers to a protein on the capsid of an adenovirus (e.g., Ad26, Ad35, rAd48, rAd5HVR48 vector), which is involved in determining the serotype and / or tropism of a particular adenovirus. Adenovirus capsid proteins generally include fiber protein, penton protein, and / or hexon protein. As used herein, the "capsid protein" with respect to a particular adenovirus, such as "Ad26 capsid protein", can be, for example, a chimeric capsid protein comprising at least a portion of the Ad26 capsid protein. In certain embodiments, the capsid protein is the complete capsid protein of Ad26. In certain embodiments, the hexon, penton, and fiber belong to Ad26.

[0068] One of ordinary skill in the art will recognize that elements derived from multiple serotypes can be combined in a single recombinant adenovirus vector. Thus, chimeric adenoviruses can be generated that combine the desired properties from different serotypes. Accordingly, in some embodiments, the chimeric adenoviruses of the present invention can combine the lack of pre-existing immunity of a first serotype with properties such as temperature stability, assembly, anchoring, production yield, redirection, or improved infection, stability of DNA in target cells, and the like. See, for example, WO 2006 / 040330 with respect to the chimeric adenovirus Ad5HVR48 (which comprises an Ad5 backbone with a partial capsid from Ad48), and also see, for example, WO 2019 / 086461 with respect to the chimeric adenoviruses Ad26HVRPtr1, Ad26HVRPtr12, and Ad26HVRPtr13 (which respectively comprise an Ad26 virus backbone with partial capsid proteins of Ptr1, Ptr12, and Ptr13).

[0069] In certain preferred embodiments, the recombinant adenovirus vector useful in the present invention is mainly or entirely derived from Ad26 (i.e., the vector is rAd26). In some embodiments, the adenovirus is replication-deficient, for example, because it contains a deletion in the E1 region of the genome. For adenoviruses derived from non-group C adenoviruses such as Ad26 or Ad35, the E4-orf6 coding sequence of the adenovirus is typically exchanged with the E4-orf6 of a human subgroup C adenovirus such as Ad5. This allows such adenoviruses to proliferate in well-known complementing cell lines that express the E1 gene of Ad5, such as 293 cells, PER.C6 cells, and the like (see, for example, Havenga, et al., 2006, J Gen Virol 87:2135-43; WO 03 / 104467). However, such adenoviruses cannot replicate in non-complementing cells that do not express the E1 gene of Ad5.

[0070] The preparation of recombinant adenovirus vectors is well known in the art. The preparation of rAd26 vectors is described, for example, in WO 2007 / 104792 and Abbink et al., (2007) Virol 81(9):4654-63. Exemplary genomic sequences of Ad26 are 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.

[0071] Generally, the vectors useful in the present invention are produced using nucleic acids (e.g., plasmids, cosmids or baculovirus vectors) containing a complete recombinant adenovirus genome. Accordingly, 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 RNA, or in the form of DNA obtained by cloning or produced synthetically. The DNA can be double-stranded or single-stranded.

[0072] The adenovirus vectors useful in the present invention are generally replication-deficient. In these embodiments, the virus is rendered replication-deficient by deletion or inactivation of regions essential for viral replication, such as the E1 region. The region can be substantially deleted or inactivated, for example, by inserting a gene of interest into the region, such as a gene encoding a stable prefusion PIV3 F protein (usually linked to a promoter), or a gene encoding a fragment of the prefusion PIV3 F protein (usually linked to a promoter). In some embodiments, the vectors of the present invention can contain deletions in other regions, such as the E2, E3 or E4 regions, or insertions of heterologous genes linked to a promoter within one or more of these regions. For E2- and / or E4-mutated adenoviruses, E2- and / or E4-complementing cell lines are generally used to generate recombinant adenoviruses. Mutations in the E3 region of adenovirus do not require complementation by a cell line, since E3 is not essential for replication.

[0073] Packaging cell lines are generally used to produce sufficient amounts of adenovirus vectors for use in the present invention. Packaging cells are cells that contain those genes that have been deleted or inactivated in the replication-deficient vector, thus allowing the virus to replicate in the cells. Suitable packaging cell lines for adenoviruses with deletions in the E1 region include, for example, PER.C6, 911, 293 and E1 A549.

[0074] In a preferred embodiment of the present invention, the vector is an adenovirus vector, and more preferably an rAd26 vector, most preferably an rAd26 vector having at least a deletion in the E1 region of the adenovirus genome, such as the vector described in Abbink, J Virol, 2007.81(9): p. 4654-63, which reference is incorporated herein by reference. Generally, the nucleic acid sequence encoding the prefusion PIV3 F protein is cloned into the E1 and / or E3 regions of the adenovirus genome.

[0075] Host cells containing a nucleic acid molecule encoding a prefusion PIV3 F protein also form part of the present invention. The prefusion PIV3 F protein may be produced by recombinant DNA techniques involving the expression of the molecule in a host cell such as Chinese hamster ovary (CHO) cells, tumor cell lines, BHK cells, human cell lines such as HEK293 cells, PER.C6 cells, or yeast, fungi, insect cells, etc., or transgenic animals or plants. In certain embodiments, the cells are from a multicellular organism, and in certain embodiments, they have a vertebrate or invertebrate origin. In certain embodiments, the cells are mammalian cells. In certain embodiments, the cells are human cells. Generally, the production of a recombinant protein, such as the prefusion PIV3 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 cells under conditions conducive to the expression of the nucleic acid molecule, and allowing the expression of the protein in the cells. The nucleic acid molecule encoding the protein in an expressible form may be in the form of an expression cassette, and generally requires sequences capable of effecting nucleic acid expression, such as enhancers, promoters, polyadenylation signals, etc. Those skilled in the art know that various promoters can be used to obtain gene expression in host cells. The promoter can be constitutive or regulatable and can be derived from various sources, including viral, prokaryotic or eukaryotic sources, or be artificially designed.

[0076] Cell culture media are available from various suppliers, and a suitable medium can be routinely selected for the host cells to express the protein of interest, here the prefusion PIV3 F protein. The suitable medium may or may not contain serum.

[0077] A "heterologous nucleic acid molecule" (also referred to herein as a 'transgene') is a nucleic acid molecule that is not naturally present 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 accomplished, for example, by placing the nucleic acid encoding the transgene under the control of a promoter. Further regulatory sequences may be added. Many promoters can be used for expressing the transgene and are known to the person skilled in the art, for example these promoters may include viral, mammalian, synthetic promoters, etc. Non-limiting examples of suitable promoters for obtaining expression in eukaryotic cells are the CMV promoter (US 5,385,839), such as the CMV immediate early promoter, for example comprising nt. -735 to +95 from the CMV immediate early gene enhancer / promoter. A polyadenylation signal, such as the bovine growth hormone polyA signal (US 5,122,458), may be present downstream of the transgene. Alternatively, several widely used expression vectors are available in the art and are obtainable from commercial sources, such as the pcDNA and pEF vector series from Invitrogen, pMSCV and pTK-Hyg from BD Sciences, pCMV-Script from Stratagene, etc., which can be used for recombinant expression of a protein of interest, or for obtaining suitable promoter and / or transcription termination sequences, polyA sequences, etc.

[0078] Cell culture can be of any type of cell culture, including adherent cell culture such as cells attached to the surface of a culture vessel or microcarriers, and suspension culture. Most large-scale suspension cultures are operated as batch or fed-batch processes because they are the most convenient to operate and scale up. Nowadays, continuous processes based on perfusion principles are becoming more common and are also suitable. Suitable culture media are also well known to the person skilled in the art and can generally be obtained in large quantities from commercial sources or customized according to standard protocols. The culture can be carried out, for example, in culture dishes, roller bottles or bioreactors, using batch, fed-batch, continuous systems, etc. Suitable conditions for culturing cells are known (see, for example, Tissue Culture, Academic Press, Kruse and Paterson, editors (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)).

[0079] The present invention further provides a composition comprising a prefusion PIV3 F protein as described herein, and / or a fragment thereof, and / or a nucleic acid molecule and / or a vector. The present invention thus provides a composition comprising a prefusion PIV3 F protein or a fragment thereof, which prefusion PIV3 F protein or fragment thereof exhibits epitopes that are present in the prefusion conformation of the PIV3 F protein but absent in the postfusion conformation. The present invention also provides a composition comprising a nucleic acid molecule and / or a vector encoding such prefusion PIV3 F protein or fragment. The present invention further provides a pharmaceutical composition, such as a vaccine composition, which comprises a prefusion PIV3 F protein, a PIV3 F protein fragment, and / or a nucleic acid molecule and / or a vector as described above and one or more pharmaceutically acceptable excipients.

[0080] The present invention also provides the use of a stable prefusion PIV3 F protein (fragment), nucleic acid molecule and / or vector according to the present invention for inducing an immune response against the PIV3 F protein in a subject. Further provided is a method for inducing an immune response against the PIV3 F protein in a subject, which comprises administering to the subject a prefusion PIV3 F protein (fragment), and / or a nucleic acid molecule and / or a vector according to the present invention. Also provided is a prefusion PIV3 F protein (fragment), nucleic acid molecule and / or vector according to the present invention, which is used for inducing an immune response against the PIV3 F protein in a subject. Further provided is the use of a prefusion PIV3 F protein (fragment), and / or a nucleic acid molecule and / or a vector according to the present invention for manufacturing an agent for inducing an immune response against the PIV3 F protein in a subject. The present invention particularly provides a prefusion PIV3 F protein (fragment), and / or a nucleic acid molecule and / or a vector according to the present invention, which is used as a vaccine.

[0081] The prefusion PIV3 F protein (fragment), nucleic acid molecule or vector of the present invention may be used for the prevention (prevention) and / or treatment of PIV3 infection. In certain embodiments, the prevention and / or treatment may target groups of patients vulnerable to PIV3 infection. Such groups of patients include, but are not limited to, for example, the elderly (e.g., ≥50 years old, ≥60 years old and preferably ≥65 years old), the young (e.g., ≤5 years old, ≤1 year old), pregnant women (for maternal immunization), as well as inpatients and patients who have been treated with antiviral compounds but have shown an insufficient antiviral response.

[0082] The prefusion PIV3 F protein, fragment, nucleic acid molecule and / or vector according to the present invention may be used for 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.

[0083] The present invention further provides a method for preventing and / or treating PIV3 infection in a subject by using the prefusion 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 to a subject in need thereof an effective amount of the prefusion PIV3 F protein (fragment), nucleic acid molecule, and / or vector as described above. A therapeutically effective amount refers to the amount of a protein, nucleic acid molecule, or vector that is effective for preventing, ameliorating, and / or treating a disease or condition resulting from infection by PIV3. Prevention encompasses inhibiting or reducing the spread of PIV3, or inhibiting or reducing the onset, development, or progression of one or more symptoms associated with infection by PIV3. As used herein, "ameliorating" may refer to a reduction in the visible or perceptible disease symptoms, viremia, or any other measurable manifestation of PIV3 infection.

[0084] For administration to a subject such as a human, the invention may employ a pharmaceutical composition comprising a pre-fusion PIV3 F protein (fragment), nucleic acid molecule, and / or vector as described herein, and a pharmaceutically acceptable carrier or excipient. In this context, the term "pharmaceutically acceptable" means that the carrier or excipient does not cause any unwanted or harmful effects in the subject to which it is administered at the doses and concentrations employed. 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 it is possible to utilize lyophilized preparations. The sterile solution is prepared by sterile filtration or other methods known per se in the art. The solution is then lyophilized or filled into pharmaceutical dosage containers. The pH of the solution is generally in the range of pH 3.0 to 9.5, such as pH 5.0 to 7.5. The PIV3 F protein is usually in a solution with a suitable pharmaceutically acceptable buffer, and the composition may also contain salts. Optionally, stabilizers such as albumin may be present. In certain embodiments, detergents are added. In certain embodiments, the PIV3 F protein may be formulated as an injectable preparation.

[0085] In certain embodiments, the compositions according to the invention further comprise one or more adjuvants. Adjuvants are known in the art to further increase the immune response against the applied antigenic determinants. The terms "adjuvant" and "immunostimulant" are used interchangeably herein and are defined as one or more substances that cause stimulation of the immune system. In this context, adjuvants are used to enhance the immune response against the PIV3 F protein of the invention. Examples of suitable adjuvants include aluminum salts such as aluminum hydroxide and / or aluminum phosphate; oil-in-water emulsion compositions (or water-in-oil compositions), including squalene-in-water emulsions such as MF59 (see, for example, WO 90 / 14837); saponin formulations such as QS21 and immunostimulating complexes (ISCOMs) (see, for example, US 5,057,540; WO 90 / 03184, WO 96 / 11711, WO 2004 / 004762, WO 2005 / 002620); bacterial or microbial derivatives, examples of which are monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), oligonucleotides containing CpG motifs, ADP-ribosylated bacterial toxins or mutants thereof, such as Escherichia coli heat-labile enterotoxin LT, cholera toxin CT, and the like; eukaryotic proteins (such as antibodies or fragments thereof (e.g., against the antigen itself or CD1a, CD3, CD7, CD80) and ligands for receptors (e.g., CD40L, GMCSF, GCSF, etc.), which stimulate the immune response after interaction with receptor cells. In certain embodiments, the compositions of the invention comprise aluminum as an adjuvant, for example in the form of aluminum hydroxide, aluminum phosphate, potassium aluminum phosphate, or combinations thereof, at a concentration of 0.05–5 mg per dose, for example an aluminum content of 0.075-1.0 mg.

[0086] In other embodiments, the composition does not comprise an adjuvant.

[0087] In certain embodiments, the present invention provides methods for preparing a vaccine against parainfluenza virus type 3 (PIV3), which comprise providing a PIV3 F protein (fragment), nucleic acid or vector according to the present invention and formulating it into a pharmaceutically acceptable composition. The term "vaccine" refers to an agent or composition containing an active ingredient that effectively induces a certain degree of immunity against a pathogen or disease in a subject, which results in at least a reduction (up to complete elimination) in the severity, duration or other manifestations of symptoms associated with infection by the pathogen or disease. In the present invention, the vaccine comprises an effective amount of a prefusion PIV3 F protein (fragment) and / or a nucleic acid molecule encoding the prefusion PIV3 F protein, and / or a vector containing said nucleic acid molecule, which results in an effective immune response against PIV3. This provides a method for preventing severe lower respiratory tract diseases leading to hospitalization, as well as a reduction in the frequency of complications such as pneumonia and bronchiolitis due to PIV3 infection and replication in a subject. The term "vaccine" according to the present invention implies that it is a pharmaceutical composition and thus generally includes a pharmaceutically acceptable diluent, carrier or excipient. It may or may not contain further active ingredients. In certain embodiments, it may be a combination vaccine, which further comprises other components that induce an immune response, such as other proteins against PIV3 and / or against other infectious agents, such as RSV, HMPV and / or influenza virus. The administration of the further active components may be accomplished, for example, by separate administration or by administering a combined product of the vaccine of the present invention with the further active components.

[0088] Administration of the composition according to the present invention can be carried out using standard administration routes. Non-limiting embodiments include parenteral administration, such as intradermal, intramuscular, subcutaneous, transdermal or mucosal administration, such as intranasal, oral, etc. In one embodiment, the composition is administered by intramuscular injection. A person skilled in the art knows the various possibilities of administering a composition, such as a vaccine, in order to induce an immune response against the antigen in the vaccine.

[0089] As used herein, the subject is preferably a mammal, such as a rodent, such as a mouse, a cotton rat, or a non-human primate, or a human. Preferably, the subject is a human subject.

[0090] The protein, fragment, nucleic acid molecule, vector and / or composition may also be administered as a prime or a boost in a homologous or heterologous prime-boost regimen. If a booster vaccination is performed, generally, such a booster vaccination is administered to the same subject one week to one year, preferably two weeks to four months, after the first administration of the composition (which is referred to as 'primary vaccination' in such cases). In certain embodiments, the administration comprises a prime and at least one booster administration.

[0091] The present invention further provides a method for preparing a vaccine against PIV3, which comprises providing a recombinant human adenovirus of serotype 26 comprising a nucleic acid encoding a prefusion PIV3 F protein or a fragment thereof as described herein, propagating the recombinant adenovirus in a culture of host cells, isolating and purifying the recombinant adenovirus, and placing the recombinant adenovirus in a pharmaceutically acceptable composition. In certain embodiments, provided herein is a method for producing adenovirus particles comprising a nucleic acid molecule encoding a PIV3 F protein or a fragment thereof (transgene). The method comprises (a) contacting a host cell of the present invention with an adenovirus vector of the present invention, and (b) growing the host cell under conditions in which adenovirus particles comprising the transgene are produced. Recombinant adenoviruses can be prepared according to well-known methods and propagated in host cells, which requires cell culture of host cells infected with adenovirus. The cell culture can be any type of cell culture, including adherent cell culture such as cells attached to the surface of a culture vessel or a microcarrier, and suspension culture.

[0092] Most large-scale suspension cultures operate as batch or fed-batch processes because they are most amenable to operation and scale-up. Nowadays, continuous processes based on perfusion principles are becoming more common and are also suitable (see, for example, WO 2010 / 060719 and WO 2011 / 098592, both of which are incorporated herein by reference and describe suitable methods for obtaining and purifying large amounts of recombinant adenovirus).

[0093] The present invention further provides an isolated recombinant nucleic acid that forms the genome of a recombinant human adenovirus of serotype 26 and that comprises a nucleic acid encoding a PIV3 F protein or a fragment thereof as described herein.

[0094] Additionally, the proteins of the present invention may be used as diagnostic tools, for example, to test the immune status of an individual by determining the presence of antibodies in the serum of such an individual that are capable of binding to the proteins of the present invention. The present invention thus also relates to an in vitro diagnostic method for detecting the presence of a PIV3 infection in a patient, the method comprising the steps of: a) contacting a biological sample obtained from the patient with a protein according to the present invention; and b) detecting the presence of an antibody-protein complex.

[0095] The present invention is further illustrated in the following examples. The examples do not limit the present invention in any way. They merely serve to illustrate the present invention.

[0096] Examples

[0097] Example 1: Instability of soluble PIV3 F extracellular domain protein

[0098] The plasmid encoding the extracellular domain of wild-type PIV3 F protein was synthesized at Genscript and codon-optimized, with the transmembrane and cytoplasmic tails replaced by a C-tag (SEQ ID NO:2). The construct was cloned into pCDNA2004 and sequenced by standard methods involving site-directed mutagenesis and PCR widely known in the art. The protein was expressed in the expi293F cell system. 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 collected and cells and cell debris were removed by centrifugation at 300 g for 5 minutes. The clarified supernatant was then sterile filtered using a 0.22 µm vacuum filter and stored at 4 °C until use.

[0099] Quantitative Octet measurements using the prefusion-specific monoclonal antibody PIA174 (Stewart-Jones et al., PNAS 115(48) 12265-12270, 2018) immobilized to an anti-human IgG sensor were used to measure the detection of the extracellular domain of PIV3 F protein in the crude supernatant using Biolayer Interferometry (BLI). Although there was a low but clear signal for wild-type (i.e., unstabilized) PIV3 preF protein on the day of harvest (day 0), it was undetectable after 20 days of storage at 4 °C ( Figure 2 ; wild type).

[0100] Example 2: Stabilizing mutations analyzed by Biolayer Interferometry and analytical SEC

[0101] To stabilize the unstable prefusion conformation of PIV3 F protein, the C-terminus of the extracellular domain of PIV3 F was fused to the GCN4 trimerization motif (SEQ ID NO:3), and the amino acid residue Asp at position 452 was mutated to Asn (D452N). Next, as Figure 2 indicated, additional mutations were introduced in this background. Plasmids encoding the extracellular domain of recombinant PIV3 F protein equipped with a C-tag were expressed in Expi293F cells, and 3 days after transfection, the binding of the supernatant to PIA174 was tested using quantitative Octet Figure 2)。The variants showed binding to the pre-fusion trimer-specific Mab PIA174 on the day of harvest and maintained the binding after storage at 4 °C for 20 days. The D452N mutation and the addition of GCN4 stabilized the pre-fusion conformation. Compared to the construct with only the D452N mutation, 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 the crude cell supernatant and also retained the pre-fusion conformation in the supernatant stored at 4 °C for 20 days.

[0102] On the day of harvest, the cell culture supernatants of different PIV3 F constructs with stabilizing mutations were analyzed using analytical size-exclusion chromatography (SEC). Figure 3 )。Combined with an online Nanostar DLS reader (Wyatt), an ultra-high performance liquid chromatography system (Vanquish, Thermo Scientific) coupled with an Optilab μT-rEX Refractive Index Detector (Wyatt) and a μDAWN TREOS instrument (Wyatt) were used to perform analytical SEC experiments. The clarified crude cell culture supernatant was applied to a column (Sepax catalog #231300-4615) with a corresponding guard column (Sepax) equilibrated in a running buffer (150 mM sodium phosphate, 50 mM NaCl, pH 7.0) at 0.35 mL / min. When analyzing the supernatant samples, the μMALS detector was offline and the analytical SEC data were analyzed using the Chromeleon 7.2.8.0 software package. As shown for the antibody binding studies described above, the SEC analysis also showed an increase in trimer content after the introduction of the stabilizing mutations. Compared to the wild type (SEQ ID NO:2) or a variant with only the GCN4 trimerization domain and the D452N substitution (PIV171432; SEQ ID NO:4), the variants with additional stabilizing substitutions showed a higher trimer content according to the analytical SEC of the culture supernatants. Figure 3 )。Compared to the soluble F variant with D452N and the C-terminal GCN4 domain, the variants with additional stabilizing substitutions S41P, (Q89M+Q222I), V165P, N167P, L168P, Q198L, F335P, (S186C+A195C), and (G85C+L221C) showed a higher trimer content according to the analytical SEC of the culture supernatants. Figure 3 )。

[0103] Example 3:Analysis of additive and synergistic stabilizing mutations by biolayer interferometry

[0104] To further stabilize the prefusion conformation of the unstable extracellular domain of PIV3 F protein, constructs with additional mutations at amino acid residue positions 41, 89, 165, 167, 168, 198, 204, 222, 335, 367, and / or 436 were prepared in the D452N background (all constructs thus contained the D452N mutation). Plasmids encoding these recombinant PIV3 F protein extracellular domains (with their C termini fused to GCN4 (SEQ ID NO:3) and equipped with a C-tag) were expressed in Expi293F cells, and 3 days after transfection, the binding of the supernatant to PIA174 was tested using the quantitative Octet ( Figure 4 ). Variants showed binding to the prefusion trimer-specific Mab PIA174 on the day of harvest. In addition, many double mutants had higher binding than each individual single mutation at positions 41, 165, 167, 168, 198, 204, 335, 367, and 436 in the D452N background or the double mutation at 89 + 222, indicating an additive or even synergistic stabilizing effect.

[0105] To further stabilize the extracellular domain of the PI V3F protein with the D452N+(Q89M+Q222I)+L168P mutation (i.e., PIV200309, SEQ ID NO:76), the previously mentioned stabilizing mutations were added in different combinations. Plasmids encoding these recombinant PIV3 F protein extracellular domains (with their C termini fused to GCN4 and equipped with a C-tag) were expressed in Expi293F cells, and 3 days after transfection, the supernatant was diluted 5-fold in mock transfection medium and the binding to PIA174 was tested using the quantitative Octet ( Figure 5 ). Variants 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).

[0106] Example 4: As analyzed by biolayer interferometry and analytical SEC, the stabilizing mutations in the stalk allowed removal of the GCN4 trimerization domain while retaining the trimeric architecture.

[0107] To stabilize the metastable trimeric prefusion conformation of the extracellular domain of the PIV3 F protein in the absence of GCN4, the amino acid residues at positions 470 and 477 were mutated in the stalk region (residues 452 - 481) of the PIV3 protein. A plasmid encoding the extracellular domain of the recombinant PIV3 F protein equipped with a C-tag was expressed in Expi293F cells, and 3 days after transfection, the binding of the supernatant to PIA174 was assayed using a quantitative Octet test ( Figure 6 A). The PIV3 backbone used was stabilized with the D452N + Q89M + Q222I + L168P mutation that lacks the GCN4 trimerization domain (PIV200941). Variants of PIV200941 that included 470V and / or 477V showed binding to the trimer-specific Mab PIA174, while constructs with wild-type amino acids at positions 470 and 477 showed only a small amount of prefusion trimers in the supernatant. Thus, the addition of the 470V and 477V mutations stabilizes the native trimeric quaternary structure of the prefusion F protein that lacks the GCN4 trimerization domain.

[0108] On the day of harvest, the cell culture supernatants of different PIV3 F constructs with the 470V and / or 477V stabilizing mutations were analyzed using analytical size exclusion chromatography (SEC) ( Figure 7 B). Compared to the variant (PIV200941; containing the D452N + Q89M + Q222I + L168P stabilizing mutation in the head domain) that lacks the stabilizing mutation in the stalk and the GCN4 trimerization domain, the variants with the 470V and / or 477V stabilizing mutations showed a higher trimer content according to analytical SEC of the culture supernatants ( Figure 6 B).

[0109] S470V and S477V were also studied in a wild-type backbone (PIV190058) that lacks the GCN4 trimerization domain and stabilizing mutations ( Figure 6 C). The introduction of S470V (PIV200960) or S477V (PIV200962) improved the binding to PIA174, indicating that these mutations stabilize the prefusion conformation without any additional mutations or heterologous trimerization domains.

[0110] Example 5: As determined by biolayer interferometry, analytical SEC, and differential scanning fluorimetry, the combination of stabilizing mutations in the head (residues 19 - 451) and stalk (residues 452 - 481) increased the expression and stability of the prefusion PIV3 F protein.

[0111] To stabilize the labile trimeric prefusion conformation of the PIV3 F protein extracellular domain in the absence of GCN4, amino acid residues at positions 41, 89, 167, 168, 222, 335, 452, 470, and / or 477 are mutated. A plasmid encoding the recombinant PIV3 F protein extracellular domain equipped with a C-tag is expressed in Expi293F cells, and 3 days post transfection, the supernatant is tested for binding to PIA174 using a quantitative Octet( Figure 7 A) and analytical SEC( Figure 7 B). In the absence of 470V and 477V, the protein runs at a lower retention time, indicating a larger protein. MALS analysis( Figure 8 ) of PIV201113 and PIV201105 showed no significant difference in molecular weight (165 vs 156 kDa respectively). The lower retention time of PIV201113 is likely due to an open stem region, increasing the apparent size of the protein, which is partially trimeric at the apex but less compact than PIV201105 which is trimeric at the base with optimized stem mutations. In the absence of the heterotrimerization domain, amino acids 470V and / or 477V are thus necessary to keep the protein in the native trimeric conformation.

[0112] In addition, the stability of the different proteins in the supernatant is determined by incubating the samples in a heating block at 4 °C, 50 °C, or 60 °C for 30 minutes. The samples are then spun at 15,000 rpm for 10 minutes to remove larger aggregates, and the supernatant is run on analytical SEC( Figure 9 ). In the absence of 470V, the protein has already lost its trimeric conformation 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 trimer peak completely disappears after 30 minutes of incubation at 60 °C.

[0113] The stability of different proteins in the supernatant was also determined by measuring the melting temperature (Tm) using differential scanning fluorimetry (DSF). For this, SYPRO Orange 5000x (S6650, Invitrogen) was diluted in PBS (1:250) to obtain a 20x working solution. For each reaction, 15 μL of the 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 subsequently read on a ViiA 7 Real-time PCR instrument. The construct with all stabilizing mutations (S41P + Q89M + Q222I + N167P + L168P + D452N + S470V + S477V + F335P) and without GCN4 had a Tm50 of 70.7 °C. (‘Backbone + F335P’ Figure 10 ). Removal of 335P decreased the Tm50 to 66.4 °C ( Figure 10 ). Further removal of 470V and / or 477V decreased the Tm50 sharply to <59 °C. Further removal of 41P or 89M + 222I decreased the Tm to 61.1 °C and 64.7 °C, respectively. This indicates that, in the absence of the GCN4 trimerization domain, especially S470V and S477V are essential for stable soluble trimers. Additionally, S41P, F335P, and Q89M + Q222I increased the melting temperature and thus further stabilized the protein.

[0114] Example 6: Characterization of the purified PIV3 F protein, as determined by SDS-PAGE, analytical SEC, and differential scanning fluorimetry.

[0115] According to the manufacturer's instructions, a set of PIV3 F designs (outlined in Figure 11 A) were transiently transfected into expi293 cells using ExpiFectamine (Life Technologies) and cultured at 37 °C and 10% CO2 for 5 days. The culture supernatant was harvested and spun at 600 g for 10 minutes to remove cells and cell debris. The spun supernatant was then filter-sterilized using a 0.22 um vacuum filter and stored at 4 °C until use. Using CaptureSelect including TMA two-step purification protocol of PIV3 F protein was performed using a C-tag affinity column, followed by size-exclusion chromatography using a HiLoad Superdex200 pg 16 / 60 column (GE Healthcare).

[0116] Figure 11 The yield in mg / L after purification for each protein design is indicated in A. The purified proteins were analyzed by SDS-PAGE under reducing and non-reducing conditions and stained with Coomassie ( Figure 11 B). The proteins ran as a single, unprocessed band, indicating that cleavage at the F2 / F1 boundary did not occur in expiHEK cells. SEC-MALS showed a clear trace with a sharp peak at the size of the PIV3 F trimer ( Figure 11 C). Differential scanning fluorimetry (DSF) showed that designs containing F335P (PIV201255 and PIV201256) showed an increase in melting temperature of 3.6 - 3.8 °C compared to their F335P-free counterparts (PIV201254 and PIV201110, respectively) ( Figure 11 D).

[0117] Example 7: As analyzed by biolayer interferometry and analytical SEC, the stabilizing mutations S470V + S477V in the HR2 stem were sufficient to form trimers.

[0118] To stabilize the prefusion conformation of the unstable trimer of the PIV3 F protein extracellular domain in the absence of GCN4, the amino acid residues at positions 470 and 477 were mutated in the stem region (residues 452 - 481) of the PIV3 protein. A plasmid encoding the extracellular domain of the recombinant PIV3 F protein equipped with a C-tag was expressed in Expi293F cells, and 3 days after transfection, the binding of the supernatant to PIA174 was assayed using the quantitative Octet test as described in Example 1 ( Figure 12 B), and the trimer content of the supernatant was analyzed using analytical size-exclusion chromatography (SEC) as described in Example 2 ( Figure 12 C). The wild-type PIV3 backbone (no stabilizing mutations, no GCN4 trimerization domain; PIV190058) did not show binding to the trimer-specific Mab PIA174 ( Figure 12 B), nor did it show a detectable trimer peak in the supernatant ( Figure 12C). In contrast, after introducing the S470V and S477V HR2 stem mutations (PIV210294), PIA174 Mab binding and a detectable trimer peak were observed, confirming that the addition of the 470V and 477V mutations stabilizes the native trimeric quaternary structure of the prefusion F protein lacking the GCN4 trimerization domain. Compared to S470V + S477V alone, subsequent introduction of various head domain mutations ( Figure 12 A) increased PIA174 binding in the quantitative Octet and improved trimer yield in analytical SEC. However, the head domain mutations alone did not produce detectable trimer binding and trimer expression in the supernatant ( Figure 12 C, dashed line), highlighting the importance of HR2 stability for the native trimeric quaternary structure of soluble PIV3 prefusion F ( Figure 12 C, solid line).

[0119] Example 8: Contributions of various mutations to the stability and yield of PIV3 prefusion F design PIV211368.

[0120] Expression of PIV3 F protein including the head stabilizing mutations S41P, Q89M + Q222I, and L168P and the stem stabilizing mutations S470V + S477V (PIV211368) was compared to PIV3 F variants in which single or double mutations were systematically removed by restoring amino acids to wild type ( Figure 13 A, indicated in bold). Plasmids encoding the extracellular domain of the recombinant PIV3 F protein without purification tags were expressed in Expi293F cells, and 3 days after transfection, the stability of different proteins in the supernatant was determined by measuring the melting temperature (Tm50) using differential scanning fluorimetry (DSF) as described in Example 5 ( Figure 13 A), and the trimer content was evaluated in analytical SEC as described in Example 2 ( Figure 13 B). Head domain mutations contributing to stability were S41P and Q98M + Q122I because they had lower melting temperatures (60.8 °C and 63.6 °C, respectively) than PIV211368 (65.8 °C) when restored to wild type. The HR2 mutations S470V and S477V similarly contributed to the thermal stability of PIV3 F, with lower melting temperatures of 50.3 °C and 58.0 °C, respectively. In contrast, the head domain L168P substitution decreased thermal stability, as indicated by an increase in the melting temperature of 67.1 °C when restored to wild type ( Figure 13 A).

[0121] The head domain mutations have little to no effect on the trimer content of PIV3 F (P41S; PIV211886), or have a positive effect (M89Q+I222Q; PIV211887 and P168L; PIV211890), as demonstrated by the reduction of the trimer peak of the wild-type rescue variant ( Figure 13 B).

[0122] In summary, in this specific stable protein design, the HR2 substitutions S470V and S477V strongly contribute to the stability of the PIV3 F protein, while the head domain mutation L168P strongly contributes 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.

[0123] Example 9: Characterization of the purified, tag-free PIV3 F protein, as determined by analytical SEC, differential scanning fluorimetry, and slow-freeze stability.

[0124] Using ExpiFectamine (Life Technologies) according to the manufacturer's instructions, PIV3 F design PIV211368, which lacks a purification tag and contains the stabilizing mutations S41P, Q89M / Q222I, L168P, S470V, and S477V, was transiently transfected into Expi293F cells and cultured for 5 days at 37 °C and 10% CO2. The culture supernatant was harvested and spun at 600 g for 10 minutes to remove cells and cell debris. The spun supernatant was then sterile filtered using a 0.22-μm vacuum filter and stored at 4 °C until use. A two-step purification protocol including ion exchange purification at pH 4.0 and polishing via size exclusion chromatography using a Superdex 200 increase 16 / 40 column was used to purify the PIV3 F protein. The trimer fractions were pooled and further characterized by SEC-MALS ( Figure 14 A). The trimer yield, molecular weight, and hydrodynamic radius are reported in Figure 14 B. Differential scanning fluorimetry (DSF) showed that the purified PIV211368 had a melting temperature of 66.5 °C ( Figure 14 B), which was slightly higher compared to the measurement of the protein in the crude cell culture supernatant ( Figure 13A). The stability of purified PIV211368 was further tested by slowly freezing the protein from 20 °C to -70 °C over a 24-hour period in various buffer compositions (FB12, PS4P4, and TS5P2). The recovery of PIV3 F trimers after slow freezing was determined in 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 tested buffers.

[0125] Example 10: Characterization of purified C-tagged PIV3F protein as determined by analytical SEC and differential scanning fluorimetry.

[0126] According to the manufacturer's instructions, PIV3 F design PIV210235 equipped with a C-tag and containing the stabilizing mutations S41P, Q89M / Q222I, S470V, and S477V was transiently transfected into Expi293 GnT1-cells using ExpiFectamine (Life Technologies) and cultured for 5 days at 37 °C and 10% CO2. The culture supernatant was harvested and spun at 600 g for 10 minutes to remove cells and cell debris. The spun supernatant was then sterile filtered using a 0.22 um vacuum filter and stored at 4 °C until use. A two-step purification protocol using a CaptureSelectTM C-tag affinity column followed by size exclusion chromatography using a Superdex200 10 / 300 column (GE Healthcare) was used to purify the PIV3 F protein. The trimer fractions were pooled and further characterized by SEC-MALS ( Figure 15 A). Trimer yield, molecular weight, and hydrodynamic radius were reported in Figure 15 B. Differential scanning fluorimetry (DSF) showed that purified PIV210235 had a melting temperature of 67.5 °C ( Figure 15 B).

[0127] Example 11: Fully single-chain protein according to the invention

[0128] Purification of PIV211368 (SEQ ID NO:237) (IEX, followed by SEC) resulted in a protein that was unexpectedly partially processed into F2 and F1 (although the cleavage site (‘RTER’) is not recognized by furin-like proteases and the native protease TMPRSS2 is not expressed in expiHEK cells), as detected by Coomassie staining following reduced SDS-PAGE ( Figure 16)。The introduction of R109Q (and T95A) results in a fully single-chain protein after IEX / SEC purification. The introduction of the E58D mutation does not affect processing (compare PIV220923 with PIV220922). As determined by DSF, all three proteins showed similar thermal stability ( Figure 17 )。The purified PIV220922 and PIV220923 proteins showed slightly higher binding to the pre-fusion specific antibody PIA174 of PIV3 ( Figure 18 )。

[0129] Table 1. Standard Amino Acids, Abbreviations, and Properties

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

Claims

1. A stable fusion of the parainfluenza virus 3 (HPIV3) F protein, which comprises F1 and F2 domains, and the F1 and F2 domains comprise the amino acid sequences of the F1 and F2 domains of the F protein of an HPIV3 strain, wherein the amino acid residue at position 41 is P, and the amino acid residue at position 89 is M, and the amino acid residue at position 222 is I, and the amino acid residue at position 168 is P, and the amino acid residue at position 470 is V, and 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.

2. The protein according to claim 1, wherein in addition, 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.

3. The protein according to any one of the preceding claims, which comprises a truncated F1 domain.

4. The protein according to claim 3, wherein the truncated F1 domain does not comprise a transmembrane region and a cytoplasmic region.

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

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

7. The protein according to any one of the preceding claims, which comprises an amino acid sequence selected from SEQ ID NO:243-250 or a fragment thereof, preferably comprises the amino acid sequence of SEQ ID NO:243 or a fragment thereof.

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

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

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

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

12. A vector which comprises the nucleic acid according to any one of claims 8-11.

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

14. The vector according to claim 13, wherein the adenovirus vector is a non-replicating Ad26 adenovirus vector with deletions in the E1 region and the E3 region.

15. A composition which comprises the protein according to any one of claims 1-7, the nucleic acid according to any one of claims 8-11 and / or the vector according to claims 12, 13 or 14.

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

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

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