Patent Application
20220185847
The present invention relates to modified metapneumovirus (hMPV) F proteins, stabilized in the pre-fusion conformation. It also relates to immunogenic compositions (vaccines) comprising these proteins for preventing and/or treating human subjects against respiratory tract infections.
Human metapneumovirus (hMPV) is a leading cause of acute respiratory tract infections in young children (0-4 years), immunocompromised patients and in elderly that can be fatal for these categories of patients (Schildgen et al. 2011. Clinical Microbiology Reviews 24(4): 734-54). Despite intensive efforts, currently there are no licensed vaccines or antivirals to prevent or treat hMPV infection. Among several vaccination strategies investigated, a subunit vaccine containing a viral protein, especially the hMPV F protein, is the most promising (Melero & Mas. 2015. Virus Res. 209: 128-35).
hMPV is an enveloped, single-stranded RNA virus of the genus Pneumovirus of the family Paramyxoviridae. The hMPV genome consists of eight genes encoding nine proteins, including three surface glycoproteins F, G and SH. Protection against hMPV is afforded mainly by neutralizing antibodies directed against the fusion (F) glycoprotein, which is highly conserved between different genotypes and shares similarities to other paramyxoviruses (see van den Hoogen et al. 2004. Emerging Infectious Diseases 10(4): 658-66; van den Hoogen et al. 2002. Virology 295(1): 119-32).
Paramyxoviral F protein is a type I integral membrane protein that spans the membrane once and contains at its N-terminus a signal peptide, which targets the ectodomain to the extracellular membrane. At the C-terminus, a hydrophobic stop-transfer domain (TM domain) anchors the protein in the membrane, leaving a short cytoplasmic tail (see
The native F protein is synthesized as an inactive precursor, designated F0 after a cleavage of the signal peptide (Yin et al. 2006. Nature 439 (7072): 38-44; Yin et al. 2005. Proc. Nat. Acad. Sci. 102(26): 9288-93; Russell et al. 1994. Virology 199(1): 160-8). To become biologically active, F0 is processed by a host protease generating two chains called F1 and F2, which remain covalently linked by disulfide bonds (Schowalter et al. 2006. Journal of Virology 80(22): 10931-41; Biacchesi et al. 2006. Journal of Virology 80(12): 5798-806; Yun et al. 2015. Scientific Reports 5: 15584). Three F1-F2 heterodimers form a mature F protein that is incorporated into the virion envelope in a metastable pre-fusion conformation (Battles et al. 2017. Nat. Commun. 8(1): 1528) and mediates fusion of the virion envelope and the host cell plasma membrane. During the fusion process, the F protein undergoes irreversible refolding from the labile pre-fusion conformation to the stable post-fusion conformation (see
Neutralizing antibodies specifically recognizing the pre-fusion hMPV F protein structure were found in human sera (Wen et al. 20012. Nat. Struct. Mol. Biol. 19: 461-463; Ngwuta et al. 2015. Science Translational Medicine 7(309): 309; Rossey et al. 2018. Trends in Microbiology 26(3): 209-19) indicating that the pre-fusion F protein could be a favorable vaccine candidate (Melero & Mas. 2015. Virus Res. 209: 128-35). One clear disadvantage of the pre-fusion over post-fusion F protein conformation is its instability. Previous attempts to produce stabilized pre-fusion F protein employed thorough structural analysis and computer modeling. In particular, one group described design of a highly stable pre-fusion RSV F protein, capable to provide protective response in rats (see Krarup et al. 2015. Nat. Commun. 6: 8143). Another group disclosed construction of stabilized pre-fusion forms of the hMPV F protein, which elicited neutralizing antibodies in mice immunized with those proteins (see WO2016/103238).
Crystal structures of the F protein in pre-fusion and post-fusion conformations were determined for hMPV, RSV and other paramyxoviruses (see e.g. Battles et al. 2017. Nat. Commun. 8(1): 1528). In spite of general structural similarities, it was revealed that the pre-fusion hMPV F protein possesses unique structural features that confer substantial functional and immunological differences between the F proteins of hMPV and RSV.
Despite significant progress in understanding a mechanism of action, structure and immunogenic properties of the hMPV F protein, no F protein based vaccine is on the market. Therefore, the objective of this invention is to provide novel modified pre-fusion hMPV F protein candidates for development of a human vaccine against a respiratory tract infection.
The present disclosure provides recombinant immunogenic human metapneumovirus (hMPV) F proteins and fragments thereof (herein referred to as the hMPV F proteins) capable to elicit neutralizing antibodies and protect against hMPV infection. A native coding sequence of the hMPV F protein was modified to generate stable pre-fusion conformation. Such modifications were designed based on three-dimensional (3D) homology models included as a part of the present invention. The invention further includes methods of producing the recombinant immunogenic proteins and methods of using the immunogenic proteins for prevention and/or treatment of hMPV infection in humans.
In one aspect, the present disclosure provides a modified hMPV F protein or a fragment thereof, stabilized in the pre-fusion conformation, comprising a single-chain polypeptide composed of an F2 domain, a heterologous peptide linker and an F1 domain lacking a fusion peptide (FP), wherein the linker is positioned between the F2 and F1 domains and contains one or more cysteine residue(s) each of which forms a non-natural disulfide bond with a cysteine residue present in the F1 domain.
In one embodiment, the single-chain F protein comprises F2 domain and F1 domains connected so that the C-terminus of F2 is proximal to the N-terminus of F1. A protease cleavage site between F2 and F1 may be mutated to eliminate the cleavage of the protein precursor. In some embodiments, the F1 domain may be a truncated F1 domain, e.g. so that it lacks the fusion peptide (FP) spanning the amino acid residues at positions 103-118 of the native hMPV F protein sequence of SEQ ID NO: 1, corresponding to residues 1 to 16 of the native F1 domain sequence of SEQ ID NO: 3. Thus, the F1 domain may comprise a fragment corresponding to residues 119 to 539 of SEQ ID NO: 1 or residues 17 to 437 of SEQ ID NO: 3. In some embodiments, the single-chain polypeptide may comprise a fragment of the F1 domain corresponding to residues 119 to 490 of SEQ ID NO: 1 or residues 17 to 338 of SEQ ID NO: 3, which does not contain an anchor transmembrane (TM) domain and a cytoplasmic tail at its C-terminus. Additionally, the F1 and F2 domains may be joined via a heterologous peptide linker containing e.g. five residues comprising at least one cysteine residue, preferably the linker of SEQ ID NO: 4.
In yet one embodiment, the single-chain F protein of the present invention has a stable pre-fusion conformation. On one side, the pre-fusion conformation is stabilized by abolished protease cleavage between F1 and F2 domains and lack of the free N-terminus of F1. Another feature that confers stabilization is the presence of at least one additional (including a non-natural) disulfide bond, which fixes the HRA domain inside the cavity formed by trimerization (see
In a further embodiment, the single-chain F protein may comprise one or more additional modification(s) that compensate for an altered geometry of the single-chain-containing F trimer. Preferably, said modification(s) is(are) substitution(s) at positions corresponding to positions 49, 51, 67, 80, 97, 137, 147, 159, 160, 161, 166, 177, 185, 258, 266, 294, 480 and/or 481 of the native hMPV F protein sequence of SEQ ID NO: 1. In particular, an asparagine at position 97 can be substituted for a glutamate (N97Q) or an alanine at position 185 can be substituted for a proline (A185P). Thus, the recombinant polypeptide may comprise the F2 domain comprising one or more substitution(s) at positions 31, 33, 49, 62 and/or 79 of SEQ ID NO: 2. The recombinant polypeptide may comprise an F1 domain (e.g. a truncated F1 domain lacking residues 1-16 of SEQ ID NO: 3) comprising one or more substitution(s) at positions 35, 45, 57, 58, 59, 64, 75, 83, 156, 164, 192, 378 and/or 379 of SEQ ID NO: 3.
Furthermore, some mutation(s) can compensate for a deficiency of cavity filling. Particularly, the cavity filling mutations can be selected from the list comprising the amino acid substitutions at positions 49, 67, 80, 137, 147, 159, 160, 161, 177 and 258 of the native hMPV F protein sequence of SEQ ID NO: 1. In one embodiment, the cavity filling mutations include a T49M substitution, an I67L substitution, an I137W substitution, an A147V substitution, an A159V substitution, a T160F substitution, an A161M substitution and/or an I177L substitution in SEQ ID NO: 1. Thus in some embodiments, the recombinant polypeptide may comprise an F2 domain comprising a T31M or I49L substitution in the sequence of SEQ ID NO: 2. In other embodiments, the recombinant polypeptide may comprise an (e.g. truncated) F1 domain comprising one or more substitutions in SEQ ID NO: 3 selected from 135W, A45V, A57V, T58F, A59M, I75L and/or F1561.
In another embodiment, the recombinant single-chain F protein may comprise one or more substitution(s) leading to formation of non-natural hydrogen bond(s) or salt bridge(s). For example, such substitutions include an E80N and S266D in SEQ ID NO: 1. Thus the recombinant polypeptide may comprise the F2 domain comprising a E62N substitution in the sequence of SEQ ID NO: 2. In other embodiments, the recombinant polypeptide may comprise an (e.g. truncated) F1 domain comprising the substitution S164D in SEQ ID NO: 3.
In some embodiments, the recombinant single-chain F protein may comprise further cysteine substitution(s) for creation an additional stabilizing disulfide bond(s). For example, substitutions E51C and K166C can form a non-natural disulfide bond between a cysteine residues at position 51 of the β-strand of the F2 domain and a cysteine at position 166 of the HRA α4 element of the native hMPV F protein sequence of SEQ ID NO: 1. This modification impairs a possible salt-bridge between E51 and K138 from a helix in HRA. Mutation S266D introduces a non-natural salt-bridge to K138 to compensate loss of attachment for this helix. Additionally, substitution of the vicinal residues 1480 and L481 of SEQ ID NO: 1 for cysteine allows introduction of three disulfide bonds across three protomers to make the covalently linked trimer protein. Thus in one embodiment, the recombinant polypeptide may comprise an F2 domain comprising a E33C substitution in the sequence of SEQ ID NO: 2. In other embodiments, the recombinant polypeptide may comprise an (e.g. truncated) F1 domain comprising the substitution(s) K64C, S164D, I378C and/or L379C in SEQ ID NO: 3.
In another embodiment, the recombinant single-chain F protein may comprise a modification(s) helpful for expression of a soluble recombinant protein. For instance, a substitution of a glycine for a glutamic acid residue may be present at position 294 (G294E) of the native hMPV F protein sequence of SEQ ID NO: 1, which may lead to a higher yield of the protein expression. Thus in one embodiment, the recombinant polypeptide may comprise a (e.g. truncated) F1 domain comprising a substitution G192E in SEQ ID NO: 3.
In some embodiments, the recombinant single-chain F protein may comprise combinations of two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions and/or other modifications. In some embodiments, the recombinant single-chain F protein may comprise a trimerization helper, so called foldon domain, e.g. linked to the C-terminus of the recombinant F protein subunit, that allows formation of a protein trimer. The foldon domain may derive from fibritin of T4 bacteriophage. The recombinant hMPV F protein of the present invention may be produced as mono- or hetero-trimer.
In some embodiments, the recombinant hMPV F protein may comprise or consist of an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 5 to 9 or 24 to 28. The recombinant single-chain F protein may comprise an F2 domain comprising or consisting of an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2. The recombinant single-chain F protein may comprise an F1 domain comprising or consisting of an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the recombinant single-chain F protein may comprise a truncated F1 domain comprising or consisting of an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to residues 17-437 or 17-388 of the amino acid sequence of SEQ ID NO: 3.
The recombinant hMPV proteins of the present invention are immunogenic and can induce neutralizing antibodies recognizing the native hMPV F protein. The present disclosure also includes immunogenic fragments of the recombinant hMPV proteins and the immunogenic proteins having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence of anyone of SEQ ID NOs: 5 to 9 or 24 to 28.
The present disclosure also provides isolated nucleic acid molecules encoding the modified hMPV F-proteins, vectors comprising the isolated nucleic acid molecules, host cells for recombinant expression of the modified hMPV F proteins.
The present disclosure also provides immunogenic compositions or vaccines comprising the recombinant hMPV F proteins, or isolated DNA molecules encoding the hMPV F protein or vectors of the invention, further comprising a pharmaceutically acceptable carrier and/or excipient, used with or without an adjuvant. Particularly, the disclosure provides immunogenic compositions or vaccines for stimulating an immune response in a subject, particularly an immune response, which can neutralize hMPV viruses and protect against hMPV infections. The disclosure further provides immunogenic compositions or vaccines comprising additional antigens derived from hMPV, RSV or PIV3 (parainfluenza virus type 3). The immunogenic proteins, isolated DNA molecules, vectors and immunogenic compositions or vaccines disclosed herein are suitable for use as a medicament, particularly for the prophylactic and/or therapeutic treatment of viral respiratory tract infections and associated diseases, especially infections and disease caused by hMPV.
Methods of production the recombinant hMPV F proteins, or isolated DNA molecules encoding the hMPV F protein or immunogenic compositions (vaccines) are encompassed in the present disclosure. Methods of generating an immune response in a subject, and methods of treating, inhibiting or preventing hMPV infections are also included.
In a further aspect, the present invention provides an immunogenic human metapneumovirus (hMPV) modified F protein or fragment thereof, stabilized in a pre-fusion conformation by one or more amino acid substitutions relative to a native hMPV F protein sequence; wherein the modified F protein or fragment thereof comprises a glutamine residue substituted for an asparagine residue at a position corresponding to position 97 of the native hMPV F protein sequence of SEQ ID NO: 1 (N97Q).
In a further aspect, the present invention provides an immunogenic human metapneumovirus (hMPV) modified F protein or fragment thereof, stabilized in a pre-fusion conformation by one or more amino acid substitutions relative to a native hMPV F protein sequence; wherein the modified F protein or fragment thereof comprises a glycine residue substituted for a glutamic acid residue at a position corresponding to position 294 of the native hMPV F protein sequence of SEQ ID NO: 1 (G294E).
In a further aspect, the present invention provides an immunogenic human metapneumovirus (hMPV) modified F protein or fragment thereof, stabilized in a pre-fusion conformation by one or more amino acid substitutions relative to a native hMPV F protein sequence; wherein the modified F protein or fragment thereof comprises one or more substitution(s) at positions corresponding to positions 49, 51, 67, 80, 137, 147, 159, 160, 161, 166, 177, 258, 266, 480 and/or 481 of the native hMPV F protein sequence of SEQ ID NO: 1.
In a further aspect, the present invention provides an immunogenic human metapneumovirus (hMPV) modified F protein or fragment thereof, stabilized in a pre-fusion conformation by two or more amino acid substitutions relative to a native hMPV F protein sequence; wherein the modified F protein or fragment thereof comprises the substitutions E51C and K166C relative to the native hMPV F protein sequence of SEQ ID NO: 1, and wherein the substituted cysteine residues form a non-native disulfide bond.
In a further aspect, the present invention provides an immunogenic human metapneumovirus (hMPV) modified F protein or fragment thereof, stabilized in a pre-fusion conformation by one or more amino acid substitutions relative to a native hMPV F protein sequence; wherein the modified F protein or fragment thereof comprises one or more substitutions selected from the group consisting of T49M, E80N, I137W, A147V, A159V, T160F, A161M, I67L, I177L, F258I, S266D, I480C and/or L481C relative to the native hMPV F protein sequence of SEQ ID NO: 1.
In a further aspect, the present invention provides an immunogenic human metapneumovirus (hMPV) modified F protein or fragment thereof, stabilized in a pre-fusion conformation by three or more amino acid substitutions relative to a native hMPV F protein sequence; wherein the modified F protein or fragment thereof comprises at least the substitutions T49M, A161M and I67L or I177L relative to the native hMPV F protein sequence of SEQ ID NO: 1.
In a further aspect, the present invention provides an immunogenic human metapneumovirus (hMPV) modified F protein or fragment thereof, stabilized in a pre-fusion conformation by three or more amino acid substitutions relative to a native hMPV F protein sequence; wherein the modified F protein or fragment thereof comprises one of the following substitution combinations relative to the native hMPV F protein sequence of SEQ ID NO: 1:
Unless otherwise specified herein, all amino acid positions mentioned in the present specification correspond to the amino acid positions of the native hMPV F protein sequence of SEQ ID NO: 1. The corresponding positions of such mutations in the F2 domain of SEQ ID NO: 2 and the F1 domain of SEQ ID NO: 3 can be derived directly therefrom. The F2 domain of SEQ ID NO: 2 corresponds to residues 19 to 102 of SEQ ID NO: 1. The F1 domain of SEQ ID NO: 3 corresponds to residues 103 to 539 of SEQ ID NO: 1.
By “adjuvant” is meant any substance that is used to specifically or non-specifically potentiate an antigen-specific immune response, perhaps through activation of antigen presenting cells. Examples of adjuvants include an oil emulsion (e.g., complete or incomplete Freund's adjuvant), montanide Incomplete Seppic Adjuvant such as ISA51, a squalene-based oil-in-water emulsion adjuvants such as MF59® (Novartis AG) (Ott G. et al. 1995. Pharm Biotechnol 6: 277-96) or AddaVax™ (InvivoGen), monophosphoryl lipid A (MPL) (Cluff C W. 2010. Adv Exp Med Biol 667:111-23), aluminum salt adjuvant (alum) (as described in WO 2013/083726), polycationic polymer, especially polycationic peptide, especially polyarginine or a peptide containing at least two LysLeuLys motifs, especially KLKLLLLLKLK, immunostimulatory oligodeoxynucleotide (ODN) containing non-methylated cytosine-guanine dinucleotides (CpG), e.g. CpG 1018 (Dynavax), in a defined base context (e.g., as described in WO 96/02555) or ODNs based on inosine and cytidine (e.g., as described in WO 01/93903), or deoxynucleic acid containing deoxy-inosine and/or deoxyuridine residues (as described in WO 01/93905 and WO 02/095027), especially oligo(dIdC)13 based adjuvant IC31® (Valneva SE) (as described in WO 04/084938 and Olafsdottir et al. 2009. Scand J Immunol. 69(3): 194-202), neuroactive compound, especially human growth hormone (described in WO 01/24822), a chemokine (e.g., defensins 1 or 2, RANTES, MIP1-α, MIP-2, interleukin-8, or a cytokine (e.g., interleukin-1β, -2, -6, -10 or -12; interferon-γ; tumor necrosis factor-α; or granulocyte-monocyte-colony stimulating factor), muramyl dipeptide (MDP) variants, non-toxic variants of bacterial toxins, QS-21 (Antigenics Inc.), Quill A, N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine-2-[1,2-dipalmitoyl-s-glycero-3-(hydroxyphosphoryloxy)]ethylamide (MTP-PE) and others as described in Sarkar et al. 2019. Expert Rev Vaccine: 18(5): 505-521, as well as compositions e.g. adjuvant systems, such as AF03, AS01, AS03 and AS04 (Giudice et al. 2018. Seminars in Immunology 39: 14-21). Adjuvants that transduce immunological signals via TLR3, TLR4, TLR7, TLR8, and TLR9 receptors promotes Th1-biased immunity, while signaling via TLR2/TLR1, TLR2/TLR6 and TLR5 promotes Th2-biased immunity. For instance, such adjuvants as CpG ODN, polyIC and MPL predominantly induce Th1 responsess, alum is strong inducer of Th2 response, while MF59®, Addavax™, and IC31® may induce mixed Th1 and Th2 responses. An adjuvant may be administered with an antigen or may be administered by itself, either by the same route as that of the antigen or by a different route than that of the antigen. A single adjuvant molecule may have both adjuvant and antigen properties.
Amino acid substitution refers to the replacement of one amino acid in a polypeptide with a different amino acid or with no amino acid (i.e., a deletion). As used herein, conservative substitutions are those substitutions that do not alter a basic structure and function of a protein, e.g. as the ability of the protein to induce an immune response when administered to a subject.
The following six groups are considered conservative substitutions for one another:
An antibody is polypeptide or protein that specifically binds and recognizes an antigen such as the hMPV F protein or an antigenic fragment of MPV F protein. The term “antibody” is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. Non-limiting examples of antibodies include, for example, intact immunoglobulins and variants and fragments thereof known in the art that retain binding affinity for the antigen. Examples of antibody fragments include but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen-binding fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (see, e.g., Kontermann and Dubel (Ed), Antibody Engineering, Vols. 1-2, 2nd Ed., Springer Press, 2010).
The following antibodies are have been used in the present invention: the MPE8 antibody is a monoclonal antibody that specifically binds to an epitope that is present on the surface of the hMPV F protein in the pre-fusion but not post-fusion conformation is (see Corti et al. 2013. Nature, 501:439-443). Sequences of the heavy and light variable regions of the MPE8 antibody are deposited in the GenBank with the accession Nos. AGU13651.1 and AGU13652.1, respectively. The MF1 antibody recognizes the 6HB domain of the post-fusion hMPV F protein as described in Rodriguez, 2015 (J Virol Methods 224: 1-8). The DS7 antibody described in Williams et al., 2007 (J Virology 81(15): 8315-24) binds to both the pre- and post-fusion hMPV F protein conformations.
A cavity-filling mutation is an amino acid substitution that fills a cavity within the protein core of the hMPV F protein. Cavities are essentially voids within a folded protein where amino acids or amino acid side chains are not present. In several embodiments, a cavity filling amino acid substitution is introduced to fill a cavity in the hMPV F protein ectodomain core present in the pre-fusion conformation.
A foldon domain is an amino acid sequence that naturally forms a trimeric structure and may also be referred to as a trimerization helper domain. In some examples, a foldon domain can be included in the amino acid sequence of a disclosed recombinant protein so that the antigen will form a trimer. In one example, a foldon domain is the T4 bacteriophage-derived foldon domain including the amino acid sequence set forth as e.g. GYIPEAPRDGQAYVRKDGEWVLLSTF (SEQ ID NO:10). The foldon domain may, for example, be cleaved from a purified protein, for example by incorporation of a thrombin cleavage site adjacent to the foldon domain.
A glycosylation site is an amino acid sequence on the surface of a polypeptide, such as a protein, which accommodates the attachment of a glycan. An N-linked glycosylation site is triplet sequence of NX(S/T) in which N is asparagine, X is any residues except proline, and (S/T) is a serine or threonine residue. A glycan is a polysaccharide or oligosaccharide. Glycan may also be used to refer to the carbohydrate portion of a glycoconjugate, such as a glycoprotein, glycolipid, or a proteoglycan.
The term “heterologous” means originating from a different genetic source. An amino acid sequence that is heterologous to a protein or virus originated from a source other than the protein or virus in which it is present or expressed. In one specific, non-limiting example, a heterologous peptide linker present in a recombinant polypeptide between two domains refers to a peptide sequence that is not naturally present in the wild type polypeptide between those two domains, e.g. the peptide linker is an artificial sequence linking the two domains in the recombinant polypeptide.
Homologous proteins have a similar structure and function, for example, proteins from two or more species or viral strains that have similar structure and function in the two or more species or viral strains. Homologous proteins share similar protein folding characteristics and can be considered structural homologs. Homologous proteins typically share a high degree of sequence conservation, such as at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence conservation, and a high degree of sequence identity, such as at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
hMPV F Protein
An hMPV F (fusion) protein is an envelope glycoprotein that facilitates fusion of viral and cellular membranes. In nature, the hMPV F protein is synthesized as a single polypeptide precursor approximately 540 amino acids long, which includes the N-terminal signal peptide (approximately the first 18 residues) that directs localization to the endoplasmic reticulum where the signal peptide is cleaved off. The remaining polypeptide, designated F0, constitutes the F protein monomer (protomer), which is processed at a protease cleavage site between positions 102 and 103 in the native F protein sequence of SEQ ID NO: 1 generating two disulfide-linked fragments, F1 and F2. The F2 fragment originates from the N-terminal portion of the F precursor and includes approximately residues 19-102 of SEQ ID NO: 1. The larger of these fragments, F1, includes the C-terminal portion of the F precursor (approximately residues 103-539 of SEQ ID NO: 1) including an extracellular/lumenal region (residues 103-490), a transmembrane domain (residues 491-513), and a cytoplasmic domain (residues 514-539) at the C-terminus. The extracellular portion of the hMPV F protein is the F ectodomain, which includes the F2 domain (approximately the hMPV F protein positions 19-102) and the F1 ectodomain (approximately the hMPV F protein positions 103-490). Three F2-F1 protomers oligomerize in the mature F protein trimer, which adopts a metastable pre-fusion conformation that is triggered to undergo a conformational change to a post-fusion conformation upon contact with a target cell membrane. This conformational change exposes a hydrophobic sequence, known as the fusion peptide (FP), located at the N-terminus of the F1 domain, which associates with the host cell membrane and promotes fusion of the membrane of the virus, or an infected cell, with the target cell membrane. Three hMPV F ectodomains may form a protein complex of three hMPV F protomers. The present invention relates to a modified hMPV F protein or fragment thereof, i.e. a recombinant polypeptide comprising one or more non-natural amino acid mutations with respect to a wild-type, native or naturally-occurring hMPV F protein sequence that stabilize the pre-fusion conformation.
hMPV F0 Polypeptide
The F0 polypeptide is a precursor of the hMPV F protein remained after cleavage of the signal peptide, which consists of the F2 domain and F1 domain including the F1 extracellular domain, transmembrane domain and cytosolic tail. The native F0 polypeptide is processed at a protease cleavage site separating F1 and F2 (approximately between positions 102 and 103 of SEQ ID NO: 1), resulting in the F1 and F2 polypeptide fragments (domains).
hMPV F1 Domain
The hMPV F1 domain is a part of the amino acid sequence of the hMPV F protein. As used herein, “F1 domain” refers to both native F1 sequences and F1 sequences including modifications (e.g. amino acid substitutions, insertions, or deletions). The native F1 domain (SEQ ID NO: 3) includes approximately residues 103-539 of the native hMPV F protein, and includes (from N- to C-terminus) an extracellular/lumenal region (residues 103-490 of SEQ ID NO: 1), a transmembrane domain (residues 491-513 of SEQ ID NO: 1), and a cytosolic domain (residues 514-539 of SEQ ID NO: 1) at the C-terminus. Several embodiments include an F1 domain modified from a native F1 sequence, for example an F1 domain that lacks a fusion peptide (e.g. residues 103-118 of SEQ ID NO: 1). In some embodiments, the F1 domain is an F1 ectodomain, i.e. lacks the transmembrane and cytosolic domain, for example the F1 domain corresponds to residues 103 to 490 or 119 to 490 of SEQ ID NO: 1. In further embodiments, the F1 domain includes one or more amino acid substitutions that stabilize a recombinant single-chain F protein (containing the F1 domain) in a pre-fusion conformation.
hMPV F2 Domain
The hMPV F2 domain is a part of the amino acid sequence of the hMPV F protein. As used herein, “F2 domain” refers to both native F2 polypeptides and F2 polypeptides including modifications (e.g. amino acid substitutions) from the native sequence, for example, modifications designed to stabilize a recombinant F protein (including the modified F2 polypeptide) in a hMPV F protein pre-fusion conformation. The native F2 domain (SEQ ID NO: 2) includes approximately residues 19-102 of SEQ ID NO: 1. In the native mature hMPV F protein, the F2 domain is linked to the F1 domain by two disulfide bonds.
hMPV Fusion Peptide (FP)
The hMPV fusion peptide is a part of the amino acid sequence of the hMPV F protein. The fusion peptide may be residues 103-118 of SEQ ID NO: 1, i.e. the N-terminal residues 1 to 16 of the F1 domain of SEQ ID NO: 3.
hMPV F Protein Pre-Fusion Conformation
The hMPV F protein pre-fusion conformation is a structural conformation adopted by the hMPV F protein prior to triggering of the fusogenic event that leads to transition of the hMPV F protein to the post-fusion conformation and following processing into a mature hMPV F protein in the secretory system. The three-dimensional structure of an exemplary hMPV F protein in a pre-fusion conformation is discussed herein and for example in WO 2016/103238. The pre-fusion conformation of hMPV F protein is similar in overall structure to the pre-fusion conformation of the F protein of other paramyxoviruses (such as RSV), though with some significant differences. In several embodiments, a recombinant hMPV F protein stabilized in the pre-fusion conformation specifically binds to an antibody (such as MPE8 antibody, see WO 2016/103238) specific for the trimeric form of the hMPV F protein in the pre-fusion, but not post-fusion, conformation.
Single-Chain hMPV F Protein
The single-chain hMPV F protein of the present invention is a recombinant hMPV F protein ectodomain (also used herein as a single-chain polypeptide) that is expressed as a single polypeptide chain including a (modified) hMPV F1 domain and a (modified) hMPV F2 domain. The single-chain hMPV F protein can typically trimerize to form a trimeric hMPV F protein subunit, preferentially being fused to a trimerization helper domain, e.g. foldon. In some embodiments, the recombinant single-chain hMPV F polypeptide does not include a protease cleavage site between the F1 domain and F2 domain and is not cleaved into separate F1 domain and F2 domain polypeptides when produced in cells. In one embodiment, F1 domain and F2 domain are linked with a heterologous peptide linker to generate the single-chain construct.
An immune response is a response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In one embodiment, the response is specific for a particular antigen (an “antigen-specific response”). In one embodiment, an immune response is a T cell response, such as a CD4+ response or a CD8+ response. In another embodiment, the response is a B cell response, and results in the production of specific antibodies. “Priming an immune response” refers to pre-treatment of a subject with an adjuvant to increase the desired immune response to a later administered immunogenic agent. “Enhancing an immune response” refers to co-administration of an adjuvant and an immunogenic agent, wherein the adjuvant increases the desired immune response to the immunogenic agent compared to administration of the immunogenic agent to the subject in the absence of the adjuvant.
An immunogen is a compound, composition, or substance that can stimulate production of antibodies or a T cell response in an animal, including compositions that are injected or absorbed into an animal. An immunogen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous antigens, such as the disclosed recombinant hMPV F proteins. An immunogen can include one or more epitopes. In some embodiments, an immunogen can be a recombinant hMPV F protein or immunogenic fragment thereof, a protein nanoparticle or virus-like particle including the recombinant hMPV F protein or immunogenic fragment thereof, or nucleic acid or vector encoding the recombinant hMPV F protein or immunogenic fragment thereof, that is capable of inducing an immune response in a mammal, such as a mammal infected or at risk of infection with a pathogen. Administration of an immunogen to a subject can lead to protective immunity and/or proactive immunity against a pathogen of interest.
An immunogenic composition is a composition comprising an immunogen that induces a measurable CTL response against an antigen, or induces a measurable B cell response (such as production of antibodies) against an antigen, included on the immunogen or encoded by a nucleic acid molecule included in the immunogen. In one example, an immunogenic composition is a composition that includes the disclosed recombinant hMPV F proteinor immunogenic fragment thereof, which induces a measurable CTL response against the hMPV virus, or induces a measurable B cell response (such as production of antibodies) against the hMPV F protein when administered to a subject. An immunogenic composition can include isolated nucleic acids encoding an immunogenic protein that can be used to express the immunogenic protein and thus to elicit an immune response against this protein. Thus, in another example, an immunogenic composition is a composition that includes a nucleic acid molecule encoding the disclosed recombinant hMPV F protein or immunogenic fragment thereof, that induces a measurable CTL response against the hMPV virus, or induces a measurable B cell response (such as production of antibodies) against the hMPV F polypeptide when administered to a subject. For in vivo use, the immunogenic composition will typically include an immunogenic polypeptide or nucleic acid molecule encoding an immunogenic polypeptide in a pharmaceutically acceptable carrier and may also include other agents, such as an adjuvant. Any particular polypeptide, such as a disclosed recombinant hMPV F protein or a nucleic acid encoding the protein, can be readily tested for its ability to induce a CTL or B cell response by art-recognized assays.
An “isolated” biological component has been substantially separated or purified away from other biological components, such as other biological components in which the component naturally occurs, such as other chromosomal and extrachromosomal DNA, RNA, and proteins. Proteins, peptides and nucleic acids that have been “isolated” include proteins purified by standard purification methods. The term also embraces proteins or peptides prepared by recombinant expression in a host cell as well as chemically synthesized proteins, peptides and nucleic acid molecules. Isolated does not require absolute purity, and can include protein, peptide, or nucleic acid molecules that are at least 50% isolated, such as at least 75%, 80%, 90%, 95%, 98%, 99%, or even 99.9% isolated. The modified hMPV F proteins disclosed herein that are stabilized in a pre-fusion conformation are isolated from hMPV F proteins in a post-fusion conformation, for example, are at least 80% isolated, at least 90%, 95%, 98%, 99%, or even 99.9% isolated from hMPV F proteins in a post-fusion conformation.
A linker is a bi-functional molecule that can be used to link two molecules into one contiguous molecule, for example, to link two domains in a single polypeptide. Preferably, the linker is a peptide linker. The linker may be of any suitable length, e.g. 1 to 20, 1 to 15, 1 to 10, 1 to 5 or less amino acid residues. The linker may comprise or consist of e.g. alanine, serine, glycine, cysteine and/or valine residues. Preferably, the linker may comprise at least one cysteine residue.
A native or natural (herein used interchangeably) polypeptide, sequence, residue or disulfide bond is one that has not been modified, for example, by selective mutation to focus the antigenicity of the antigen to a target epitope, or to introduce a disulfide bond into a protein that does not occur in the native protein. Native or natural proteins, residues or sequences are also referred to as wild type proteins, residues or sequences. A non-native or non-natural disulfide bond is a disulfide bond that is not present in a native protein, for example a disulfide bond that forms in a protein due to introduction of one or more cysteine residues into the protein by genetic engineering. Likewise, a non-natural cysteine residue in a domain is a cysteine residue that is not present at that position in a wild type, native or natural sequence.
A neutralizing antibody reduces the infectious titer of an infectious agent by binding to a specific antigen on the infectious agent. In some examples, the infectious agent is a virus. In some examples, an antibody that is specific for hMPV F protein neutralizes the infectious titer of hMPV. In some embodiments, the neutralizing antibody binds to and inhibits the function of related antigens, such as antigens that share at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity antigenic surface of antigen. With regard to an antigen from a pathogen, such as a virus, the antibody can bind to and inhibit the function of an antigen from more than one class and/or subclass of the pathogen. For example, with regard to hMPV, the antibody can bind to and inhibit the function of an antigen, such as hMPV F protein from more than one group. In one embodiment, broadly neutralizing antibodies to hMPV are distinct from other antibodies to hMPV in that they neutralize a high percentage of the many types of hMPV in circulation.
Pharmaceutically acceptable carriers are used to formulate the immunogenic hMPV F protein for clinical administration. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition, 1995, describes compositions and formulations suitable for pharmaceutical delivery of the disclosed immunogens. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. In particular embodiments, suitable for administration to a subject the carrier may be sterile, and/or suspended or otherwise contained in a unit dosage form containing one or more measured doses of the composition suitable to induce the desired anti-MPV immune response. It may also be accompanied by medications for its use for treatment purposes. The unit dosage form may be, for example, in a sealed vial that contains sterile contents or a syringe for injection into a subject, or lyophilized for subsequent solubilization and administration or in a solid or controlled release dosage.
A polypeptide is any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). “Polypeptide” applies to amino acid polymers including naturally occurring amino acid polymers and non-naturally occurring amino acid polymer as well as in which one or more amino acid residue is a non-natural amino acid, for example an artificial chemical mimetic of a corresponding naturally occurring amino acid. A “residue” refers to an amino acid or amino acid mimetic incorporated in a polypeptide by an amide bond or amide bond mimetic. A polypeptide has an amino terminal (N-terminal) end and a carboxy terminal (C-terminal) end. “Polypeptide” is used interchangeably with peptide or protein, and is used herein to refer to a polymer of amino acid residues. In many instances, one or more polypeptides can fold into a specific three-dimensional structure including surface-exposed amino acid residues and non-surface-exposed amino acid residues. In some instances, a protein can include multiple polypeptides that fold together into a functional unit. For example, the mature hMPV F protein on the cell surface includes three F2/F1 heterodimers that trimerize in to a multimeric protein. “Surface-exposed amino acid residues” are those amino acids that have some degree of exposure on the surface of the protein, for example such that they can contact the solvent when the protein is in solution. In contrast, non-surface-exposed amino acids are those amino acid residues that are not exposed on the surface of the protein, such that they do not contact solution when the protein is in solution. In some examples, the non-surface-exposed amino acid residues are part of the protein core.
A recombinant polypeptide (or protein) is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of amino acid residues, for example, by genetic engineering techniques. In several embodiments, a recombinant single-chain polypeptide is encoded by a heterologous (for example, recombinant) nucleic acid that has been introduced into a host cell, such as a bacterial or eukaryotic cell. The nucleic acid can be introduced, for example, on an expression vector having signals capable of expressing the protein encoded by the introduced nucleic acid or the nucleic acid can be integrated into the host cell chromosome.
Sequence identity Sequence identity is frequently measured in terms of percentage identity: the higher the percentage, the more identical the two sequences are. Homologs, orthologs, or variants of a polypeptide will possess a relatively high degree of sequence identity when aligned using standard methods.
Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman (Adv. Appl. Math. 2:482, 1981); Needleman & Wunsch (Mol. Biol. 48:443, 1970); Pearson & Lipman (Proc. Natl. Acad. Sci. USA 85:2444, 1988); Higgins & Sharp (Gene, 73:237-44, 1988); Higgins & Sharp (CABIOS 5:151-3, 1989); Corpet et al. (Nuc. Acids Res. 16:10881-90, 1988); Huang et al. (Computer Appls in the Biosciences 8:155-65, 1992); Pearson et al. (Meth. Mol. Bio. 24:307-31, 1994) and Altschul et al. (J. Mol. Biol. 215:403-10, 1990), presents a detailed consideration of sequence alignment methods and homology calculations. Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is present in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. Preferably, the percentage sequence identity is determined over the full length of the sequence. For example, a peptide sequence that has 1166 matches when aligned with a test sequence having 1554 amino acids is 75.0 percent identical to the test sequence (1166÷1554*100=75.0). The percent sequence identity value is rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The length value will always be an integer.
The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al. 1990. Mol. Biol. 215:403) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and on the internet, for use in connection with the sequence analysis programs BLASTP, BLASTN, BLASTX, TBLASTN and TBLASTX. A description of how to determine sequence identity using this program is available on the NCBI website on the internet. The BLAST and the BLAST 2.0 algorithm are also described in Altschul et al. (Nucleic Acids Res. 25: 3389-3402, 1977). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov). The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands. The BLASTP program (for amino acid sequences) uses as defaults a word length (W) of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff. 1992. Proc. Natl. Acad. Sci. USA 89:10915-10919).
Homologs and variants of a polypeptide are typically characterized by possession of at least about 75%, for example at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity counted over at least 50, 100, 150, 250, 500 amino acid residues of the reference sequence, over the full length of the reference sequence or over the full length alignment with the reference amino acid sequence of interest. Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. For sequence comparison of nucleic acid sequences, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters are used.
One example of a useful algorithm is PILEUP. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle (Mol. Evol. 35: 351-360, 1987). The method used is similar to the method described by Higgins & Sharp (CABIOS 5:151-153, 1989). Using PILEUP, a reference sequence is compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. PILEUP can be obtained from the GCG sequence analysis software package, e.g., version 7.0 (Devereaux et al. 1984. Nuc. Acids Res. 12: 387-395).
As used herein, reference to “at least 80% identity” refers to “at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity” to a specified reference sequence, e.g. to at least 50, 100, 150, 250, 500 amino acid residues of the reference sequence or to the full length of the sequence. As used herein, reference to “at least 90% identity” refers to “at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity” to a specified reference sequence, e.g. to at least 50, 100, 150, 250, 500 amino acid residues of the reference sequence or to the full length of the sequence.
A therapeutically effective amount is the amount of agent, such as a disclosed immunogen or immunogenic composition, that is sufficient to prevent, treat (including prophylaxis), reduce and/or ameliorate symptoms and/or underlying causes of a disorder or disease, for example to prevent, inhibit and/or treat hMPV infection. In some embodiments, a therapeutically effective amount is sufficient to reduce or eliminate a symptom of a disease, such as hMPV infection. For instance, this can be the amount necessary to inhibit or prevent viral replication or to measurably alter outward symptoms of the viral infection. In general, this amount will be sufficient to measurably inhibit virus replication or infectivity. In one example, a desired response is to inhibit, reduce or prevent hMPV infection. The infection does not need to be completely eliminated, reduced or prevented for the method to be effective. For example, administration of a therapeutically effective amount of the agent can decrease the infection (as measured by infection of cells, or by number or percentage of infected subjects) by a desired amount, for example by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable hMPV infection) as compared to a suitable control.
It is understood that to obtain a protective immune response against a pathogen can require multiple administrations of the immunogenic composition. Thus, a therapeutically effective amount encompasses a fractional dose that contributes in combination with previous or subsequent administrations to attaining a protective immune response. For example, a therapeutically effective amount of an agent can be administered in a single dose, or in several doses, for example daily, during a course of treatment (such as a prime-boost vaccination treatment). However, the therapeutically effective amount can depend on the subject being treated, the severity and type of the condition being treated, and the manner of administration. A unit dosage form of the agent can be packaged in a therapeutic amount, or in multiples of the therapeutic amount, for example, in a vial (e.g., with a pierceable lid) or syringe having sterile components.
A vaccine is a pharmaceutical composition that elicits a prophylactic or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Typically, a vaccine elicits an antigen-specific immune response to an antigen of a pathogen, for example a viral pathogen, or to a cellular constituent correlated with a pathological condition. A vaccine may include a polynucleotide (such as a nucleic acid encoding a disclosed antigen), a peptide or polypeptide (such as a disclosed antigen), a virus, a cell or one or more cellular constituents. In one specific, non-limiting example, a vaccine reduces the severity of the symptoms associated with hMPV infection and/or decreases the viral load compared to a control. In another non-limiting example, a vaccine reduces hMPV infection compared to a control.
In one aspect, this disclosure provides a novel modified hMPV F protein stabilized in a pre-fusion conformation. The present disclosure also provides a method for generating stabilized pre-fusion F proteins based on crystal structures and homology modelling. For the analysis of structural basis of stabilizing modification(s), a series of structures of available fusion proteins of hMPV and homologous fusion proteins were used. Among them, the crystal structure models of the pre-fusion hMPV F protein ectodomain (PDB:5WB0) and the post-fusion hMPV F protein ectodomain (PDB:5L1X), the crystal structure models of the pre-fusion RSV F protein ectodomain (PDB:4DAB) and the post-fusion RSV F protein ectodomain (PDB:3RRR). The so-called trimerization helper domain by using foldon domain was modeled into the homology model based on structural data in PDB:2IBL, 1OX3 and 1AVY.
The native mature hMPV F protein is composed of two polypeptides F2 and F1 covalently linked by two disulfide bonds. The maturation process includes one cleavage of the F0 precursor by a trypsin-like protease resulted in generating the free N-terminus of the F1 domain the fusion peptide FP, which interacts with the target cellular membrane and triggers the conformational changes. After the cleavage the relocation of the C-terminal part of F1 into the hydrophobic region of the inner trimeric cavity occurs. This may enhance the stability of the metastable pre-fusion state until a trigger event initiates refolding into the post-fusion conformer (see
The modelling of a single-chain F protein was performed aiming to obtain a stabilized pre-fusion conformation and simultaneously keep a maximum structural similarity to the native hMPV F protein (see
For achieving a 3D structure similarity of the single-chain F ectodomain to the native pre-fusion hMPV F protein, a loop between the F1 domain and F2 domain was designed. In one embodiment, the loop is constructed by insertion of a heterologous peptide linker. In preferred embodiments, the size and the composition of the linker is optimized for the stability, antibody binding qualities and yield of the antigen. In one embodiment, length of the linker can be between 2 and 10 amino acid residues, preferably between 2 and 5 residues, more preferably between 3 and 5 residues, even more preferably 4 or 5 residues, the most preferably 5 residues. In another embodiment, the linker may be inserted between the amino acid residue at positions 95 to 102, preferably at positions 102, most preferably to the glycine residue at position 102 of SEQ ID NO: 1.
In some embodiments, the linker may be composed of one or more cysteine, glycine, alanine, phenylalanine, valine and/or serine residue(s). The liker may comprise one, two or three alanine residue(s), one or two valine residue(s) and one or two glycine residue(s). For instance, an alanine can be at position 1, 2, 3, 4, and/or 5 of the linker; a glycine can be at position 2, 3 and/or 4 of the linker; and a valine preferably can be at position 3 and/or 5 of the linker. Some non-limiting examples of the linker together with additional modifications of F2 and F1 are provided in Table 1.
In a preferred embodiment, the linker comprises at least one (e.g. one) cysteine residue. The cysteine may be at any position of the linker, preferably at position 1 or 3, that corresponds to position 103 and 105 of SEQ ID NO: 1. More preferably, the cysteine is at position 1 of the linker (i.e. at the N-terminal of the linker, preferably adjacent to the F2 domain) that corresponds to position 103 of the native hMPV F protein sequence of SEQ ID NO: 1. Even more preferably, the linker is CGAGA, CGAGV, CGAAV, AGCGA, CAAAV, CAAFV or CGAGA. In the most preferred embodiment, the linker is CGAGA (SEQ ID NO: 4).
In some embodiments, the pre-fusion conformation of the single-chain F protein may be covalently stabilized by introducing at least one non-natural intra- or inter-protomer disulfide bond. For instance, a non-natural disulfide bond may be introduced between a cysteine residue of the heterologous peptide linker located between the F2 and F1 domains and a cysteine residue of the F1 domain. The first cysteine residue can be at any position of said linker, for example, at position 1, 2, 3 or 4, preferably at position 1, which corresponds to position 103 of SEQ ID NO: 1. Alternatively, the first cysteine residue can be introduced in the F2 domain at position 96, or 101, or 102 of SEQ ID NO: 1. In the most preferred embodiment, the cysteine at position 103 forms a non-natural disulfide bond with the cysteine substitution of the alanine at position 338 of the native hMPV F sequence of SEQ ID NO: 1. This S—S-bond can stabilize the pre-fusion conformation of the single-chain F protein by fixing the loop between F2 and F1 within the hydrophobic trimeric cavity. Such loop fixation mimics the positioning effect of the internalized cleaved N-termini of F1 in the native hMPV protein. In some embodiments, the single-chain hMPV F protein may comprise further cysteine substitution(s) that can introduce non-native inter-protomer disulfide bonds, e.g. to stabilize a protein trimer by linking it covalently.
In yet one embodiment, the single-chain hMPV F protein lacks amino acid residues 1 to 16 at the N-terminus of the F1 domain of SEQ ID NO: 3, encompassing the entire or partial sequence of the fusion peptide FP. Preferably, the single-chain hMPV F protein lacks the amino acid residues at positions 103-118 of the native hMPV F protein sequence of SEQ ID NO: 1. The deletion of FP further stabilizes the pre-fusion conformation of the single-chain hMPV F protein.
In some embodiments, the single-chain hMPV F protein may comprise one or more further modification(s). On the one hand, the additional modification may compensate an altered geometry of the single-chain hMPV F protein. On the other hand, the additional modification may further stabilize the pre-fusion conformation. The additional modifications can comprise one or more amino acid substitutions, insertions and/or deletions. Among modifications, the conservative substitutions may be, but not necessarily are, preferred. The following groups of substitutions are considered conservative:
In one embodiment, the additional modification, which stabilized the single-chain hMPV F protein, is the substitution of a glutamine residue for an asparagine residue at position 97 (N97Q) of the native hMPV F protein sequence of SEQ ID NO: 1.
In another embodiment, the additional modification of the single-chain F ectodomain may comprise one or more cavity filling substitution(s), including but not limited to substitutions at positions 49, 67, 137, 159, 147, 160, 161 and/or 177 relative to the native hMPV F protein sequence of SEQ ID NO: 1. In particular, the cavity filling substitution can be selected from, but is not limited to, a T49M substitution, an I67L substitution, an I137W substitution, an A147V substitution, an A159V substitution, a T160F substitution, an A161M substitution or I177L substitution. Additionally, combinations of two or more cavity filling substitutions are possible. In one particular embodiment, the combination comprises the T160F and I177L substitutions. In another particular embodiment, the combination comprises the T49M, I67L and A161M substitutions. In yet particular embodiment, the combination comprises the T49M, A161M, I137W, A147V, A159V and I177L substitutions.
Rigidification of the HRA α3 by cavity filling. In native and cleaved F protein the N-terminal part of F1 bears the HRA containing domain, a long extended helix in the thermodynamically more stable post-fusion F protein, but folded with several distinct small helices, even bearing a beta hairpin element, in the pre-fusion conformation (“loaded spring”). Additional contacts of this element may allow for stabilization of the protein in pre-fusion conformation. To fill up a cavity beneath the HRA α3 two small residues were replaced with the space-filling aliphatic residue methionine at positions T49M and A161M to form a complementary pair packing together and to strengthen the aliphatic fixation of this surface-located helix. Mutations at the position 161 have been reported for the hMPV F pre-fusion ectodomain by Battles et al., 2017 leading either to non-expressing F protein subunits (A161F) or to subunits poorly reacting with the MPE8 antibody (A161L) (see Battles et al. 2017. Nat. Commun. 8(1): 1528).
Covalent attachment of the HRA α4 by a disulfide-bond. HRA α4 extends to the tip of the F protein and is situated C-terminal of the beta-hairpin element following HRA α3 in the pre-fusion structure. All of these elements participate in the transformation to the long alpha-helical element in the post-fusion conformation, which includes a movement away from the head domain towards the host cell membrane in the fusion process of the virus. Here a disulfide bridge is introduced between the HRA α4 helical element (K166C) to a long beta-strand provided by the F2 portion (E51C). This change modifies two charged residues, which participate in a polar network including contacts to HRA α3. An additional modification, S266D, was introduced to allow for a partial reconstitution of the disturbance in the salt-bridge network by this disulfide bond-forming mutation.
Rigidification of the HRA α2-α3 by introduction of tryptophan. HRA α2-α3 forms a bent substructure on the pre-fusion hMPV F protein (while being part a long helix in the post-fusion conformation). Rigidification of this bend would hinder transformation to the post-fusion conformation. For this bend similarly folded substructures can be observed, in which the hMPV F protein 1137 is tryptophan in the analog position and providing a more densely packed substructure e.g. in a crystal structure of the N-terminal part of cleaved Protein C Inhibitor bound to Heparin (PDB:3DY0, W271 of chain A). Based on the homology modeling and molecular simulation, two further mutations A147V and A159V were introduced to provide extra space filling in this substructure to force the tryptophan side chain into the orientation observed in the Protein C Inhibitor structure, which allows additional stabilization of the tryptophan side-chain amide with a polar contact to S149 (an asparagine in Protein C inhibitor). Also, the analog positions to A147 and A159 provide residues with space-filling side-chains.
In some embodiments, the single-chain hMPV F protein may comprise one or more further stabilizing substitution(s), for example, substitutions leading to formation of a non-natural hydrogen bond(s), variant core packing or a salt bridge(s). In particular, the modified single-chain hMPV F protein may comprise the E80N, F258I and/or G294E substitutions. The E80N substitution can establish an inter-protomer H-Bond to D224′ (the prime denoting the neighbor protomer) and reduce repulsion with D209. On the other hand, the E80N substitution enhances the recombinant expression of the single-chain hMPV F protein. Another modification, which is helpful for increasing a yield of the recombinant protein, is the G294E substitution.
In some embodiments, the substitution of the vicinal residues 1480 and L481 for cysteine residues allows introduction of three disulfide bonds across the three protomers in the form of a covalent ring. The covalently linked trimer is supposed to be more stable than the foldon trimerized particle. Formation of a functional ring requires that all three disulfide bonds, or in case of multiple rings at least one disulfide bond between each protomer, are formed. The distance of the ring to the foldon domain is short and the foldon attachment position optimized for a more rigid geometry.
In preferred embodiment, the following substitution combinations are as follows:
N97Q, R102G and G294E (L7F_A 1_23) (e.g. as present in SEQ ID NO: 5)
N97Q, R102G, T160F, I177L and G294E (sF_A1_K_L7) (e.g. as present in SEQ ID NO: 6);
N97Q, R102G, T49M, I67L, A161M, E80N, F258I and G294E (L7F_A1_31) (e.g. as present in SEQ ID NO: 7);
N97Q, R102G, T49M, I67L, A161M, E51C, K166C, S266D, G294E, 1480C and L481C (L7F_A1_33) (e.g. as present in SEQ ID NO: 8), and
N97Q, R102G, T49M, A161M, I137W, A159V, A147V I177L and G294E (L7F_A1_4.2) (e.g. as present in SEQ ID NO: 9).
In some embodiments, the modified single-chain hMPV F proteins of the invention differ from the native hMPV F protein in that they do not possess a transmembrane domain and a cytoplasmic tail. Nevertheless, in some embodiments, the modified single-chain hMPV F proteins can form mono- or hetero-trimers. In order to form a trimer a trimerization helper domain, so called foldon, may be inserted in the C-terminal part of the F ectodomain. Addition of the trimerization helper, which retains the soluble state to the C-terminus of the subunit ectodomain, supports formation of a stable trimeric and soluble protein trimer.
In one embodiment, the foldon domain may derive from fibritin of T4 bacteriophage and comprises the sequence of SEQ ID NO: 10. In another embodiment, the fibritin foldon may be modified by insertion of one or more N-glycosylation site(s) (motif NxT/S, wherein “x” any amino acid residue except proline), which could help to hide hMPV non-specific epitope(s). Some non-limiting examples of modified foldon domain sequences are as following:
Alternatively, the foldon domain may possess structural elements from the GCN4 leucine zipper (Harbury et al. 1993. Science 262:1401) or monomers of self-assembling nanoparticles allowing attachments around a C3 axis (e.g. ferritin and lumacine synthase).
In another embodiment, the foldon domain is attached to the C-terminus of the F protein, replacing its transmembrane and cytosolic domains. The glycine residue at the N-terminus of the foldon may be attached to the F1 domain directly or via a peptide linker of a various length, which may include at least one protease site. Longer linkers allow to decouple the movement of the foldon domain, but are less potent to support keeping the helices of the HRB region (stalk) at a defined position. The helices could undergo movements with transversal displacement and bending of the trimerization helper domains into an angle off-axis of the main c3 axis of the particle. Shorter linkers allow more rigid attachment of the foldon domain with stronger fixating effect on the three helices of the stalk domain.
In particular, the foldon domain can be attached via an alanine residue inserted after the S482 of the native hMPV F protein sequence of SEQ ID NO: 1. This allows to keep S482 as C-terminal helix cap and to reproduce the local geometry of the foldon interface. Such geometry may be achieved by the foldon attachment via a short linker, for example, the short linker called “VSL” or “VSA”, consisting of the sequence ILSA (SEQ ID NO: 34) and CCSA (SEQ ID NO: 35), respectively. Alanine (A483) therein is in an analog position to the alanine at n−2 position to the tyrosine in the crystal structure PDB:1AVY (corresponding to position 2 in SEQ ID NO: 10) and shows similar contacts to the tyrosine sidechain in structure models. Additionally, the short linker “VSL” or “VSA” may be used in combination with other mutations, e.g. amino acid substitutions in the close vicinity to the linker. For example, the combination of the linker “VSA” with the substitutions C480 and C481 allows to covalently link three protomers via formation of the disulfide ring across the three protomers. In this geometry the cysteine residues of the disulfide ring are kept in spacial proximity, and therefore formation of the fully closed rings, which increases the overall stability of the protomer trimer is supported. An example for a less rigid foldon attachment retains residues 483-485 of SEQ ID NO: 1 (ILSSAE or CCSSAE with a disulfide ring). Some examples of modified foldon linkers forming more than one cysteine ring are shown below:
Other non-limiting examples of short linkers are: GG, SG, GS, GGG, GGA, GGS, SGG, SSG, SGS, SGA, GGA, SSA and SGGS. Such linkers may be used in combinations with cleavage sites, introduced by e.g. replacement of A496. The cleavage site is preferably a thrombin cleavage site, the TEV-cleavage site (Tobacco etch virus protease) or the Xa-cleavage site (Factor Xa) disclosed in Table 2.
In some embodiments, for easier purification of the recombinant protein the single-chain polypeptide may comprise any purification tag sequences known in the prior art. Examples of polypeptides that aid purification include, but are not limited to, a His-tag, a myc-tag, an S-peptide tag, a MBP tag, a GST tag, a FLAG tag, a thioredoxin tag, a GFP tag, a BCCP, a calmodulin tag, a streptavidin tag, an HSV-epitope tag, a V5-epitope tag and a CBP tag. The proteins of the present invention preferably comprise the His and/or streptavidin tags having the sequences of SEQ ID NO: 11 and SEQ ID NO: 12, respectively.
The non-limiting examples of combinations that may be applied are shown in Table 3 that may allow forming a parallel three-helix-bundle with two disulfide rings. Trimerization could occur with sequence portion containing 480-495 residues, but can be facilitated by the presence of the foldon domain. Availability of cysteine rings allows forming the disulfide bonds making covalent connection between three protomers. After that the trimerization helper function becomes obsolete and the folder could be cleaved off with the advantage that the immunogenic side-effects from a heterologous sequence (and e.g. non-hMPV) can be avoided.
In some embodiments, the recombinant hMPV F protein may comprise or consist of an amino acid sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence any of SEQ ID NOs: 5 to 9 or 24 to 28, e.g. wherein the percentage sequence identity is determined over the full length of the reference sequence. The recombinant single-chain hMPV F protein may comprise an F2 domain comprising or consisting of an amino acid sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2. The recombinant single-chain F protein may comprise an F1 domain comprising or consisting of an amino acid sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3, preferably with respect to at least residues 17-437 or 17-388 of SEQ ID NO: 3.
The present application provides isolated nucleic acid molecules encoding the recombinant hMPV proteins of the present invention. The nucleic may acid encode e.g. a polypeptide comprising for example a) a (modified) F1 domain of the hMPV F protein; b) a (modified) F2 domain of the hMPV F protein, c) a heterologous peptide linker located between F1 and F2 domains; d) a trimerization helper domain; and, optionally, e) a purification tag, wherein the F1 and F2 domains are covalently linked by at least one non-natural disulfide bond introduced between a cysteine in the linker and a cysteine in the F1 domain. The nucleic acids encoding the proteins of the present invention may comprise or consist of the sequences of SEQ ID NOs 19 to 23.
The present application also includes isolated nucleic acid molecules encoding proteins having at least 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence any of SEQ ID NOs 5 to 9 or 24 to 28. The present application also includes isolated nucleic acid molecules having at least 85%, 90%, 95%, 98% or 99% sequence identity to the sequence of SEQ ID NOs 19 to 23, e.g. wherein the percentage sequence identity is determined over the full length of the reference sequence.
The present application also provides vectors comprising the isolated nucleic acid molecules for expression of the recombinant proteins of the present invention. The present invention also provides expression systems designed to assist in expressing and providing the isolated polypeptides. The present application also provides host cells for expression of the recombinant hMPV F proteins of the present invention. The host cell may be a prokaryote. The prokaryote may be e.g. E. coli. The host cell may be an eukaryotic cell.
The recombinant single-chain hMPV F proteins of the present invention are immunogenic and can induce neutralizing antibodies recognizing the native hMPV F protein. The present disclosure also includes immunogenic fragments of the recombinant hMPV proteins and immunogenic proteins having at least 85% sequence identity to the proteins of SEQ ID NOs 5 to 9 or 24 to 28.
The present disclosure also provides immunogenic compositions or vaccines comprising the recombinant hMPV F proteins, or isolated DNA molecules encoding the hMPV F protein, or vectors of the invention, comprising an acceptable carrier and/or excipient or stabilizers known in the art (see generally Remington, 2005. The Science and Practice of Pharmacy, Lippincott, Williams and Wilkins). An immunogenic composition is any composition of material that elicits an immune response in a mammalian host when the immunogenic composition is injected or otherwise introduced. The immune response may be humoral, cellular, or both. A booster effect refers to an increased immune response to an immunogenic composition upon subsequent exposure of the mammalian host to the same immunogenic composition. A humoral response results in the production of antibodies by the mammalian host upon exposure to the immunogenic composition.
The immunogenic compositions or vaccines may further comprise an adjuvant. The adjuvant can be selected based on the method of administration and may include mineral oil-based adjuvants such as Freund's complete and incomplete adjuvant, Montanide incomplete Seppic adjuvant such as ISA, oil in water emulsion adjuvants such as the Ribi adjuvant system, oil-in-water emulsion adjuvants such as MF59® (Novartis AG) or Addavax™ (InvivoGen) (Ott G. et al. 1995. Pharm Biotechnol 6: 277-96), monophosphoryl lipid A (MPL) (Cluff C W. 2010. Adv Exp Med Biol 667:111-23), aluminum salt adjuvant (alum) (e.g., as described in WO 2013/083726), syntax adjuvant formulation containing muramyl dipeptide (MDP), polycationic polymer, especially polycationic peptide, especially polyarginine or a peptide containing at least two LysLeuLys motifs, especially KLKLLLLLKLK, immunostimulatory oligodeoxynucleotide (ODN) containing non-methylated cytosine-guanine dinucleotides (CpG), e.g. CpG 1018 (Dynavax), in a defined base context (e.g. as described in WO 96/02555) or ODNs based on inosine and cytidine (e.g. as described in WO 01/93903), or deoxynucleic acid containing deoxy-inosine and/or deoxyuridine residues (as described in WO 01/93905 and WO 02/095027), especially oligo(dIdC)13 (as described in WO 01/93903 and WO 01/93905), IC31® (Valneva SE) as described in WO 04/084938 and Olafsdottir et al. 2009 (Scand J Immunol. 69(3): 194-202), neuroactive compound, especially human growth hormone (described in WO 01/24822) and others described in Sarkar I. et al. 2019 (Expert Rev Vaccine: 18(5): 505-521), or combinations thereof, such as AF03, AS01, AS03 and AS04 described in Giudice G D et al. 2018 (Seminars in Immunology 39: 14-21). Some combinations are according to the ones e.g. described in WO 01/93905, WO 02/32451, WO 01/54720, WO 01/93903, WO 02/13857, WO 02/095027 and WO 03/047602. In one preferred embodiment, the adjuvant is aluminium hydroxide or aluminium salt that induces strong antibody and Th2-biased immune response. In another preferred embodiment, the adjuvant is an adjuvant or composition of adjuvants that induce mixed Th1/Th2 responses, such as MF59® or Addavax™ MPL/alum and IC31®.
The present disclosure also provides pharmaceutical compositions comprising the recombinant hMPV F proteins of the invention, further comprising a pharmaceutically acceptable carrier and/or excipient. The pharmaceutical composition may further comprise pharmaceutically acceptable carriers and/or excipients. The pharmaceutically acceptable carriers and/or excipients may include buffers, stabilizers, diluents, preservatives, and solubilizers (see Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition, 1975).
Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations that are administered. Carriers, excipients or stabilizers may further comprise buffers. Examples of excipients include, but are not limited to, carbohydrates (such as monosaccharide and disaccharide), sugars (such as sucrose, mannitol, and sorbitol), phosphate, citrate, antioxidants (such as ascorbic acid and methionine), preservatives (such as phenol, butanol, benzanol; alkyl parabens, catechol, octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, resorcinol, cyclohexanol, 3-pentanol, benzalkonium chloride, benzethonium chloride, and m-cresol), low molecular weight polypeptides, proteins (such as serum albumin or immunoglobulins), hydrophilic polymers amino acids, chelating agents (such as EDTA), salt-forming counter-ions, metal complexes (such as Zn-protein complexes), and non-ionic surfactants (such as TWEEN™ and polyethylene glycol).
The immunogenic compositions of the present invention elicit an immune response in a mammalian host, including humans. The immune response may be either a cellular dependent response or an antibody dependent response or both. These immunogenic compositions are useful as vaccines and may provide a protective response against the hMPV infection.
The disclosure further provides immunogenic compositions or vaccines comprising one or more additional antigen(s) derived from at least one different infectious virus, especially virus that causes a respiratory tract infection, such as hMPV, RSV (Respiratory Syncytial Virus), PIV3 (ParaInfluenza Virus type 3), influenza virus or a coronavirus (such as SARS-CoV, SARS-CoV-2, MERS or alike). Preferably, the additional antigen is the RSV F protein, PIV3 F protein, influenza hemagglutinin or coronavirus S-protein.
The immunogenic recombinant proteins, isolated DNA or RNA molecules, vectors and immunogenic compositions or vaccines disclosed herein are suitable for use as a medicament, particularly for the prophylactic and/or therapeutic treatment of viral respiratory tract infections and associated diseases, especially infections and disease caused by hMPV.
Methods of production of the recombinant hMPV F proteins or isolated nucleic acid (DNA or RNA) molecules encoding the hMPV F protein or immunogenic compositions (vaccines) are encompassed in the present disclosure. Methods of generating an immune response in a subject and methods of treating, inhibiting or preventing respiratory tract infections, especially caused by hMPV, are also included.
The invention will now be described by way of examples only with reference to the following non-limiting embodiments.
Design of the mutated hMPV F proteins in the stabilized pre-fusion conformation was done based on homology models derived from available crystal structures of the pre-fusion RSV F (PDB:4JHW and 4MMV, also 4MMU, 4MMS), pre-fusion PIV-5 F (PDB:SGIP), pre-fusion hMPV F (PDB:SWB0) and post-fusion hMPV F (PDB:5L1X) proteins, representing models of different transition phases of the fusion process. The foldon domain was adapted from the model PDB:2IBL. Model construction and structural analysis was performed by using the open-source version of PyMol structure editor package (Schrodinger LLC, https://github.com/schrodinger/pymol-open-source). Models were refined with the NAMD modeling package (Phillips et al. 2005. J Comput Chem. 26(16):1781-802) and the charmm36 forcefield (McKerell et al. 1998. J Phys Chem B. 102(18):3586-3616) or Gromacs (Berendsen et al. 1995. Comp. Phys. Comm. 91:43-56; Hess et al. 2008, J Chem Theory Comput. 4, 435-447; www.gromacs.org)/OPLS-AA (Jorgensen W L, Yale Univ.). Candidate models were typically refined in a protocol applying after in vacuo relaxation in a NVT, NPT simulation sequence for a total of 13 ns, with application of three cycles of symmetry annealing (adapted from Anishkin et al. 2010. Proteins. 78(4): 932-949) followed by free sampling and energy minimization.
The native hMPV F protein can be selected from any hMPV strain and any serotype represented by the sequences of SEQ ID NOs 1, 13 to 18, or variants thereof. In certain exemplary embodiments, the hMPV F protein derives from the strain NL/1/00, serotype A1, represented by SEQ ID NO: 1 and strain CAN97-83, serotype A2, represented by SEQ ID NO: 14.
The plasmid pVVS 1371 used for cloning contains:
The coding sequence of the wild type F protein was isolated from the hMPV strain NL/1/00, sublineage A1 and was codon-optimized for expression in CHO cells. The coding sequences of the wild type and modified F proteins were cloned into pVVS1371 plasmid for transient or stable protein expression in CHO cells.
Briefly, the coding sequences were cloned between the chimeric intron and the bGH A polyadenylation site of the pVVS1371 vector using the restriction sites SalI and PacI. The vector and the synthetized coding sequence (synthesis was done by GeneArt) were digested with SalI and PacI before purification on an agarose gel. The fragments were ligated with T4 DNA ligase and the ligation product was used to transform Max efficiency DH5a competent cells. Selected clones were tested for designed mutations by sequence analysis.
The protein expression is based on transient transfection of CHO cells using a MaxCyte® STX Scalable Transfection System device and following experimental recommendations of the supplier. Briefly, prior to electroporation, CHO cells are pelleted, suspended in MaxCyte® electroporation buffer and mixed with corresponding expression plasmid DNA. The cell-DNA mixture is transferred to a cassette processing assembly and loaded onto the MaxCyte® STX Scalable Transfection System. Cells are electroporated using the “CHO” protocol preloaded in the device and immediately transferred to culture flasks and incubated for 30 to 40 minutes at 37° C. with 8% CO2. Following the recovery period, cells are resuspended at high density in EX-CELL ACF CHO medium (Sigma Aldrich). Post-electroporation cell culture is carried out at 37° C., with 8% CO2 and orbital shaking.
The production kinetics consist of decreasing the culture temperature to 32° C. and feeding the transfected cells daily with a fed-batch medium developed for transient protein expression in CHO cells (CHO CD EfficientFeed™ A (ThermoFischer Scientific), supplemented with yeastolate, glucose and glutaMax). After about 7 to 14 days of culture, cell viability is checked and conditioned medium is harvested after cell clarification corresponding to two runs of centrifugation at maximum speed for 10 minutes. Clarified product is filtered through a 0.22 μm sterile membrane and stored at −80° C. before protein purification.
At day 7 post transfection, cells are washed once in PBS and fixed for 10 minutes in 4% paraformaldehyde at room temperature. Fixed cells are permeabilized in BD Perm wash for 15 minutes at room temperature and incubated with the primary antibody diluted in BD Perm wash for 1 hour at 4° C. Finally, a secondary antibody coupled to a fluorescent marker is added for 1 hour at 4° C. and stored in PBS at 4° C. until analysis by flow cytometry (MacsQuant Analyzer, Miltenyi Biotec). As the primary antibody the MPE8 N113S antibody (PRO-2015-026-01) specifically recognizing the pre-fusion conformation of the hMPV F protein, or the DS7 IgG1 antibody (PRO-2016-003) recognizing both pre- and post-fusion hMPV F protein have been used. The fluorescent FITC secondary antibody was goat anti-mouse IgG+IgM (JIR 115-096-068).
Frozen supernatant is brought to a room temperature and dialyzed with a standard grade regenerated cellulose dialysis membrane Spectra/Por® 1-7 CR (MWCO: 3.5 kDa) (Spectrum) against PBS. Subsequently, it is equilibrated with 50 mM Na2HPO4 buffer at pH 8.0, 300 mM NaCl and purification of the protein is performed using Immobilized Metal ion Affinity Chromatography (IMAC) followed by gel filtration chromatography.
For IMAC, agarose resin containing Ni2+ (His GraviTrap) is packed into chromatography columns by the manufacturer (GE Healthcare). The resin is washed with two volumes of deionized water and equilibrated with three volumes of equilibration and wash buffer (20 mM sodium phosphate, pH 7.4, with 0.5 M sodium chloride and 20 mM imidazole) as indicated by the manufacturer. After sample loading the column is washed with 10 mL of wash buffer. The His-tagged protein is eluted from the column using 3-10 column volumes of elution buffer as indicated by the manufacturer (50 mM sodium phosphate, pH 8.0, with 0.5 M sodium chloride and 500 mM imidazole). Eluate is then filtered on a 0.22 μm filter and dialyzed twice in Slide-A-lyzer™ Dialysis cassettes against a storage buffer (50 mM Na2HPO4, 300 mM NaCl, 5 mM EDTA, pH 8.0) before being aliquoted and stored at −20° C.
Analysis of the purity, size and aggregation of the recombinant proteins is performed by size exclusion chromatography (SE-HPLC) and SDS-PAGE SE-HPLC (Shimadzu) is run on the column SUPERDEX200 (GE Healthcare). For SDS-PASGE proteins are diluted in sample buffer (0.08 M Tris-HCl at pH 8.8, 2% SDS, 10% glycerol, 0.01% bromophenol blue), boiled for five minutes in the presence of beta-mercaptoethanol (or DTT) and electrophoretically separated on Criterion XT 4-12% Bis-Tris glycine polyacrylamide gels (BioRad) (SDS-PAGE). The gels are stained in a solution of Coomassie blue (Instant blue, Sigma Aldrich). The excess stain is removed with water and the bands are visualized using the Imager 600 (Amersham).
Determination of a conformation profile by sandwich ELISA
Medium binging plates (Greiner) are coated with the human IgG1 DS7 capture antibody (Williams et al., 2007) at 200 ng/well and incubated overnight at 4° C. The plates are saturated for 2 hours at 37° C. with PBS 0.05% Tween 20 and 5% dried-skimmed milk under agitation (saturation buffer). The liquid is removed from the wells and plates are incubated for 1 hour at 37° C. with 2.5 ng/well of the purified proteins of interest diluted in the saturation buffer. After washing, 5-fold serial dilution in saturation buffer of mouse antibody MPE8 N113S (Corti et al., 2013) directed against pre-fusion hMPV F protein or mouse antibody MF1 (Melero, personal communications) directed against post-fusion hMPV F protein are incubated for 1 hour at 37° C. Then the immune complexes are detected by incubation for one hour at 37° C. with secondary α-Ig species-specific antibody conjugated with peroxidase HRP Goat Anti-Mouse IgG (Covalab #lab0252) followed by 50 μL of peroxidase substrate (TMB, Sigma). The colorimetric reaction is stopped by adding 3 N H2504 and the absorbance of each well is measured at 490 nm with a spectrophotometer (MultiSkan).
Groups of five to ten BALB/c mice are immunized three times with three weeks interval (e.g. days 0, 14 or 21 and 28 or 42) subcutaneously with the recombinant F proteins (used in different experiments in amounts from 0.06 μg to 6.0 μg per mouse) with or without different adjuvant, particularly alum, alum+MPL, IC31 ®, Addavax™ (InvivoGen). Sera are collected by retro-orbital bleeding. One to four weeks after the last vaccination, blood is drawn and sera are prepared. Evaluation of the Th1/Th2 type immune response is performed by determining IgG1/IgG2a subtypes in the sera by indirect ELISA as described below.
The recombinant F protein is diluted in carbonate/bicarbonate buffer at pH 9.6, and 50 ng of the protein per well is added to 96-well high binding plate (50 μL/well, Greiner). The plates are incubated overnight at 4° C. The wells are saturated for 30 minutes at room temperature with 150 μL of PBS 0.05% Tween 20 and 5% dried skimmed milk (saturation buffer). The liquid is removed from the wells and plates are incubated for 1 hour at room temperature with 50 μL/well of the sera of immunized mice at different dilutions (5-fold serial dilution) in saturation buffer. After washing 3 times with PBS 0.05% Tween 20, the immune complexes are detected by incubation for one hour at room temperature with 50 μl of secondary anti-IgG1 or IgG2a mouse-specific antibody conjugated with peroxidase followed by 50 μL of peroxidase substrate (TMB, Sigma). The colorimetric reaction is stopped by adding orthophosphoric acid and the absorbance of each well is measured at 490 nm with a spectrophotometer (MultiSkan).
Briefly, the plaque reduction neutralization test (PRNT) is used to determine a serum/antibody titer of an immunized subject required to reduce the number of hMPV virus plaques by 50% (PRNT50) as compared to a control serum/antibody. The PRNT50 is carried out by using monolayers of cells that can be infected with hMPV. Sera from subjects are diluted and incubated with the live hMPV virus. Plaques formed on cell monolayers are counted and compared to the number of plaques formed by the virus in the absence of serum or a control antibody. A threshold of neutralizing antibodies of 1:10 dilution of serum in a PRNT50 is generally accepted as evidence of protection (Hombach et. al. 2005. Vaccine 23: 5205-5211).
The 24-well plate is seeded with LLC-MK2 cells, 1.2×105 cells per well, under 1 mL of EMEM (Lonza), 2 mM L-Gln (Ozyme), 5% FBS and 1% NeAA (Ozyme) buffer and incubated 24 hours at 37° C. and 5% CO2. The hMPV A1 virus (Valneva MVB 1611-009 bank) is diluted to 50 pfu/62.5 μL (per well) or 800 pfu/mL. Equal volumes of the virus and serum dilution (2×62.5 μL) are combined and incubated at 37° C. with 5% CO2 for approximately 1 hour. After plate washing with PBS, 125 μL of the virus/serum mixture is added to each well and the plate is incubated for 2 hours at 37° C. under shaking (100 rpm). Then, 225 μL/well of the overlay solution (EMEM, 2 mM Gln, 0.75% methylcellulose) is added and the plate is incubated 5 days at 37° C. with 5% CO2. For cell fixation, 225 μL/well of 4% PFA/PBS is added, incubated for 10 minute at room temperature and subsequently washed 3 times with PBS. For cell permeabilization, 0.5 ml of PBS, 0.5% Tween, 0.1% BSA is added to each well and incubated 30 minutes at 4° C. Primary antibody (human DS7-PRO-2016-003) (0.91 μg/mL) is diluted in PBS to 5 μL/mL final concentration and 150 μL of primary antibody dilution is added per well and incubated 45 minutes at 37° C. under shaking. After removing the primary antibody and washing the plate 3 times with the blocking buffer, 150 μL/well of the diluted secondary antibody—anti-human-HRP (UP783493)—are added and the plate is incubate for another 45 minutes at 37° C. under shaking. Then, the secondary antibody are removed and the plate is washed 3 times with the blocking buffer. For staining, 150 μL of DAB 1× substrate per well is added. After 1 hour incubation at room temperature cells are counted manually under the microscope.
In the experiments where all protein candidates were tested alongside, the IC50 values were highest for the L7F_A1_23 candidate adjuvanted with Addavax™ and for the L7F_A1_23 candidate adjuvanted with IC31® (see
The assay for hMPV foci quantification was developed based on the methods published in Williams et al., 2005. J Virology 79(17):10944-51; Williams et al., 2007. J Virology 81(15):8315-24; and Cox et al., 2012. J. Virology 86(22):12148-60. Briefly, confluent cultures of Vero cells or LLC-MK2 cells in 24-well plates are infected with 50 μL/well of the hMPV virus pre-incubated for 30 minutes at room temperature in the presence or absence of mouse sera diluted in the medium. The mouse IgG2a DS7 monoclonal antibody is used for detection of hMPV. After a period of two hours of virus adsorption at 37° C., 1.5% methylcellulose overlay containing EMEM medium supplemented with 2 mM L-Gln and 20 to 50 μg/mL of trypsin is added. At day 6 post-infection, the supernatant is removed and the cells are washed twice with PBS. Cell monolayers are fixed and stained with the human IgG1 DS7 antibody. Foci are counted and 50% plaque reduction titers are calculated taking into account relevant negative and positive controls. Cell images are captured with a Zeiss microscope using a 2.5× or 10× objective. Results of the immunostaining are expressed as focus forming units per milliliter, or FFU/mL.
The hMPV A 1 and A2 isolates, grown on LLC-MK2 cells, are used in animal challenge experiments. BALB/c mice are immunized three times in two weeks interval with adjuvanted recombinant F protein, as described previously, and on day 42 post-immunization they are challenged intranasally with around 1×106pfu of the hMPV. Four to five days later, the animals are sacrificed and individual serum samples are taken and frozen. Lung tissue samples are harvested, weighed and homogenized for determination of viral titer. Viral load in lung tissues is determined by virus foci immunostaining, as described above. Alternatively or additionally, RT-qPCR is used to determine viral load in the harvested tissues.
It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described embodiments. We claim all such modifications and variations that fall within the scope and spirit of the claims below. All publications described in the present application are incorporated herein by reference.
| Number | Date | Country | Kind |
|---|---|---|---|
| 19175413.4 | May 2019 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/063973 | 5/19/2020 | WO | 00 |
