Heat-stable oligomeric recombinant polypeptides, presenting at least one antigenic epitope of the pre-fusion Respiratory Syncytial Virus (RSV) F protein, comprising the RSV F protein ectodomain, functionally deleted in the HRB region, transmembrane and cytoplasmic domains replaced with a heterologous trimerization domain, and absent two functional multibasic furin cleavage sites, are useful as antigenic components in immunogenic compositions useful in methods of inducing an immune response and vaccinate against RSV infections.
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0. 14. A heat-stable oligomeric recombinant polypeptide presenting at least one antigenic epitope of the pre-fusion Respiratory Syncytial Virus F protein, said polypeptide comprising
the Respiratory Syncytial Virus F protein ectodomain, which is extended with a heterologous trimerization domain that replaces the deleted Respiratory Syncytial Virus F protein transmembrane and cytoplasmic domains; and
wherein the two multibasic furin cleavage sites in said Respiratory Syncytial Virus F protein ectodomain are mutated by the substitution of all arginine residues in said sites with lysine residues, thereby rendering said furin cleavage sites defective.
1. A heat-stable oligomeric recombinant polypeptide presenting at least one antigenic epitope of the pre-fusion Respiratory Syncytial Virus F protein, said polypeptide comprising
the Respiratory Syncytial Virus F protein ectodomain,
from which the HRB region is deleted, and
from which the transmembrane and cytoplasmic domains are deleted and replaced with which Respiratory Syncytial Virus F protein ectodomain is extended with a heterologous trimerization domain, that replaces the deleted Respiratory Syncytial Virus F protein transmembrane and cytoplasmic domains;
and
wherein the two multibasic furin cleavage sites in said Respiratory Syncytial Virus F protein ectodomain are mutated by the substitution of all lysine arginine residues in said sites with arginine lysine residues, thereby rendering said furin cleavage sites defective.
2. The heat-stable oligomeric recombinant polypeptide according to
3. The heat-stable oligomeric recombinant polypeptide according to
4. The heat-stable oligomeric recombinant polypeptide according to
5. The heat-stable oligomeric recombinant polypeptide according to
6. The heat-stable oligomeric recombinant polypeptide according to
7. The heat-stable oligomeric recombinant polypeptide according to
8. An immunogenic composition comprising the oligomeric recombinant polypeptide of
9. An immunogenic composition according to
10. An immunogenic composition according to
11. An immunogenic composition according to
12. A recombinant expression vector comprising a nucleotide sequence encoding the polypeptide forming the heat-stable oligomer of
13. A method of inducing an immune response in a subject to RSV comprising administering to said subject an immunogenic composition according to
0. 15. The heat-stable oligomeric recombinant polypeptide according to claim 14, wherein said heterologous trimerization domain is a GCN4 leucine zipper trimerization motif.
0. 16. The heat-stable oligomeric recombinant polypeptide according to claim 14, further comprising a LysM peptidoglycan binding domain linked to the carboxy-terminal end of said trimerization domain.
0. 17. The heat-stable oligomeric recombinant polypeptide according to claim 14, further comprising a triple Strep-tag.
0. 18. The heat-stable oligomeric recombinant polypeptide according to claim 14, wherein said ectodomain is a soluble ectodomain.
0. 19. The heat-stable oligomeric recombinant polypeptide according to claim 14, wherein said antigenic epitope is recognized by a pre-fusion specific monoclonal antibody AM22 or D25, or AM22 and D25.
0. 20. An immunogenic composition comprising the oligomeric recombinant polypeptide of claim 14.
0. 21. An immunogenic composition according to claim 20, further comprising an adjuvant.
0. 22. An immunogenic composition according to claim 20, in wherein said oligomeric recombinant polypeptide is bound, covalently or non-covalently, to a carrier particle.
0. 23. An immunogenic composition according to claim 22, wherein said carrier particle is a bacterium-like particle.
0. 24. A recombinant expression vector comprising a nucleotide sequence encoding the polypeptide forming the heat-stable oligomer of claim 14.
0. 25. A method of inducing an immune response in a subject to RSV comprising administering to said subject an immunogenic composition according to claim 20.
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The invention relates to the field of medicine and in particular to vaccines, and, more particularly, recombinant proteins that are useful in vaccines to immunize against Respiratory Syncytial Virus (RSV).
Human Respiratory Syncytial Virus (hRSV) causes acute upper and lower respiratory tract infections and is a major cause for hospitalization of infants in the first year of life. Re-infection with RSV occurs frequently and sterilizing immunity is never firmly established. RSV also causes a significant disease burden and mortality in the elderly, comparable to influenza.
hRSV is an enveloped negative strand RNA virus belonging to the subfamily Pneumovirinae of the family Paramyxoviridae. Other members of this subfamily are bovine RSV (bRSV) and human metapneumovirus (hMPV). The hRSV particle contains two major glycoproteins, which are the key targets of neutralizing antibodies: the attachment protein G and the fusion protein F (review by Collins P L and J A Melero. 2011. Progress in understanding and controlling respiratory syncytial virus: still crazy after all these years. Virus Res 162:80-99). There are two RSV serotypes (A and B), which differ more in their G than F proteins. The F protein appears to be a more efficient neutralizing and protective antigen compared to G. This may be related to the high carbohydrate content of the G protein, which may shield the protein from immune recognition. In addition, the G protein is also secreted from infected cells, in which form it may function as an antigen decoy. The F protein not only functions to fuse viral and host membranes, but also plays a major role in virus-cell attachment. Neutralizing antibodies targeting F may therefore interfere with virus-cell attachment and/or with virus-cell fusion.
The RSV F protein is a type I membrane protein that is synthesized as an inactive precursor protein (named ‘F0’) that assembles into trimers. This precursor protein is cleaved by furin-like proteases into the forms named ‘F2’, ‘p27’ and ‘F1’ during its transport through the secretory route. Homotrimers of F2 and F1, which are covalently linked via disulfide bridges, form the metastable pre-fusion active structure. The F1 contains heptad repeats A and B (referred to as HRA and HRB), the fusion peptide (FP) and the transmembrane (TM) domain, the latter two positioned at opposite sides of the molecule. Upon virus-cell attachment, conformational changes in the RSV F protein lead to the insertion of the hydrophobic fusion peptide into a host cell membrane. Subsequently, this fusion intermediate refolds into a highly stable post-fusion structure. The assembly of this latter structure is dictated by the assembly of a six-helix bundle (6HB). This 6HB contains HRA and HRB of each monomer in an anti-parallel conformation, as a result of which the transmembrane domain, located downstream of HRB, and the fusion peptide, located upstream of HRA, are positioned in adjacent positions and fusion of the viral and host membranes is achieved. Recent studies have elucidated the structure of the F protein in its post-fusion conformation (McLellan J S et al. 2011. J Virol 85:7788-96; Swanson K A et al. 2011. Proc Natl Acad Sci USA 108:9619-24).
hRSV vaccine development has been haunted by the disastrous results obtained with the formalin-inactivated virus vaccine that was tested in the 1960s. Disease severity and hospital admission rates were increased in vaccinated children, who were naturally infected with RSV later, and several deaths occurred. The mechanism of this vaccine-induced disease enhancement remains incompletely understood, but appears associated with low induction of neutralizing antibodies and recruitment of eosinophils. Next to this effort, a large number of RSV vaccine strategies has been explored with varying success, including live attenuated RSV strains, subunit vaccines and viral vectored vaccines (Groothuis J R et al. 2011. Prevention of serious respiratory syncytial virus-related illness. I: Disease pathogenesis and early attempts at prevention. Adv Ther 28:91-109; Hurwitz J L. 2011. Respiratory syncytial virus vaccine development. Expert Rev Vaccines 10:1415-33). Obviously, successful RSV vaccines should induce protective immunity, but no immunopathology.
Currently, the only available option to prevent RSV-mediated disease is the passive administration of the commercially available RSV neutralizing monoclonal antibody Palivizumab. This product is used as prophylaxis for RSV infection and recognizes a highly conserved epitope in the F protein (Beeler J A and K van Wyke Coelingh. 1989. Neutralization epitopes of the F glycoprotein of respiratory syncytial virus: effect of mutation upon fusion function. J Virol 63:2941-50; Groothuis J R et al. 2011. Prevention of serious respiratory syncytial virus-related illness. II: Immunoprophylaxis. Adv Ther 28:110-25). However, due to its high cost the use of Palivizumab is restricted to infants considered at high risk of developing severe respiratory disease.
Although there is a need for a vaccine for protection of the general population, there is currently no approved vaccine against RSV available. Many vaccine candidates based on the main RSV neutralizing antigen, which is the F protein, failed due to problems with stability, reproducibility and potency.
Although the post-fusion form of RSV F was shown to contain neutralizing epitopes (McLellan J S et al. 2011. Structure of respiratory syncytial virus fusion glycoprotein in the postfusion conformation reveals preservation of neutralizing epitopes. J Virol 85:7788-96; Swanson K A et al. 2011. Structural basis for immunization with postfusion respiratory syncytial virus fusion F glycoprotein (RSV F) to elicit high neutralizing antibody titers. Proc Natl Acad Sci USA 108:9619-24) Magro and coworkers showed that antibodies specific for the pre-fusion form of F account for most of the neutralizing activity found in human sera (Magro M et al. 2012. Neutralizing antibodies against the preactive form of respiratory syncytial virus fusion protein offer unique possibilities for clinical intervention. Proc Natl Acad Sci USA 109:3089-94). Furthermore, RSV neutralizing antibodies were identified that recognize F, but that do not recognize recombinant soluble ectodomains thereof that are presumably in the post-fusion conformation (WO 2008/147196; US 2012/0070446; McLellan J S et al. 2011. J Virol 85:7788-96; Swanson K A et al. 2011. Proc Natl Acad Sci USA 108:9619-24; Gonzalez-Reyes L et al. 2001. Cleavage of the human respiratory syncytial virus fusion protein at two distinct sites is required for activation of membrane fusion. Proc Natl Acad Sci USA 98:9859-64; Ruiz-Arguello M B et al. 2002. Effect of proteolytic processing at two distinct sites on shape and aggregation of an anchorless fusion protein of human respiratory syncytial virus and fate of the intervening segment. Virology 298:317-26; Ruiz-Arguello M B et al. 2004. Thermostability of the human respiratory syncytial virus fusion protein before and after activation: implications for the membrane fusion mechanism. J Gen Virol 85:3677-87), see FIG. 2. These antibodies, which presumably recognize pre-fusion F or an intermediate form between pre- and post-fusion F, and not post-fusion F, were shown to more effectively neutralize RSV than Palivizumab, which recognizes all forms of F protein.
Attempts to modify the RSV F protein for use in an immunological composition have been reported (WO2010/149743; WO2012/158613).
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The present invention relates to a heat-stable oligomeric complex of a recombinant polypeptide presenting at least one antigenic epitope of the pre-fusion Respiratory Syncytial Virus (RSV) F protein, said polypeptide comprising the RSV F protein ectodomain from which the HRB region is functionally deleted and from which the transmembrane and cytoplasmic domains are deleted and replaced with a heterologous trimerization domain, and wherein the two multibasic furin cleavage sites in said ectodomain are deleted or mutated, thereby rendering said sites defective. The invention further relates to an immunogenic composition comprising the aforesaid heat-stable oligomeric recombinant polypeptide and a method of inducing an immune response in a subject to RSV F comprising administering to said subject said immunogenic composition.
The design and evaluation of recombinant soluble F proteins of RSV has suffered considerably by a lack of tools to demonstrate the conformational status of a recombinant F protein. Based on the available literature the inventors of the present invention hypothesized that the post-fusion form of F should comprise a stable 6HB and that it should be recognized by post-fusion specific antibodies, but not by pre-fusion specific antibodies. In contrast, the pre-fusion form of the F protein should not carry the 6HB, will be recognized by pre-fusion specific antibodies, but not by post-fusion specific antibodies. Using this knowledge, it was reasoned that such pre-fusion specific antibodies would enable the identification of RSV F protein mutants that are stable (resistant to for instance heat) and remain in their pre-fusion conformation also under stressful conditions. Such stable pre-fusion mutants could in a next step be used in vaccines against RSV because they would—in their stable—conformation give an immune response (in vivo) and give rise to neutralizing antibodies that would be able to neutralize the virus carrying pre-fusion state F proteins. The present invention discloses that the inventors were indeed able to identify a recombinant RSV F protein mutant that is stably maintained in its pre-fusion conformation and is therefore useful in RSV vaccines.
The present invention may be understood with reference to the following definitions.
‘Functionally deleted’ means a deletion of a sequence of amino acids (which may be referred to as a ‘domain’) from a natural protein sequence such that the function of the deleted domain is lost, and the properties of the protein are thereby altered.
‘Heat-stable’ means that a polypeptide retains its three dimensional conformation in aqueous solution over a range of temperatures, and thereby retains its properties, including for example such polypeptide's antigenic properties. Preferred temperature ranges are from about 5 degrees C. to about 60 degrees C. A more preferred range is from about 10 degrees C. to about 80 degrees C. A most preferred range is from about 20 degrees C. to about 100 degrees C. Exemplary conformations are those characterized as a pre-fusion state.
“Oligomer” or ‘oligomeric complex’ means polypeptide that consists of two, three, or four polypeptide monomers, such as a dimeric, trimeric or tetrameric complexes of essentially the same polypeptide monomers. Most preferred oligomeric polypeptides according to the present invention are trimeric polypeptides.
‘Post-fusion conformation’ or ‘post-fusion state’ means a three-dimensional protein configuration that differs from that configuration taken by the polypeptide or polypeptide oligomer upon initial expression or oligomeric assembly, and results from the interaction of such polypeptide or oligomer from enzymatic action and/or physical contact with other proteins or proteinaceous assemblies, such as a cell membrane. The RSV F proteins that form a post-fusion state are RSV F proteins that include an HRA-HRB 6HB.
‘Pre-fusion conformation’ or ‘pre-fusion state’ means a three-dimensional protein configuration taken by the polypeptide or polypeptide oligomer upon initial expression or oligomeric assembly. RSV F proteins form RSV F protein oligomers that exhibit a pre-fusion configuration prior to fusing with the cell membrane. Pre-fusion RSV F proteins include the following characteristics: the HRA region is packed against domain III in the RSV F head region and/or the HRB region forms a trimer coil-coil stalk in proximity to domains I and II rather than associating with the HRA region in the context of the 6-helix bundle (6HB).
‘Purified’ protein or polypeptide means a protein or polypeptide isolated from other components of the polypeptide production system such that the amount of protein relative to other macromolecular components present in a composition is substantially higher than that present in a crude preparation. In general, a purified protein or polypeptide will be at least about 50% homogeneous and more preferably at least about 75%, at least about 80%, at least about 90%, at least about 95% or substantially homogeneous.
The RSV F ectodomain protein sequence is exemplified by the sequence of SEQ ID NO: 18. References to the sequence numbering and identifiable domains of the RSV F protein will herein be made to SEQ ID NO: 18. However, other strains of RSV may also be used to generate equivalent recombinant polypeptides.
The present invention relates to recombinant soluble proteins that mimic the pre-fusion state of human RSV F protein. See
Preferably, the heat-stable oligomeric polypeptide according to the present invention is stable at room temperature. More preferably, the polypeptide is stable at temperatures up to 40° C., and even more preferably, the polypeptide is stable at temperatures up to 60° C., and yet in an even more preferred embodiment, the heat-stable recombinant polypeptide of the invention is stable at temperatures up to 70° C. In a most preferred aspect, the heat-stable recombinant polypeptide according to the present invention remains stable at temperatures of about 96° C. for at least about 5 to about 15 minutes.
The heat-stable recombinant polypeptide according to the invention comprises a functional deletion of the HRB region. This is different from what has been performed in the art and as shown herein, for instance by introducing (cysteine or alanine) mutations in the HRB region (WO 2012/158613). Such mutants cannot—similar to what has been shown intra—form 6HB structures, but still result in labile conformations. Such labile conformations are prevented by removing the HRB region from the RSV F protein as disclosed herein. The polypeptide of the present invention has a functional deletion of the HRB region of the RSV F protein such that the HRB region can no longer perform its natural function, for instance in building the 6-helix bundle (6HB) rendering the protein unable to form a post-fusion conformation. The deletion of the HRB region preferably comprises the amino acids of SEQ ID NO: 10. It will be appreciated by the person skilled in the art that such deletion may be slightly smaller and/or slightly bigger on either side of the HRB region, and/or may be shifted by a small number of amino acids. Nonetheless, such deletions will still render a functional deletion of the HRB region and, in combination with the mutations of the furin cleavage sites and the addition of a heterologous trimerization motif, provide a heat-stable polypeptide as shown herein.
The furin cleavage sites of the RSV protein may be mutated by different methods known in the art, for instance by replacement of the arginine residues by any other type of amino acid, or by deletions of the crucial residues. In a preferred embodiment, the mutation of the furin cleavage sites comprises the replacement of all arginine residues with lysine residues.
The heat-stable oligomeric polypeptide of the invention comprises a heterologous trimerization domain selected from the group consisting of: GCN4 leucine zipper trimerization motif, the trimerization motif from influenza virus HA protein, SARS spike, HIV gp41, NadA, ATCase and foldon sequence. In a preferred embodiment, said heterologous trimerization domain is a GCN4 leucine zipper trimerization motif. Leucine zipper motifs such as GCN4, as well as other trimerization motifs, induce the formation of trimeric-coiled coils, in which three alpha-helices are coiled together like the strands of a rope. Such trimerization motifs have been used in the art to generate RSV vaccines based on RSV F proteins (WO 2010/149743 and WO 2012/158613).
The heat-stable oligomeric polypeptide according to the invention is preferably recognized by the pre-fusion specific monoclonal antibodies AM22 and D25, the preparation and characteristics of which are both described in WO2008/147196, the recognition indicating that pre-fusion specific antigenic epitopes are available.
The polypeptides of the present invention are suitable as antigenic component(s) of a vaccine that protects against infection and disease caused by human RSV (serotype A and B). By employing the present F protein mutation/deletion strategy to F proteins of other viruses belonging to the sub-family Pneumovirinae, immunogenic polypeptides protective against infection caused by these similar viruses, such as bovine RSV or human metapneumovirus, may be prepared.
The invention further relates to an immunogenic composition that is an effective vaccine to immunize against RSV infections. In a preferred embodiment, said immunogenic composition comprises an adjuvant to boost immunogenicity. In yet another aspect, the invention relates to a recombinant expression vector comprising a nucleotide sequence encoding the heat-stable polypeptide according to the invention.
The recombinant proteins of the present invention may also be used in diagnostics assays, with which one may measure the antibody response specifically targeted against the pre-fusion form of the F protein of RSV (or relatives thereof). This response may be of predictive value with respect to disease. Furthermore, the recombinant proteins of the present invention may be used to test the quality of the antibody response induced by a candidate vaccine, and may be used to generate conformation-specific antibodies which may be used as therapeutics, to study the epitopes present on a candidate vaccine, or to control the antigenicity of a (candidate) vaccine.
In another preferred embodiment, the invention relates to a heat-stable oligomeric complex of a recombinant polypeptide according to the invention that further comprises a LysM peptidoglycan binding domain as a tag. For easy purification and detection of the recombinant polypeptide, the polypeptide preferably comprises a triple Strep-tag. In yet another preferred aspect, the ectodomain within the polypeptide is a soluble ectodomain.
The heat-stable oligomeric complex of a recombinant polypeptide of the present invention is in a pre-fusion conformation that is antigenic, and can be confirmed by the use of antibodies that neutralize and recognize certain epitopes in the RSV F protein. In a preferred aspect, the heat-stable recombinant polypeptide according to the invention comprises an available epitope that is recognized by monoclonal antibody AM22. Whether AM22 recognizes the polypeptide can be easily checked by common methods used in the art, and as disclosed herein.
The present invention is useful in the field of medicine, and in particular in the field of vaccines against RSV infections. The heat-stable polypeptides of the present invention can be used in immunogenic compositions that may be applied in vaccination programs, in regions that present storage and handling challenges such as the third world, to protect (human) subjects who are at risk of developing disease caused by RSV.
Because the polypeptides of the present invention are stable in their pre-fusion conformation, presenting pre-fusion specific epitopes recognized by potent neutralizing antibodies, epitopes absent in polypeptides folded in the post-fusion conformation, the polypeptides are capable of inducing superior pre-fusion specific neutralizing antibodies that protect against RSV infection. The importance of prefusion-specific VN antibodies in naturally RSV infected humans has been demonstrated by Magro Metal. (2012. Neutralizing antibodies against the preactive form of respiratory syncytial virus fusion protein offer unique possibilities for clinical intervention. Proc Natl Acad Sci USA 109:3089-94). Hence, the present invention also relates to an immunogenic composition comprising the purified heat-stable oligomeric polypeptide of the present invention, and optionally further comprises excipients commonly used in vaccine preparations.
It should be understood that the present polypeptides used in the immunogenic compositions of the present invention do not include the signal peptide, which is co-expressed with the heat-stable polypeptide, but which is cleaved from the polypeptide before leaving the production cell. In a preferred aspect, although not strictly necessary, the additional (non-RSV F protein) tag sequences are removed by enzymatic digestion during or after the purification process. Preferably, an immunogenic composition according to the present invention further comprises an adjuvant to further boost the immune response.
The present invention also relates to recombinant expression vectors comprising the nucleotide sequences encoding the heat-stable polypeptides of the present invention. Moreover, the invention relates to a method of inducing an immune response in a subject to RSV F comprising administering to said subject an immunogenic composition according to the invention, and to methods of vaccinating human subjects against RSV infections by applying the immunogenic compositions as disclosed herein. The invention also relates to the use of a heat-stable polypeptide according to the invention for the manufacture of a medicament for the prophylaxis or treatments of RSV infections or diseases that follow an RSV infection. The invention further relates to heat-stable recombinant polypeptides and/or recombinant expression vectors according to the invention for use in vaccines against RSV infections.
Immunogenic compositions according to the present invention may be prepared, tested for immunogenicity, efficacy and safety employing the technology disclosed in published PCT application WO2012/128628, hereby incorporated by reference. Vaccine formulations may be based on particles derived from inactivated Lactococcus lactis bacteria, a safe bacterium traditionally used in the food industry, such as for the production of cheese (described elsewhere as Gram-positive Enhancer Matrix or Bacterium-Like Particles and herein referred to as “BLPs”). BLPs are obtained by the acidic heat treatment of L. lactis bacteria, resulting in non-living spherical particles that predominantly consist of a peptidoglycan surface, the preparation of BLPs is disclosed in WO 02/101026. The antigenic polypeptides of the present invention may be loaded onto the BLPs, which employs the non-covalent coupling technology referred to as, Protan technology, disclosed in U.S. Pat. No. 7,312,311, which is hereby incorporated by reference. The resulting antigen-associated BLPs constitute the final vaccine that may be delivered to humans via the mucosal layers of the nose (e.g. drops or spray) or mouth (e.g. capsule, tablet or liquid), without the need for an injection.
This example describes the generation of different F protein constructs and their characterization with respect to antibody binding and gel electrophoretic mobility. This analysis demonstrates that the tools and assays are suitable to demonstrate the conformational state of a RSV F protein. Also, a method is disclosed that shows the production of recombinant soluble proteins that mimic the pre-fusion state of human RSV F.
In an initial step, a comparative analysis of the virus neutralizing capacity was performed of the MAbs that were used herein (
The inventors of the present invention and others previously demonstrated that recombinant soluble class I fusion proteins can be stably maintained in their pre-fusion conformation by the addition of artificial trimerization domains (de Vries R P et al. 2010. The influenza A virus hemagglutinin glycosylation state affects receptor-binding specificity. Virology 403:17-25; Wei C J et al. 2008. Comparative efficacy of neutralizing antibodies elicited by recombinant hemagglutinin proteins from avian H5N1 influenza virus. J Virol 82:6200-8; Yang X et al. 2000. Characterization of stable, soluble trimers containing complete ectodomains of human immunodeficiency virus type 1 envelope glycoproteins. J Virol 74:5716-25; Yin H S et al. 2006. Nature 439:38-44). Use was made of a similar construct that was used previously to express recombinant soluble bioactive influenza A virus HA protein to express soluble F proteins (Cornelissen L A et al. 2010. A single immunization with soluble recombinant trimeric hemagglutinin protects chickens against highly pathogenic avian influenza virus H5N1. PLoS One 5:e10645; de Vries R P et al. 2011. Only two residues are responsible for the dramatic difference in receptor binding between swine and new pandemic H1 hemagglutinin. J Biol Chem 286:5868-75; de Vries R P et al. 2010. Virology 403:17-25).
In the expression constructs used in the present invention (overview provided in
Gene construction and cloning was performed as follows: Two variants of a cDNA clone corresponding to residues 26 to 515 of the F protein of a European isolate of RSV serotype A (Genbank accession number JX015498.1) were synthesized using human-preferred codons by GenScript USA Inc. While one cDNA clone encoded the wild type F protein ectodomain, the other clone encoded a F protein ectodomain in which the arginine residues in the two multibasic furin cleavage sites are mutated into lysines (RARR to KAKK and RKRR to KKKK). Each cDNA was cloned into the pCD5 expression vector for efficient expression in mammalian cells (de Vries R P et al. 2010. Virology 403:17-25). The pCD5 vector had been modified such that the F protein-encoding sequences were cloned in frame downstream of a DNA sequence coding for a CD5 signal peptide and when indicated upstream of sequences encoding the heterologous GCN4 isoleucine zipper trimerization motif and the specified tag. The tag either consisted of a triple Strep-tagII (IBA, Germany) or of a LysM peptidoglycan binding domain (van Roosmalen M L et al. 2006. Mucosal vaccine delivery of antigens tightly bound to an adjuvant particle made from food-grade bacteria. Methods 38:144-9; WO2012/128628) followed by a triple StreptagII. Two codon-optimized DNA fragments encoding the variable heavy and light chains of antibody AM22 (US 2012/0070446 A1) were synthesized by GenScript USA, Inc. and cloned in-frame into pCAGGS mammalian expression vectors containing human IgG1 heavy and light constant domains, respectively.
Expression of the F protein ectodomains was achieved by transient transfection as follows: pCD5 expression vectors containing RSV F ectodomain-encoding sequences were transfected into HEK293T cells using polyethyleneimine I (PEI) in a 1:5 w/w ratio (μg DNA: μg PEI). At 6 h post transfection, the transfection mixture was replaced by 293 SFMII expression medium (Invitrogen), supplemented with sodium bicarbonate (3.7 g/L), glucose (2.0 g/L), Primatone RL-UF (3.0 g/L), penicillin (100 units/ml), Streptomycin (100 μg/ml), glutaMAX (Gibco), and 1,5% dimethylsulfoxide. Tissue culture supernatants were harvested 5-6 days post transfection. F proteins were either purified using Strep-tactin Sepharose beads according to the manufacturer's instructions (IBA, Germany) for further analysis of the protein. The AM22 expression vectors were co-transfected at a 1:1 ratio into HEK293T cells similarly as described above. The cell culture media were clarified by centrifugation and the AM22 antibody was purified with protein A sepharose beads using standard conditions. The concentration of purified protein was determined by using a Nanodrop 1000 spectrophotometer (Isogen Life Sciences) according to the manufacturer's instructions.
Expression and secretion of recombinant proteins were confirmed by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE; 10% NuPAGE BisTris, Invitrogen) followed by western blotting using anti-Strep-tag antibody conjugated with horse radish peroxidase (HRP) (StrepMAB-classic-HRP, IBA), Palivizumab (Synagis®, Abbott Laboratories) followed by HRP-conjugated anti-human IgG antibody (ITK Southern Biotech). This latter antibody was also used to confirm expression of recombinant antibody AM22. Prior to SDS-PAGE analysis, the samples were resuspended in Laemmli sample buffer (LSB) that either did or did not contain 5% 2-mercaptoethanol (Sigma), and when indicated heated at 96° C. for 5-15 minutes.
The results are given in
In contrast, while the Fwt and Fwt-GCN proteins clearly ran at a lower position in the gel than the Flys and Flys-GCN under reducing conditions, the difference in the electrophoretic mobility appeared much smaller in the absence of reducing agents, in agreement with the F2 part still being attached to the F1 part via disulfide bridges also in the furin-cleaved proteins. The small difference in electrophoretic mobility between the cleaved and non-cleaved F proteins that was still noticeable is most likely explained by the dissociation of the glycosylated p27 sequence from the cleaved proteins. Interestingly, the electrophoretic mobility of the cleaved F proteins was dramatically changed when the preparations were not heated prior to electrophoresis under non-reducing conditions. In contrast to the non-cleaved proteins (Flys and Flys-GCN, the electrophoretic mobility of which was not much affected) the majority of the Fwt and Fwt-GCN proteins migrated at a much higher position in the gel. The migration of these latter proteins is explained by the cleaved F proteins adopting a stable post-fusion conformation, characterized by the presence of an extremely stable 6HB, resistant to SDS unless the protein preparations are heated. These results indicate that the large majority of the soluble, cleaved F ectodomains adopts a post-fusion conformation. The post-fusion conformation is not prevented when the ectodomain is extended with an artificial trimerization domain. However, the stable post-fusion conformation is not formed when the F proteins are not cleaved.
To confirm and extend these observations, a subsequent experiment was performed in which the purified F proteins were subjected to limiting proteolysis followed by SDS-PAGE under non-reducing conditions. Despite the fact that the furin-cleavage sites in Flys and Flys-GCN had been mutated by substitution of the arginines by lysines, these positions are still sensitive to trypsin digestion. Treatment of the Flys and Flys-GCN proteins with trypsin will thus result in cleavage of these proteins and possibly in formation of the SDS-resistant higher-order structure corresponding to the post-fusion conformation of the F protein.
Purified F proteins were (mock-) treated with varying amounts of TPCK treated trypsin from bovine pancreas (Sigma) for 30 min at 23° C. The samples were next put on ice and trypsin inhibitor (Sigma) was added, after which they were analyzed by SDS-PAGE as described above. Protein bands were visualized by general staining using the Colloidal Blue Staining kit (Invitrogen). The results are shown in
The reactivity of the F protein preparations with the RSV F specific MAbs Palivizumab (Synagis®), AM22 and 131-2a were probed using an ELISA format. For this, 96-well Nunc maxisorp plates were overnight coated with different F protein preparations (50 ng per well) at 4° C. After blocking and extensive washing, the plates were incubated with limiting dilutions of Palivizumab (Synagis®, starting with 1 in 500 dilution of a 3 mg/ml stock), AM22 (starting with a 200 fold dilution of a 0.7 mg/ml stock), 131-2a (Millipore, starting with a 500 fold dilution of a 1 mg/ml stock), or anti-strep (StrepMAb classic from IBA, starting with a 500 fold dilution of the stock). After extensive washing, the plates were incubated with HRP conjugated goat-anti-human IgG antibodies (ITK Southern Biotech) or HRP conjugated rabbit-antimouse IgG antibodies (DAKO) at a 1:500 dilution for 1 h at RT. Detection of HRP reactivity was performed using tetramethylbenzidine substrate (BioFX) and a ELISA plate reader (EL-808 from Biotek). The results are given in
All F proteins were coated with similar efficiencies as demonstrated by the binding of MAb specific for the Strep tag (Anti-Strep panel). Palivizumab displayed a concentration-dependent binding to all F protein preparations in agreement with the assumption that this antibody recognizes the F protein regardless of its conformational state. In contrast, AM22 was not able to bind Fwt, in agreement with the assumption that this antibody is not able to bind a protein in post-fusion conformation. However, intermediate binding was observed when the cleaved protein was extended with the trimerization motif (Fwt-GCN) or when cleavage was prevented (Flys). The highest reactivity was observed when these two features were combined (Flys-GCN). Trypsin treatment of Flys and Flys-GCN prior to coating of the wells resulted in reduced AM22 reactivity, which subsequently was comparable to the reactivity observed with Fwt and Fwt-GCN. Reactivity of Palivizumab with the F proteins was not affected by the trypsin treatment. MAb 131-2a efficiently bound to all F protein preparations.
These results show that binding of neutralizing antibody AM22 differs between different F protein preparations: Fwt, which adopts the post-fusion conformation, is hardly detected, while the highest reactivity was observed for Flys-GCN. From these results it was concluded that the majority of Fwt is in the post-fusion conformation (6HB+, 131-2a+, AM22−). Fwt-GCN is probably present in a mixture of conformations (6HB+, 131-2a+, AM22+/−). In the absence of cleavage, the F proteins do not adopt the post-fusion conformation (no 6HB). Their reactivity with both 131-2a and AM22 indicates that these non-cleaved proteins are in some form of intermediate state.
Next, the characteristics of F proteins, in which 4 cysteine residues were introduced into their HRB domain (designated Fwt-cys; Fwt-cys-GCN; Flys-cys; Flys-cys-GCN;
To confirm and extend these observations, a subsequent experiment was performed in which the purified F proteins were subjected to limiting proteolysis followed by SDS-PAGE under non-reducing conditions. The results are shown in
The reactivity of the cysteine mutant F proteins preparation was investigated with the RSV F specific MAbs Palivizumab, AM22 and 131-2a using the ELISA format (number 1-4 in
To study the effect of mutations in the HRB domain in more detail, mutant F proteins were produced in which the same amino acids as outlined in example 2 (see also Magro M et al. 2012. Proc Natl Acad Sci USA 109:3089-94) were substituted by alanines rather than cysteines (
The disclosed data show that a set of assays was developed with which the conformation of recombinant soluble F proteins can be determined. With these assays four conformational states of the F protein can be discriminated that are schematically shown in
1) Pre-fusion F (6HB−, 131-2a−, AM22+), see
2) Intermediate 1 (6HB−, 131-2a+, AM22+), see
3) Intermediate 2 (6HB−, 131-2a+, AM22−), see
4) Post-fusion F (6HB+, 131-2a+, AM22−), see
In the examples above it is demonstrated that F proteins in the pre-fusion conformation are antigenically different from F proteins that have other conformations. The immune response specifically targeted against pre-fusion F epitopes, may be hampered by the presence of non-pre-fusion specific epitopes, the corresponding antibodies of which do not neutralize the virus (e.g. 131-2a). Although Fwt-cys-GCN and Flys-cys-GCN are in the pre-fusion conformation, these proteins are not considered suitable for the production of pre-fusion F proteins at a larger scale, primarily because these proteins are expressed at very low levels. To solve this, the inventors set out to device an alternative strategy to express high levels of recombinant F proteins that contain pre-fusion specific epitopes and lack post-fusion specific epitopes. The data show that GCN4 is able to confer the pre-fusion state onto recombinant proteins, but this only efficiently occurs when the HRB stem in the pre-fusion state is stabilized (e.g. in Fwt-cys-GCN and Flys-cys-GCN). This instability may even be increased by other mutations in HRB (such as the alanine mutations described herein).
The inventors hypothesized that there might be some form of inherent instability in HRB, which makes it possible for HRB to dissociate and subsequently to interact with the extended HRA. Rather than to try to stabilize HRB via the introduction of intermolecular disulfide bonds, which severely reduces protein expression levels, it was decided to remove the HRB region (and its inherent instability) altogether. However, in the complete absence of HRB it was expected that epitopes that are normally not available in the pre-fusion state would be exposed, either directly by removal of HRB (e.g. epitope of 131-2a) or because the resulting stem-less protein may behave as a monomer rather than a trimer. Furthermore, the data indicate that in the absence of cleavage the AM22 epitope and probably other pre-fusion specific epitopes are better maintained in the recombinant F protein (compare Flys-cys-GCN and Fwt-cys-GCN). In view of these considerations, a recombinant protein was generated, in which HRB was removed, the GCN4 trimerization motif was added and the furin cleavage site was mutated (
These recombinant proteins were analyzed by SDS-PAGE using reducing and non-reducing conditions (
Next, cleavage of these recombinant proteins was induced by trypsin treatment to study the effect of protein digestion on the protein conformation. As a control Flys-GCN was taken and proteins were run on non-reducing gels. The results are shown in
The reactivity of the Flys-ΔHRB and Flys-ΔHRB-GCN proteins with the monoclonal antibodies was tested in an ELISA as outlined above (
Haijema, Bert Jan, Rottier, Petrus Josephus Marie, de Haan, Cornelis Alexander Maria
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