VACCINE

专利摘要:
The present disclosure relates to immunogenic compositions comprising human picornavirus peptides derived from structural proteins of the virus, constructs comprising the peptides, the peptides themselves and their use in the prevention of picornavirus infection and disease. Specific VP4 and VP1 peptides are described.
公开号:BE1022174B1
申请号:E2014/0161
申请日:2014-03-13
公开日:2016-02-24
发明作者:Guy Baudoux；Mathieu Boxus；Brigitte COLAU；Martine Marchand
申请人:Glaxosmithkline Biologicals S.A.；
IPC主号:

专利说明:

VACCINE
Background of the invention
The present disclosure relates to the field of human vaccines. More particularly, the present disclosure relates to pharmaceutical and immunogenic compositions for the prevention or treatment of human picornavirus infection or disease, particularly human rhinovirus infection (HRV).
Picornaviridae are one of the largest viral families, consisting of 14 genera, six of which include human pathogens. The well-known picornaviruses are enteroviruses (including polio and rhinovirus), foot-and-mouth disease virus (FMDV) and hepatitis A virus (HAV). Other members of the picornavirid family are coxsackievirus, echovirus, human parechovirus and Aichi virus. Picornaviridae cause diseases such as the common cold, gastroenteritis, hepatitis, pneumonia, poliomyelitis, meningitis, foot-and-mouth disease. Although infections are often benign, some strains can cause pandemic outbreaks with meningitis and / or paralysis.
Rhinoviruses are the leading cause of acute upper respiratory tract infections in humans, known as the common cold. They are also the most common viral cause of severe exacerbations of chronic respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD). Currently, there are more than 100 serotypes of HRV. There is little or no cross-protection between these serotypes because of the existence of specific immunodominant neutralizing epitopes and no vaccine has been developed to date. A vaccine against rhinovirus that should be able to protect against multiple serotypes therefore represents a large medical need that remains to be filled.
Short summary
The present disclosure relates to vaccines against human picornaviruses which contain antigens conferring protection against different picornaviruses, either from different serotypes or strains of the same picornavirus, or from different members of the picornavirus family. Specific embodiments relate to vaccines against human enteroviruses, particularly rhinoviruses, containing antigens that confer protection against different enterovirus or HRV serotypes. The vaccines contain picornavirus peptides from conserved regions of picornavirus structural proteins, which generate a cross-reaction or cross-neutralization response to confer cross-protection against a series of picornaviruses, for example against a series of HRVs of different serotypes. . The invention provides an immunogenic composition comprising a first and a second peptide derived from a structural protein of a picornavirus, said peptides each being capable of inducing an immune response by cross neutralization against two or more picornaviruses, and a diluent, excipient or pharmaceutically acceptable carrier.
Certain new picornavirus and rhinovirus peptides of VP4 and VP1 are also provided here.
In another aspect, the invention provides a picornavirus peptide consisting of at most 20 amino acids from the N-terminus of VP4, said peptide comprising amino acids 1 to 16 of VP4 or a variant of amino acids 1 to 16 having 1 to 4 additions or deletions of amino acids at either end.
In another aspect, the invention provides a picornavirus peptide consisting of at most 40 amino acids from the N terminal region of VP1, said peptide comprising amino acids 32 to 45 or a variant of amino acids 32 to 45 having 1 to 4 additions or deletions of amino acids at either end.
In another aspect, the invention provides a chimeric polypeptide particle comprising a backbone polypeptide capable of forming a particle and at least one peptide comprising an epitope of a picornavirus structural polypeptide.
In another aspect, the invention provides an immunogenic composition comprising a chimeric polypeptide peptide or particle of the invention, together with a pharmaceutically acceptable diluent, excipient or carrier.
In another aspect, the invention provides the use of an immunogenic composition herein described in the prevention or treatment of picornavirus infection such as HRV infection. The invention further provides the use of an immunogenic composition described herein for the manufacture of a medicament for the prevention or treatment of picornavirus infection such as HRV infection.
In another aspect, the invention provides a method for inducing neutralizing antibodies against a picornavirus such as HRV in humans, comprising administering to a human an immunogenic composition as described herein.
In another aspect, the invention provides a method of inducing antibodies resulting in cross-neutralization against picornaviruses such as HRV in humans, comprising administering to a human an immunogenic composition described therein .
In another aspect, the invention provides a method of preventing picornavirus infection or picornavirus disease associated with picornavirus infection, such as HRV infection or HRV disease associated with HRV, said method comprising administering to a human an immunogenic composition as described herein.
In another aspect, the invention provides a method for preparing an immunogenic composition, said method comprising combining (i) two or more picornavirus peptides of picornavirus structural proteins, said peptides being respectively capable of inducing a response. cross-neutralizing immunity against two or more picornaviruses or picornavirus serotypes, and (ii) a pharmaceutically acceptable diluent, excipient or vehicle.
In another aspect, the invention provides a method for preparing an immunogenic composition, said method comprising combining (i) a chimeric polypeptide particle comprising one or more picornavirus peptides derived from picornavirus structural proteins; and (ii) a pharmaceutically acceptable diluent, excipient or carrier.
Brief description of the drawings
Figure 1 shows a schematic diagram of the picornavirus genome.
Figure 2 shows peptide-specific antibodies generated in rabbits immunized with KLH-conjugated VP1 peptides.
Figure 3 shows peptide-specific antibodies generated in rabbits immunized with VP4 peptides, KLH-conjugated, or chimeric constructs of hepatitis B surface antigen (HBsAg).
Figure 4 shows peptide-specific antibodies generated in rabbits immunized with full-length VP4 in the form of a concatamer.
Figure 5 shows neutralizing antibodies against various layers of HRV, induced in rabbits immunized with KLH-conjugated VP1 peptides.
Figure 6 shows neutralizing antibodies against various strains of HRV, induced in rabbits immunized with KLH-conjugated VP4 peptides or in a chimeric construct with HBsAg or with full length VP4 concatamers.
Figure 7 shows antibodies specific for VP4 region 1 to 16 in rabbits immunized with full-length VP4 1-31 or VP4, relative to VP4 1-16.
Figure 8 shows an alignment of amino acids 32 to 45 of VP1 of different HRV serotypes of clade A aligned on HRV14.
Figure 9 shows an alignment of amino acids 32 to 45 of VP1 of different HRV clade B serotypes aligned on HRV14.
Figure 10 shows an alignment of amino acids 32 to 45 of VP1 of different CLV serovar HRV serotypes aligned on HRV14.
Figure 11 shows an alignment of amino acids 1 to 16 of VP4 of different HRV serotypes of clade A aligned on HRV14.
Figure 12 shows an alignment of amino acids 1 to 16 of VP4 of different cloned B HRV serotypes aligned on HRV14.
Figure 13 shows an alignment of amino acids 1 to 16 of VP4 of different clad C HRV serotypes aligned on HRV14.
Figure 14 shows an alignment of the N-terminal residues of the VP1 proteins of some picornaviruses. The peptides similar to HRV14 32-45 are labeled in the box.
Figure 15 shows an alignment of the VP4 proteins of the selected picornaviruses. The peptides are similar to the HRV14 VP4 1-16 peptide labeled in the box.
Figures 16 to 24 show the nucleotide and amino acid sequences and plasmids for the VP4-S chimeric polypeptide constructed in Example 2 below.
Detailed Description Introduction 1
The present disclosure relates to compositions and methods for the prevention and treatment of picornavirus infection, particularly to picornavirus of the enterovirus genus, more particularly a human enterovirus such as human rhinovirus (HRV).
Rhinoviruses are non-enveloped viruses and are composed of a capsid consisting of four viral proteins VP1, VP2, VP3 and VP4. VP1, VP2 and VP3 constitute the major part of the capsid of the protein. The much smaller VP4 protein, about 70 amino acids in length, has a more extensive structure and is at the interface between the capsid and the genomic RNA. The capsid consists of 60 copies of each of these proteins assembled into an icosahedron.
The rhinovirus genome consists of straight-chain, single-stranded linear RNA between 7.2 and 8.5 kb in length. The structural proteins are encoded in the 5 'region of the genome (from the 5' end: VP4, VP2, VP3 and VP1) and non-structural at the 3 'end, as is the case for all picornaviruses. . RNA is translated into a single polyprotein that is co-translational and post-translational cleaved into four structural proteins and seven nonstructural proteins. Nonstructural genes are involved in the treatment of the viral genome, viral replication and the cessation of protein production in the host cell.
Currently, there are more than 100 serotypes of HRV. Based on the nucleotide identity and sensitivity of antiviral compounds, HRVs were
classified in clades A, B, C and possibly D (Rollinger & Schmidtke, 2011, Palmenberg, Rathe & Liggett, 2010), see Table 1.
Table 1
In addition, receptor specificity of the host cell was used to further classify these viruses into major and minor groups. Serotypes that use the intercellular adhesion molecule 1 (ICAM-1) receptor (HRV-A serotypes 62 and all B serotypes) are part of the major receptor group and the remaining 12 HRV-A serotypes use members of the the receiver family of
low density lipoproteins (LDL) and are part of the minor receptor group. Therefore, the terms "major HRV-A", "minor HRV-A" and "major HRV-B" are used.
Serotypes are further classified according to the antigenic sites they use to escape the host's immune system. For the major receptor group, four primary neutralizing immunogenic (NIm) sites were assigned to prominent regions on the outer capsid proteins VP1, VP2 and VP3. They are known as NIm-IA, NIm-IB, NIm-II and NIm-III. For the minor receptor serotypes, there are three distinct antigenic sites A, B and C which are located in the same vicinity as the NIm sites (described by Lewis-Rogers et al 2009). Antibodies induced with recombinant HRV-14 or 89 VP1 proteins or a peptide spanning amino acids 147 to 162 of HRV14 VP1 have been shown to exhibit specific cross-neutralizing activity (McCray & Werner, 1998; Edlmayr et al. , 2011). It has been observed that the rhinovirus capsid structure is dynamic and found to oscillate between two different structural states: one in which VP4 is deeply buried and the other in which the N terminations of VP4 and VP1 are accessible to proteases (Lewis et al 1998). Antibodies directed against the 30 N-terminal amino acids of VP4 but not of VP1 have been shown to successfully neutralize viral infectivity in vitro (Katpally et al 2009). Antibodies to the N-terminal VP4 amino acids were found to neutralize HRV14, HRV16 and HRV29. In addition, antibodies directed against a consensus sequence of the first 24 residues of rhinovirus VP4 also have cross-neutralizing activity (Katpally et al, 2009).
Other occurrences of peptides and / or rhinovirus epitopes can be found in the literature, in Niespodziana et al 2012 for which a response against an N-terminal 20-mer of VP1 was not a neutralizing response, that is, that is, a non-protective epitope; Miao et al 2009 - MAbs generated against the N-terminal part of the highly conserved VP1 enterovirus are useful for recognizing a wide range of enteroviruses; WO 2006/078648 for peptide vaccines against HRV derived from transiently exposed VP4 regions, particularly amino acids 1-31 or 1-24 of VP4; WO 2011/050384 relating to peptides of the N-terminal region of VP1 comprising amino acids 1-8; WO 2008/057158 relating to rhinovirus Nim IV, in particular a peptide comprising amino acids 277-283 or 275-285 of the carboxy-terminal region of VP1, in particular HRV-14.
The availability of a vaccine against HRV is a particular challenge because of the large number of serotypes of the virus and the lack of a protective response generated in individuals infected with one serotype against infection by another serotype. An important aspect of an HRV vaccine that provides protection against a sufficient number of HRV serotypes to confer effective protection against HRV infection is the provision of epitopes of more than one HRV structural protein, by example, VP4 and VPl. Another important aspect is the provision of peptides that are conserved among HRV serotypes. Another important aspect is the provision of peptides that generate a neutralizing antibody response. In this document are proposed HRV peptides and combinations of HRV peptides of different structural proteins of HRV and constructs containing peptides and peptide combinations. By providing peptides that are conserved among HRV serotypes, the inventors have also discovered peptides that are remarkably preserved among picornaviruses in general.
Accordingly, the present disclosure relates to picornavirus structural protein peptides that are selected to be capable of inducing a cross-neutralizing immune response against different picornaviruses that may be different picornaviruses or different serotypes of the same picornavirus, for example, different serotypes of rhinoviruses. These peptides can be distributed in many ways including peptides coupled or conjugated to vector proteins such as CRM197 or in a chimeric construct, to a polypeptide in which the peptide or peptides are inserted, for example a polypeptide that forms a particle such as than a viral-like particle or a subviral particle.
In one embodiment, a combination of picornavirus peptides is provided, which comprises first and second peptides of different picornavirus structural proteins. For example, the first and second peptides may be VP4 and VP1 picornaviruses. Advantageously, the peptides are short peptides of at most 20 amino acids, although they may be longer than that. In one embodiment, the peptides are derived from the N-terminal region of the structural proteins.
In one embodiment, the first and second peptides are derived from a human enterovirus and the enterovirus peptides are capable of inducing a cross-neutralizing immune response against two or more enteroviruses. In a particular embodiment, one or both of the first and second peptides are derived from a human rhinovirus and the rhinovirus peptides are capable of inducing a cross-neutralizing immune response against two or more rhinovirus serotypes. that is, against the rhinovirus serotype from which the peptide is derived and at least one other rhinovirus serotype.
In one embodiment, the first peptide comprises amino acids 32 to 45 of VP1 or a variant of amino acids 32 to 45 of VP1 having 1 to 4 amino acid deletions or deletions at either end and or 1 to 2 substitutions or additions or deletions of amino acids or in the peptide sequence.
In a particular embodiment, the VP1 peptide is a human rhinovirus peptide and, in particular, comprises the peptide having a sequence selected from: HRV14 (B): 32-PILTANETGATMPV-45 [SEQ ID NO: 1] HRV8 ( AM): 32-PALDAAETGHTSSV-45 [SEQ ID NO: 2] HRV25 (Am): 32-PILDAAETGHTSNV-45 [SEQ ID NO: 3] HRV_C_ ° 26: 32-QALGAVEIGATADV-45 [SEQ ID: 4] or a variant thereof having 1 to 4 additions or deletions of amino acids at either end and / or 1 to 2 amino acid substitutions or additions or deletions in the peptide sequence.
In one embodiment, the second peptide has amino acids 1 to 16 of VP4 or a variant of amino acids 1 to 16 of VP4 having 1 to 4 amino acid additions or deletions at either end and or 1 to 2 substitutions or additions or deletions of amino acids in the peptide sequence.
In a particular embodiment, the VP4 peptide is a human rhinovirus peptide and, in particular, comprises the peptide having a sequence selected from: HRV14 (B): 1-GAQVSTQKSGSHENQN-16 [SEQ ID NO: 5] HRV100 ( AM): 1-GAQVSRQNVGTHSTQN-16 [SEQ ID NO: 6] HRV_C_02 6: l-GAQVSRQSVGSHETMI-16 [SEQ ID NO: 7] or a variant thereof having 1 to 4 amino acid additions or deletions at either end and / or 1 to 2 substitutions or additions or deletions of amino acids in the peptide sequence. The invention also provides individual picornavirus peptides, for example, rhinovirus peptides having the sequences set forth in SEQ ID Nos. 1 to 7 and variants thereof as described herein.
When a variant of a peptide sequence has 1 to 4 amino acid additions or deletions at either end and / or 1 to 2 amino acid substitutions or additions or deletions in the peptide sequence , it means that the variant has at least one amino acid difference with respect to the reference peptide sequence, which can comprise between 0 and 4 additions or deletions of amino acids on one end and between 0 and 4 additions or deletions on the other end and between 0 and 2 substitutions or additions or deletions of amino acids in the sequence.
In one embodiment, a picornavirus peptide provided in this document consists of at most 20 amino acids of the N-terminus of VP4, said peptide comprising amino acids 1 to 16 of VP4 or a variant of amino acids 1 to 16 having 1 with 4 additions or deletions of amino acids at either end and / or 1 to 2 amino acid substitutions or additions or deletions in the peptide sequence. In a particular embodiment, the VP4 peptide consists of amino acids 1 to 16 of VP4 or a variant having one or two or three or four additions or deletions or substitutions of amino acids. Other specific VP4 peptides include, for example, amino acids 1 to [16-20], amino acids 2 to [17-21], 3 to [18-22], 4 to [19-23], at [2024], where it will be understood that the numbers in square brackets include all the numbers individually in the indicated range. Advantageously, the VP4 peptide consists of 16 contiguous amino acids of at most VP4. It will be appreciated that the numbering of the VP4 peptide as used herein is methionine independent of the starting codon.
In another embodiment, a picornavirus peptide consists of at most 40 amino acids of the N-terminal region of VP1, said peptide comprising amino acids 32 to 45 of VP1 or a variant of amino acids 32 to 45 having 1 to 4 additions or deletions of amino acids at either end and / or 1 to 2 substitutions or additions or deletions of amino acids in the peptide sequence. In a particular embodiment, the VP1 peptide consists of amino acids 32 to 45 of VP4 or a variant having one or two or three or four additions or deletions or substitutions of amino acids. The VP1 peptides include, for example, amino acids [5-35] to 45, [6-35] to 46, [7-35] to 47, [8-35] to 48, [9-35] to 49 and similarly 32 to [45-72], 33 to [45-73], 34 to [45-74], 35 to [45-75] and 36 to [45-76], where the numbers in brackets include all numbers in the specified range individually. These peptides may be combined in an immunogenic composition described herein. These picornavirus peptides in general, or viruses of the enterovirus and rhinovirus genus in particular, are a feature of the present invention, individually and in combination as first and second peptides.
In one embodiment, the picornavirus peptide or peptides are coupled to a carrier protein such as CRM197. Suitable carrier proteins include CRM197, non-typeable Haemophilus influenza derivative D, PhtD, PhtDE, adenylatecyclase, tetanus toxoid (TT), tetanus toxoid fragment C, nontoxic mutants of toxoid tetanus, diphtheria toxoid (DT), pneumolysin (Ply), exotoxin A (ExoA) and nanoparticles such as synthetic nanoparticles. Other suitable carrier proteins include picornavirus proteins, e.g. nonstructural HRV proteins such as viral protease, polymerase and other proteins involved in replication of picornavirus or other viruses. Advantageously, the carrier protein is a non-structural picornavirus protein such as HRV, conferring a further benefit of an immune response against the nonstructural protein. The first and second peptides in the immunogenic composition described herein may be coupled to the same or different carrier proteins that may be selected from the above list. When coupled to the same carrier protein, the peptides can be coupled separately to the same carrier protein and then the coupled peptides can be combined or the peptides can be first mixed together and then coupled to the carrier protein.
In another embodiment, the peptide or peptides are combined with or inserted into a polypeptide to provide a chimeric polypeptide construct. In this embodiment, the immunogenic composition comprises at least one chimeric polypeptide construct comprising a backbone polypeptide and a peptide or peptides. If two or more peptides are present, these may be in the same chimeric polypeptide construct or in separate chimeric polypeptide constructs that may have the same or different backbone polypeptide backbone. Advantageously, the chimeric polypeptide construct forms a particle such as a virus-like particle. The backbone polypeptide may be any suitable polypeptide, such as structural or nonstructural polypeptides of viruses such as human papillomavirus (HPV), rhinovirus, hepatitis B virus, EV-71, influenza virus or the like. a norovirus.
In some embodiments, the peptides are present on an exposed region of the particle by being inserted into an appropriate region of the backbone polypeptide, for example a surface-exposed loop, for example in the "a" loop of the antigen of the Hepatitis B surface (HBsAg) or the N-terminal or C-terminal region of HBsAg comprising one of the termini. In some embodiments, two of the same or different HRV peptides are inserted into different sites in a single polypeptide such as the "a" loop and the N-terminal or C-terminal region of the HBsAg polypeptide, thereby giving a HBsAg chimeric polypeptide with double peptide insertion. In a particular embodiment, a VP1 peptide as described herein, such as a VP1 32-45 peptide or a variant thereof, is inserted into the "a" loop of HBsAg and a VP4 peptide as described in this document, such as a VP4 1-16 peptide or a variant thereof, is inserted into the N-terminal region of the same HBsAg polypeptide or vice versa. In another aspect of the disclosure there is provided a chimeric polypeptide particle comprising a backbone polypeptide capable of forming a particle and at least one peptide comprising an epitope of a picornavirus structural polypeptide. The backbone polypeptide can be, for example, HBsAg, HPV L1 or a rhinovirus structural protein or any other viral particle, advantageously one that is capable of forming a particle such as a VLP.
In a particular embodiment, the particle is a chimeric HBsAg comprising an HBsAg polypeptide or a fragment thereof, into which are inserted one or more VP4 picornaviruses or VP1 peptides as described herein. In one embodiment, chimeric HBsAg comprises two or more picornavirus structural protein peptides that may be the same or different. Advantageously, the peptides are each capable of inducing a cross-neutralizing immune response against two or more different picornaviruses, for example two or more rhinovirus serotypes. Advantageously, the chimeric chimeric HBsAg polypeptide forms a virus-like particle. In one embodiment, the chimeric HBsAg particle is proposed in which a VP4 peptide as described in this document, advantageously a VP4 peptide which contains an epitope of a picornavirus capable of inducing a cross-neutralization immune response, for example a VP4 peptide comprising VP4 1-16, for example, VP4 1-31 or VP1-24 or VP1-16, is fused to HBsAg at the N-terminus or in the "a" loop of HBsAg. In another embodiment there is provided a chimeric HBsAg particle in which a VP1 peptide as described herein, advantageously, a VP4 peptide which contains an epitope of a picornavirus capable of inducing a cross-neutralizing immune response, e.g. VP1 peptide comprising VP1 32-45 is fused to HBsAg at the N-terminus or in the "a" loop of HBsAg. In another embodiment, a double peptide chimera is proposed in which both the VP1 and VP4 peptides as described herein are inserted into HBsAg, one at the N-terminus and the other in the loop "a". In one embodiment, the VP1 and VP4 peptides are from a rhinovirus.
The immunogenic compositions provided herein may further comprise an adjuvant which may be, for example, a mineral salt such as an aluminum salt, for example, aluminum hydroxide. In another embodiment, the adjuvant comprises 3-deacylated monophosphoryl lipid A (3D-MPL). In another embodiment, the adjuvant comprises QS21.
Another aspect of the present disclosure relates to nucleic acid molecules that encode a chimeric peptide or polypeptide as described above. These nucleic acids may be present in a prokaryotic or eukaryotic expression vector. Suitable expression vectors include, for example, a yeast such as Pichia pastoris. Recombinant nucleic acids, for example, expression vectors, can be introduced (e.g., by infection, transfection, or transformation) into host cells. These host cells are also one aspect of this disclosure. These host cells can be used to produce the chimeric polypeptides, for example by replicating the host cell under conditions suitable for the expression of the recombinant polypeptide. Optionally, the polypeptide may then be isolated and / or purified, for example, prior to formulation into an immunogenic composition. Any of the chimeric peptides or polypeptides described herein may be used in medicine, for example, as immunogenic compositions (such as vaccines) for the prevention or treatment of picornavirus infection such as RVH. These compositions are suitable for use in methods of inducing antibodies against a picornavirus such as HRV in humans by administering the immunogenic composition to a human subject. Advantageously, the administration of the immunogenic composition to the human subject induces the development of antibodies which prevent, ameliorate or treat picornavirus infection or disease, for example, an infection or disease at HRV.
Therefore, the present disclosure also provides immunogenic compositions for use in the prevention, amelioration or treatment of a picornavirus infection or disease. These immunogenic compositions comprise a chimeric polypeptide comprising one or more picornavirus peptides as described herein, said chimeric polypeptide being in the form of a VLP particle as described above, in combination with an excipient, diluent or pharmaceutically acceptable vehicle. In some embodiments, the immunogenic composition also includes an adjuvant. Suitable adjuvants include an aluminum salt such as aluminum hydroxide, 3D-MPL and QS21. Suitable combinations of adjuvants include aluminum hydroxide and 3D-MPL; and 3D-MPL and QS21 optionally prepared with liposomes. Definitions
In order to facilitate the discussion of the various embodiments of the present disclosure, the following explanations of the terms are provided. Additional terms and explanations may be proposed in the context of this disclosure.
Unless otherwise indicated, all technical and scientific terms used in this document have the same meaning as commonly accepted by those skilled in the art to which the present description is intended. Definitions of common terms in molecular biology can be found in the books of Benjamin Lewin, Gen. V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-63202182-9); and Robert A. Meyers (eds.), Molecular Biology and Biotechnology: A Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081569-8).
The singular articles "one", "one", and "the" include plural referents unless the context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "plurality" refers to two or more. It will be further understood that all base sizes or amino acid sizes and all molecular weights or molecular weight values, referred to for nucleic acids or polypeptides, are approximate and are mentioned for purposes of description. In addition, the numerical limits mentioned with respect to the concentrations or levels of a substance such as an antigen, are understood to be approximate. Therefore, if it is indicated that a concentration is at least (for example) 200 μg, it is understood that the concentration will be understood as being at least approximately (or "about" or "~" ) 200 pg.
Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term "includes" means "included". Therefore, unless the context indicates otherwise, the word "includes" and its variants such as "understand" and "including" will be understood to imply the inclusion of a named compound or composition (eg for example, a nucleic acid, a polypeptide, an antigen) or a step or group of compounds or steps but not the exclusion of any other compound, composition, steps or groups thereof.
A "polypeptide" is a polymer in which the monomers are amino acid residues that are joined to each other by amide linkages. A "peptide" is a short amino acid sequence, for example, about 10 to 50 or 10 to 40 amino acids in length. The terms "polypeptide" or "protein" or "peptide" as used herein are meant to include any amino acid sequence and include modified sequences such as glycoproteins. The terms "polypeptide" and "peptide" are specifically intended to cover naturally occurring proteins as well as those that are recombinantly or synthetically produced. The term "fragment", with reference to a polypeptide, refers to a portion (i.e., a subsequence) of a polypeptide. The term "immunogenic fragment" refers to all fragments of a polypeptide that retain at least one predominant immunogenic epitope of the full-length reference protein or polypeptide. Orientation in the picornavirus structural proteins and the exemplified peptides refers to an N-terminal to C-terminal orientation, defined by the orientation of the amino and carboxy moieties of the individual amino acids. The polypeptides and peptides are translated from the N- or amino-terminal to the C- or carboxy-terminus.
The "structural proteins" of a virus such as a picornavirus are proteins that are components of the assembled mature viral particle and may comprise a core nucleotide protein, enzymes packaged in the viral particle, and membrane proteins. Picornavirus structural proteins such as HRV include VP1, VP2, VP3, VP4. Structural proteins do not include "non-structural proteins" of the virus that are proteins that are produced in infected cells but are not present in the mature viral particle. The "N-terminal" region of the picornavirus structural proteins refers to the N-terminal half of the full-length proteins, preferably a region in the N-terminal half of the protein and in the N-terminal region or near the N-terminal region. terminal of the full-length protein. Therefore, for VP4 which is only about 70 amino acids in length, the N-terminal region is considered to be amino acids 1 to 35 of the full-length protein or a region in amino acids 1 to 35 on the N-terminus or near the N-terminus of the full-length protein, amino acids 1 to 30 or 1 to 25 or 1 to 20 of the full-length protein or a region in amino acids 1 to 30 or 1 to 25 or 1 to 20 on the N-terminus or near the N-terminus of the full-length protein. For VP1 which is a longer protein of nearly 300 amino acids, the N-terminal region is considered amino acids 1 to 100, preferably 1 to 80 or 1 to 70 or 1 to 60 or 1 to 50 of the full-length protein or region in the 100 or 80 or 70 or 60 or 50 N-terminal amino acids and on the N-terminus or near the N-terminus of the protein.
The term "picornavirus" refers to any virus of the picornaviridae family including human and animal viruses. The term "human rhinovirus" abbreviated as HRV refers to any rhinovirus serotype of the picornavirid family that is capable of infecting humans and has been identified or yet to be identified as rhinovirus. There are several different ways of grouping HRVs as described in this document and each grouping contains multiple "serotypes" or "strains" of viruses (eg, HRV-14, HRV-8, HRV-25, etc.). categorized according to genetic similarities. In the context of the present description, the term "serotype" may be used to designate an HRV, and / or a polypeptide or peptide of the indicated HRV type.
Terms . "Polynucleotide" and "nucleic acid sequence" denote a polymeric form of nucleotides of at least 10 bases in length. The nucleotides may be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. The term includes both single and double forms of DNA. The term "isolated polynucleotide" refers to a polynucleotide that is not immediately contiguous to the two coding sequences from which it is immediately contiguous (one at the 5 'end and the other at the 3' end) in the genome existing in the natural state of the organism from which it comes. In one embodiment, a polynucleotide encodes a polypeptide. The 5 'and 3' directions of a nucleic acid are defined with reference to the binding capacity of the individual and named nucleotide units as a function of the carbon positions of the sugar deoxyribose (or ribose) cycle. The information content (coding) of a polynucleotide sequence is read in a 5 'to 3' direction. The term "carrier protein" refers to any protein to which the peptide is coupled or attached or conjugated, usually to enhance or facilitate the detection of the antigen by the immune system. The term is intended to cover both small peptides and large polypeptides (> 10 kDa). The carrier protein may comprise one or more T helper epitopes. The peptide may be coupled to the carrier protein by any means such as chemical conjugation. The term "virus-like particle" (VLP) refers to a viral capsid that resembles the structure of the external protein of the native virus but is non-infectious because it does not contain viral genetic material. Expression of viral structural proteins, known as envelope or capsid proteins or surface proteins, may result in self-assembly of VLPs. VLPs can be wrapped or not wrapped. VLPs generally have an icosahedral structure consisting of repeating identical protein subunits known as capsomeres. Capsomeres self-assemble to form VLPs. The "particles" of the chimeric polypeptide constructs are structures such as amorphous aggregates or more ordered structures, for example, a capsomer (capsomer) or a virus-like particle (VLP) or small non-VLP structures. Particles comprising VLPs, capsomeres and less ordered structures include HBsAg particles of hepatitis B virus consisting of HBV small surface antigen, HPV particles consisting of HPV L1 or L1 and L2 proteins, HPV particles. HRV consisting of VP1, VP2, VP3 and VP4 or VP1, VP2 and VP3 of HRV and particles of other viruses such as influenza virus or a norovirus or enterovirus, for example, EV-71. More recently, particles comprising VLPs have been produced from components of a wide variety of virus families including parvoviruses (e.g., adeno-associated viruses), retroviruses (e.g., HIV) and flavivirids (by example, hepatitis C virus). EV71 VLPs are described by Cheng-Yu Chung et al 2010. VLPs can be produced in various cell culture systems including mammalian cell lines, insect cell lines, yeasts, plant cells, and the like. and E. coli.
The term "heterologous" with respect to a nucleic acid, a polypeptide or other cellular component, indicates that the component exists where it is not normally found in the natural state and / or from a source or different species.
The terms "native" and "naturally occurring" refer to an element such as a protein, a polypeptide or a nucleic acid that is present in the same state as in its natural state. This means that the element has not been artificially modified. It will be understood that in the context of the present description, there are many native / naturally occurring serotypes of HRV (and HRV proteins and polypeptides), for example, obtained from different HRV serotypes existing at the time of the present invention. natural state.
A "variant" when referring to a nucleic acid or a polypeptide (for example, a VP1 or VP4 picornavirus nucleic acid or polypeptide) is a nucleic acid or a polypeptide that differs from a nucleic acid or a polypeptide. reference. Generally, the difference (s) between the variant and the reference nucleic acid or polypeptide is a proportionally small number of differences from the reference. A variant nucleic acid may differ from the reference nucleic acid to which it is compared by the addition, deletion or substitution of one or more nucleotides or by the substitution of an artificial nucleotide analog. Likewise, a variant peptide or polypeptide may differ from the reference polypeptide to which it is compared by the addition, deletion or substitution of one or more amino acids or by the substitution of an amino acid analogue. Variants of the VP1 and VP4 peptides are described more specifically and more specifically in this document.
An "antigen" is a compound, a composition or a substance that can stimulate antibody production and / or a T cell response in an animal, including compositions that are injected, absorbed or otherwise introduced into an animal. animal. The term "antigen" includes all related antigenic epitopes. The term "epitope" or the term "antigenic determinant" refers to a site or antigen to which B and / or T cells respond. The term "dominant antigenic epitopes" or "dominant epitope" refers to epitopes to which an immune response of the functionally significant host, for example, an antibody response or a T cell response is produced. Therefore, with respect to a protective immune response against a pathogen, the dominant antigenic epitopes are antigenic fractions which, when recognized by the host immune system, provide protection against a disease caused by the agent. pathogenic. The term "T cell epitope" refers to an epitope that, when bound to an appropriate MHC molecule, is specifically bound by a T cell (via a T cell receptor). A "T cell epitope" is an epitope that is specifically bound by an antibody (or a molecule of the B-cell receptor). A "neutralizing epitope" is an epitope that is capable of inducing a neutralizing immune response.
An "adjuvant" is an agent that enhances the production of an immune response in a non-specific manner. Common adjuvants include suspensions of minerals (alum, aluminum hydroxide, aluminum phosphate) on which an antigen is adsorbed; emulsions comprising water-in-oil and oil-in-water emulsions (and variants thereof including double emulsions and reversible emulsions), liposaccharides, lipopolysaccharides, immunostimulatory nucleic acids (such as CpG oligonucleotides), liposomes, agonists Toll-type receptors (in particular, TLR2, TLR4, TLR7 / 8 and TLR9 agonists) and their various combinations of these components.
An "immunogenic composition" is a composition of materials suitable for administration to a human or animal subject (e.g., in an experimental setting) that is capable of causing or inducing a specific immune response, for example against an agent pathogen such as a picornavirus. As such, an immunogenic composition comprises one or more antigens (e.g., polypeptide antigens) or antigenic epitopes. An immunogenic composition may also include one or more additional components capable of inducing or inducing or enhancing an immune response, such as an excipient, vehicle and / or adjuvant. In some instances, immunogenic compositions are administered to induce or induce an immune response that protects the subject against pathogen-induced symptoms or conditions. In some cases, pathogen-induced symptoms or diseases are prevented (or reduced or improved) by inhibiting the replication of the pathogen (eg, a picornavirus) after exposure of the subject to the agent pathogenic. In the context of the present description, the term "immunogenic composition" will be understood to include those compositions which are intended for administration to a subject or population of subjects to induce or induce a protective or palliative response against picornavirus, for example, HRV (i.e., vaccine compositions or vaccines).
An "immune response" is a response of an immune system cell such as a B lymphocyte, a T cell or a monocyte to a stimulus. An immune response may be a B-cell response that results in the production of specific antibodies such as antigen-specific neutralizing antibodies. An immune response may also be a T cell response such as a CD4 + response or a CD8 + response. In some cases, the response is specific for a particular antigen (i.e., a "specific antigen response"). If the antigen is derived from a pathogen, the specific antigen response is a "pathogen specific response". A "protective immune response" is an immune response that inhibits a pathogenic function or activity of a pathogen that reduces infection with a pathogen or that decreases the symptoms (including death) that result from infection with a pathogen. the pathogen. A protective immune response can be measured, for example, by inhibiting viral replication or plaque formation in a plaque reduction assay or ELISA neutralization assay or by measuring resistance to pathogen challenge. in vivo. An immune response is a cross-neutralization immune response when induced by an antigen of a picornavirus serotype and neutralizes not only a virus of this serotype but also a virus of a different picornavirus serotype. For example, one HRV peptide of one HRV serotype may induce a cross-neutralizing immune response against another HRV serotype. A peptide of a picornavirus may also induce a cross-neutralization response against another picornavirus. Cross-neutralization can therefore take place between viruses or between serotypes or strains of the same virus. A cross-neutralizing immune response against two or more viruses or serotypes comprises the immune response against the virus from which the antigen is. derivative and an immune response against another virus or serotype. A cross-neutralization immune response may include the generation of neutralizing antibodies that can be measured by an appropriate neutralization assay using a virus or pseudovirus to evaluate an antibody neutralizing ability.
The picornavirus peptides described herein may be said to be cross-reactive or cross-neutralized or cross-protected. Cross-reactive peptides are peptides that are capable of inducing an immune response against viruses or serotypes additional to that from which the peptide is derived. Cross-neutralizing peptides are peptides that are capable of inducing a cross-neutralizing immune response i.e., an immune response that neutralizes the virus against which the response has been induced and also against another related virus, by example, the same virus but of a different serotype or a different virus of the same family. A cross-protective peptide is a peptide that induces an immune response that can prevent infection or disease caused by the virus against which the response has been induced and also against infection or disease caused by another related virus, for example, 'a different serotype.
Proteins and structural peptides from HRS
The present invention focuses on the need for a rhinovirus vaccine and relates to the use of structural rhinovirus proteins and peptides that can stimulate an immune response against a number of HRV serotypes and thus confer protection against infection and a disease at HRV.
The rhinovirus proteins and peptides used in the invention can be selected from all HRV serotypes, for example HRV IB, 2, 3, 8, 10, 14, 26, 29, 31, 39, 47, 61, 62, 63, 66, 77, 97, 100 or other serotypes that may be untyped or non-typeable. Serotypes of particular interest include HRV 8, HRV 25 and HRV 100 serotypes of clade A, HRV 14 of clade B and HRV_C_026 of clade C. HRV serotypes A and C are associated with the diseases of the highest severity and therefore, the presence of the combination of an HRV A serotype sequence and an HRV C serotype sequence in a composition described herein is specifically contemplated.
Several three-dimensional (3D) structures of HRV capsids are available. For HRV 14, for example, a very detailed analysis was published by Arnold & Rossmann (1990). The capsid has an icosahedral organization of pseudo-symmetry T = 3. The surface of the virus is defined by 12 star-shaped pentons, one at each axis of symmetry of order 5. They are surrounded by a furrow or "canyon Of 20 ang. depth. There are also 20 triangular faces, one on each axis of symmetry of order 3. The 3D structures of HRV IA, HRV 2, HRV 3 and HRV 16 have also been determined, sometimes complexed with receptors or antibody fragments. .
The capsid dynamics of HRV 14 has been shown to resemble "breathing" (Lewis et al 1998). The capsid structure appears to oscillate between two different structural states, one observed in 3D structures in which VP4 is deeply buried and the other in which the N-termini of VP4 and VP1 are accessible to proteases. This has also been demonstrated by the accessibility of different capsid fragments over time, by proteolysis and mass spectroscopy (Lewis et al 1998). This "breathing" can be blocked by antiviral compounds binding into a pocket at the back of the capsid canyon.
Katpally et al (2009) showed that antibodies directed against a consensus sequence of the first 24 most likely residues of rhinovirus VP4 could cross-neutralize HRV 14 and 16 and that a peptide corresponding to the first 30 amino acids of HRV 14 VP4 used to produce an antiserum that neutralized HRV 16 and HRV 29. However, the inventors did not find that in fact a shorter peptide was more effective.
Therefore, the invention provides a VP4 peptide that has at most 20 amino acids from the N-terminus of VP4, in particular amino acids 1 to 16 of VP4 and its variants.
Miao et al (2009) have shown that a conserved peptide of the N-terminus of other enteroviruses, specifically Polio 1 and Cox B3, is recognized by monoclonal antibodies (MAb) generated against full-length VP1 proteins. different species of enteroviruses.
A conserved peptide equivalent of HRV VP1 is also capable of generating a cross-neutralizing antibody response against different HRV serotypes. The HRV VP1 peptide originates from the N-terminal region of VP1, in particular amino acids 32 to 45 of VP1 and its variants. By performing an alignment of VP1 and VP4 sequences of all picornaviruses, it has also been surprisingly found that there are similar peptides of HVR14 VP4 1-16 and HVR14 32-45. Thus, picornaviruses other than rhinovirus also potentially have cross-neutralizing peptides equivalent to those of HVR14 VP4 1-16 and HVR14 32-45. These picornavirus peptides are another aspect of the invention described herein.
Throughout the description, the VP1 and VP4 sequences of HRV14 are used as reference sequences to determine the region from which the VP1 and VP4 peptides originate (Palmenberg et al 2010).
The selected rhinovirus peptides are capable of inducing a cross-neutralizing immune response against HRV. This means that when properly presented, the peptides generate an immune response, e.g., an antibody response, against several HRV serotypes. Therefore, for example, the generated immune response neutralizes the HRV serotype from which the peptide originates and at least one other HRV serotype. For example, the cross-neutralization response may neutralize more than 2 or more than 5 or more than 10 different HRV serotypes. In one embodiment, the cross-neutralization response neutralizes more than 2 or more than 5 or more different HRV serotypes selected from HRV IB, 2, 3, 8, 10, 14, 26, 29, 31, 39, 47, 61, 62, 63, 66, 77, 97, 100.
Suitably, the HRV peptide is selected which has a high level of sequence identity ("homology") between HRV serotypes, which is greater than 80% between two (or more) serotypes. In some cases, the HRV peptide has more than 85% serotype sequence identity, or more than 90% serotype sequence identity, or more than 95% serotype sequence identity. Sequence identity can also be evaluated by examining the number of amino acid differences, therefore, for example, one can choose the HRV peptide that has only one or only two amino acid differences, or only one or only two conservative amino acid differences or no amino acid differences between two or more serotypes over the length of the peptide. In some embodiments, the HRV peptide is selected which has 100% sequence identity between at least two HRV serotypes, i.e.
amino acid differences. These HRV peptides may be named in this document, "consensus" sequences of VP4 or VP1.
The HRV VP4 1-16 peptide described herein, of HRV 14 has a 100% sequence identity with those of Clade B for the currently known clade B serotypes. The HRV VP4 peptide described herein, of HRV 100 (A-M) has a 100% sequence identity with those of clade A for the currently known clade A serotypes.
In a particular embodiment, the HRV peptide is a clade A consensus sequence that is identical (i.e., has a 100% sequence identity) between 2 or more HRV serotypes selected from serotypes of HRV listed in Figure 8 or Figure 11. In another embodiment, the HRV peptide is a clade B consensus sequence that is identical between
2 HRV serotypes or more, selected from the HRV serotypes listed in Figure 9 or Figure 12. For example, in an exemplary specific embodiment, the consensus sequence is identical between two or more serotypes of clade A or clade B shown in FIGS. 8 and 11, at the amino acid level 32 to 45 of VP1, or between two serotypes of clade A or of clade B presented in FIGS. 9 and 12, at the level of amino acids 1 to 16 of VP4 .
The numbering starts at amino acid 1 of the N-terminus, where the N-terminus is on the left side of a sequence and the C-terminus is on the right-hand side. It will be understood, of course, that the peptides may exhibit variability. For example, the peptides may be longer or shorter by one or two or three or four amino acids at either end, relative to the specific peptide sequences indicated. Thus, for example, if a VP4 1-16 peptide is used, it may be possible to use a peptide 1 to 14 or 1 to 15 or 1 to 17 or 1 to 18 or 2 to 14 or 2 to 15 or 2 to 16 or 2 to 17 or 2 to 18, for example, or an equivalent peptide containing one or two conservative amino acid substitutions, or one or two amino acid deletions, without modifying the immunological properties of the peptide or without eliminating the epitope. A VP4 peptide as described herein may for example start with amino acid 1, 2, 3 or 4 and terminate for example at amino acid 14, 15, 16, 17, 18, 19 or 20. Similarly, for VP1 32-45 peptide, it may be possible to use a longer peptide containing amino acids 32 to 45, for example, 32 to 43 or 32 to 44 or 32 to 46 or 32 to 47 or 30. at 45 or 31 to 45 or 33 to 45 or 34 to 44 or an equivalent peptide having one or two conservative amino acid substitutions, or one or two amino acid deletions, without modifying the immunological properties of the peptide or without eliminating the epitope. A VP1 peptide as described herein may for example start with amino acid 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 and end, for example, at the same time. amino acid 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50, using these numbers as for HRV14. It will be understood that this variability falls within the scope of the peptides described herein and that the specific peptides described herein are exemplary and are not restrictive with respect to peptides that are capable of conferring an immune response. cross-neutralization as described in this document.
HRV VP4 and VP1 cross-reactive peptides that are capable of inducing an immune response against other HRV serotypes can be identified according to the present disclosure. As shown in this document, the HRV sequences of different HRV serotypes can be aligned to identify regions with high similarity between HRV serotypes. Many sequence programs are available to perform these alignments and to identify the location of sequence homology. This may allow for the selection of HRV VP4 and VP1 peptides that are most similar between the HRV serotypes of interest and therefore potentially cross-react between some or all of the HRV serotypes.
Suitably, the peptide or peptides of HRV VP4 or VP1 are cross-reactive peptides, so that they can induce an immune response that recognizes not only the VP4 or VP1 of the HRV serotype from which the VP4 peptide or VP1 is derived but also a VP4 or VP1 peptide or protein of an HRV serotype other than that from which it is derived. Suitably, the peptide cross-reacts with 1 or 2 or more other serotypes in the same or different clade. Suitably, the peptide or peptides of HRV VP4 or VP1 used in the invention are capable of generating a cross-neutralizing immune response which is an immune response that is capable of neutralizing HRV of an HRV serotype different from the serotype of HRV from which the peptide VP4 or VP1 is derived, in the same clade or a different clade. Cross neutralization can be tested using assays well known in the art such as the assay described by Katpally et al (2009) or Phillips et al (2011) or the assay described herein in Example 1 which is adapted from these published essays.
Suitably, the VP4 or VP1 peptide can provide cross-protection and suitably comprises a cross-neutralization epitope.
Cross-protection occurs appropriately when a VP4 or VP1 peptide is capable of generating a protective immune response against infection / disease caused by at least two HRV serotypes. Cross-protection can occur when a VP4 or VP1 consensus peptide is selected and presented in the context of a carrier protein such as CRM197 or as a chimeric construct in which the peptide is inserted into a polypeptide, for example, a polypeptide of HBsAg or HPV or HRV which forms a particle such as a virus-like particle.
Cross-protection can be assessed by comparing the incidence of infection and / or disease for a group of HRV serotypes in individuals vaccinated with a given HRV VP4 or VP1 peptide or a combination thereof, at an unvaccinated group. Complete cross-protection against a serotype or group of serotypes is not necessary according to this description; indeed, any level of cross-protection provides a benefit. Suitably, the level of cross-protection observed is such that the vaccinated group has 5% less infection and / or serotype-related disease or non-vaccine HRV serotypes, compared to a comparable nonvaccinated group, more suitably, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 80%, up to 90% or even up to 100% % less infection and / or disease.
The HRV peptides and constructs VP1 and VP4 containing them can be tested for immunogenicity, cross-reactivity and cross-neutralization by standard techniques well known in the art. For example, peptides may be injected into animal or human models and a measure of antibody responses and / or cellular immune responses may be performed, for example by ELISA or cytokine analysis / measurement, respectively. Antibody screening methods are well known in the art. An ELISA test can be used to evaluate the cross-reactivity of antibodies. Antibodies can be tested for neutralization and cross-neutralization properties using a test as described in this document in Example 1.
Cross-protection against different HRV serotypes different from that of which the VP4 or VP1 peptide is derived can be identified using an animal model, for example, a mouse model (Bartlett et al., 2008).
Picornavirus peptides such as rhinovirus peptides VP1 and VP4 can be synthesized chemically by standard or recombinantly produced techniques. The peptides may be in the form of individual peptides or peptide concatamers bound in series, for example of 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 peptides or more.
Vector Proteins for Picornavirus Peptides
The picornavirus peptides described herein such as HRV peptides can be coupled to a carrier protein. Coupling can be by any suitable means, for example by expression as a construct with the carrier protein or by chemical coupling or conjugation of the peptide with the carrier protein using a chemical conjugation step. Vector proteins include CRM197 which is well known. The carrier proteins also include KLH which can be used in an immunogenic composition for an animal but not for human use. CRM197 is a non-toxic form of diphtheria toxin but can not be distinguished immunologically from diphtheria toxin. CRM197 is produced by C. diphtheriae infected with the non-toxigenic β19tox phase created by nitrosoguanidine mutagenesis of corynephage b (Uchida et al., Nature New Biology (1971) 233; 8-11). The CRM197 protein has the same molecular weight as diphtheria toxin but differs from it by a single base change in the structural gene. This results in an amino acid change from a glycine to a 52-position glutamine, rendering fragment A unable to bind to NAD and therefore non-toxic (Pappenheimer 1977, Ann Rev, Biochem 46; 69-94). , Applied Rappuoli and Environmental Microbiology Sept 1983 pp. 560-564).
Conjugation of peptides to a carrier protein can be accomplished by many different, well known chemical means. Examples of chemical methods include conjugating amino groups between the peptide and the vehicle with amino-reactive reagents such as glutaraldehyde or a bis-succinimidyl ester reagent (DSG-disuccinimidyl-glutarate or DSS-disuccinimidylsuberate (Greg T Hermanson, Bioconjugate Techniques, Academic Press, 1996, 218-220 and 194-196) or by condensation of carboxyl groups and amino groups with carbodiimide reagents (Greg T. Hermanson, Bioconjugate Techniques, Academic Press, 1996, 171). It is also possible to use a thioether linkage to conjugate peptides to carrier proteins, for example by adding a terminal thiol group moiety to the peptide, for example by adding a cysteine. and then reacting the reactive thiol group with a maleimide derivatized vector protein (see Greg T. Hermanson, Bioconjugate Techniques, Academic Press, 1996). The method involves coupling a thiolated vehicle with a sulfhydryl group on the peptide to form a disulfide bridge. The peptides may also be synthesized with an additional haloalkyl group such as an iodoalkyl or bromoalkyl group. Suitably, the bromoalkyl group is a bromoacetyl group. The use of bromoacetyl groups to bind peptides to vehicles is described in the literature (Ivanov et al., 1995, Bioconjugatechemistry, 6, 269-277). Reductive amination can also be used to conjugate an aldehyde-containing molecule with an amine-containing molecule. The peptides can also be synthesized with an additional hydrazide group. Macromolecules containing an aldehyde may also spontaneously react with hydrazide compounds to form hydrazone bonds. Hydrazides are more potent nucleophiles and react more readily with aldehydes than primary amines. The hydrazone bond is a Schiff base form that is more stable than that formed by the interaction of an aldehyde and an amine. Therefore, specific conjugation can be achieved by reductive amination using peptides having additional hydrazide (Shannessy, D.J. and Wilcheck, 1990. Analytical Biochemistry 191: 1-8).
In one embodiment, the peptides are coupled to CRM197 according to well-known chemical conjugation techniques, see for example, Mattson et al., MolBiol Reports, 17, 167-183, 1993. In one embodiment, CRM197 is purified at from
Corynebacterium and fermentation of CRM197 is re-engineered as described in WO 2006/100108. In one embodiment, the purification method comprises three chromatographic steps (Q-sepharose-XL, hydroxyapatite type I and Octyl-Sepharose) and an ultrafiltration step. The maleimide chemistry can be used to conjugate peptides having cysteine at the N or C-terminal level.
Chimeric polypeptides comprising picornavirus peptides
As another means of presenting picornavirus peptides, a chimeric polypeptide construct may be used. Advantageously, the chimeric polypeptide construct forms particles such as capsomeres or viral-like particles (VLPs) or small non-VLP type structures.
In another embodiment of the invention there is provided a chimeric polypeptide construct comprising a polypeptide that forms particles and a peptide comprising an epitope of a picornavirus structural polypeptide such as a rhinovirus structural peptide, eg VP1 or VP4. The particles can be capsomers or VLPs or small non-VLP type structures.
An example of a polypeptide that can be used in a chimeric polypeptide construct with a picornavirus peptide such as a peptide or HRV peptides is a hepatitis B surface antigen polypeptide. HBsAg has been used for years. 1980 HBsAg is also used in a malaria vaccine candidate known as RTS, S, which comprises chimeric HbsAg polypeptides having a 226 amino acid extension of the S protein. hepatitis B virus (serotype adw) fused via its N-terminus to a fragment of circumsporozoite protein (CSP) of P. falciparum, via four amino acids, Pro, Val, Thr, Asn, representing the four carboxy residues of the preS2 protein of hepatitis B virus (adw serotype). RTS, S is described in WO 93/10152. The chimeric polypeptide is expressed in a yeast strain which already carries in its genome several copies of an expression cassette for the surface antigen of hepatitis B. The strain obtained synthesizes two polypeptides, S and RTS, which spontaneously co-assemble into mixed lipoprotein particles (RTS, S) that have CSP sequences on their surface.
Advantageously, the picornavirus peptide / HBsAg polypeptide chimera forms a particle that resembles an HBsAg particle. In a particular embodiment, the S antigen polypeptide is a contiguous segment of 226 amino acids, specifying the hepatitis B virus S protein (serotype adw). The picornavirus peptide / HBsAg polypeptide chimera is advantageously constructed to spontaneously form particles. The particles may be mixed particles comprising a non-chimeric HBsAg polypeptide together with a picornavirus peptide / chimeric HBsAg polypeptide. Suitable sites for insertion of picornavirus peptides such as HRV peptide or peptides include the "a" loop, the N-terminus and the C-terminus of HBsAg. Peptides that may be included in a chimeric HBsAg include any of the peptides described herein, including VP4 peptides such as peptides 1 to 16 and VP1 peptides such as 32 to 45 and variants of one or the other. other or both and other picornavirus structural protein peptides comprising VP4 and VP1, such as rhinovirus VP4 and VP1 peptides. In a particular embodiment, the peptide in the construction of the chimeric polypeptide contains a neutralizing epitope. HRV peptides containing a neutralizing epitope can be found in the literature and include 1-31 of VP4 (Katpally et al 2009) and 147-162 of HRV14 VP1 (Edlmayret al 2011).
In the case of virus-like particles of HPV, there are particles of the viral type of HPV 16 or HPV 18 suitable. HPV L1 protein self-assembles into viral-like particles that typically resemble HPV viruses observed under an electron microscope. Usually, they consist of 72 capsomeres which themselves consist of 5 LI polypeptides in a pentamer unit. Suitably, the L1 protein is a truncated L1 protein capable of self-assembly, for example in VLP capsomeres. Suitably, L1 is truncated to eliminate a nuclear localization signal. Suitably, truncation is a C-terminal truncation. Suitably, C-terminal truncation removes less than 50 amino acids, for example, less than 40 amino acids. In a particular embodiment, the C-terminal truncation removes 34 amino acids from HPV 16 and 35 amino acids from HPV 18. The location of the peptide or picornavirus / HRV peptides in a chimeric HPV L1 polypeptide described herein is important. A location of the picornavirus peptide is a location of the exposed loops or the arm entering the C-terminus of the L1 protein. Inner loops and arm are found when L1 is in the form of capsomeres or virus-like particles (Chen et al 2000).
In any of the embodiments described herein, the HRV peptide may be located at a position selected from the following regions of the L1 sequence, the locations corresponding to the reference sequence of HPV 16 and HPV 18 L1 or an equivalent position in another HPV L1 sequence: (i) BC loop in amino acids 50 to 61 (ii) DE loop in amino acids 132 to 142, for example, amino acids 132 to 141, in particular, amino acids 137 to 138 (iii) looped EF in amino acids 172 to 182, for example 176 to 182, in particular 176 to 179 (iv) FG loop in amino acids 271 to 290, for example 272 to 275, in particular 272 to 273 (v) HI loop in amino acids 345-359, for example 347 to 350, in particular 349 to 350 (vi) C-terminus in amino acids 429 to 445, for example 423 to 440, in in particular, 423 to 424, 431 to 433 or 437 to 438 for HPV 16, and 424 to 425, 432 at 433 or 439 to 440 for HPV 18.
In one embodiment described herein, the picornavirus peptide may be inserted into the polypeptide sequence without removing the amino acids from the polypeptide. Alternatively, the picornavirus peptide can be inserted into the polypeptide sequence with the removal of one or more amino acids from the polypeptide sequence at the insertion position, for example 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids of the polypeptide sequence can be removed at the site where the peptide is inserted. Accordingly, the picornavirus peptide may replace one or more amino acids in the polypeptide sequence, for example, the picornavirus peptide may replace a polypeptide sequence of length equivalent to that of the picornavirus peptide sequence.
When two or more picornavirus peptides are present in a chimeric picornavirus peptide / polypeptide construct, they may be different picornavirus peptides of the same picornavirus or peptides of the same picornavirus but of different serotypes, in which case they may come from a corresponding region in different picornaviruses or different regions in different picornaviruses. For example, when HRV peptides are present in a chimeric peptide / polypeptide HRV peptide construct, they may be different HRV peptides of the same HRV serotype or they may be peptides of different HRV serotypes, in which case they may come from the corresponding region in the different serotypes of HRV or different regions of the different serotypes of HRV.
In one embodiment, the picornavirus peptide such as an HRV peptide is inserted at a site that allows the assembly of a supramolecular set of chimeric polypeptides, for example in polypeptide particles such as viral-like particles. (VLPs) or capsomeres or small non-VLP type structures. For example, in the case of chimeric HPV particles, to retain the VLP structure, the picornavirus peptide is inserted into the L1 polypeptide at a site that does not interfere with the sites involved in forming the disulfide bridges that are involved in the conservation of inter-capsomere interactions and therefore, the conformation of VLPs. Usually, the chimeric VLPs are similar in size or identical to the native VLPs, that is, in the case of HPV, the chimeric VLPs are similar in size or identical to the VLPs in which the L1 protein is full length or truncated but does not contain picornavirus peptide. The chimeric HPV VLPs may be of the order of 50 nm in diameter. In other embodiments, small non-VLP structures between 20 and 35 nm are formed.
In one embodiment comprising two or more picornavirus peptides in a polypeptide, the picornavirus peptides may be inserted into identical or different sites in the polypeptide sequence. When picornavirus peptides are inserted at the same site, this can be done in the same loop and in the same hypervariable region of the same loop. It may be advantageous to have a short amino acid extension between the picornavirus peptides, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids between the picornavirus peptides.
Optionally, a spacer segment of one or more amino acids such as glycine residues may also be included in the N or C terminus of the picornavirus peptide. For example, the peptides may further comprise one or two or three spacer amino acids added at the amino or carboxy terminus (or between the bound peptides in which two or more picornavirus peptides are present). Generally, the spacer segment will not have specific biological activity other than joining the immunogenic peptide to the polypeptide sequence or maintaining a minimal distance or other spatial relationship between them. A spacer segment may be necessary or useful to maintain the correct conformation of the polypeptide particle and / or an effective or improved presentation of the inserted picornavirus peptide relative to the absence of a spacer segment. Any of the picornavirus peptides may be modified, for example, by the insertion (addition), deletion or substitution of one or more amino acids. For example, HRV peptides may incorporate amino acids that differ from the HRV sequence of the native (i.e., naturally occurring) sequence of HRV VP4 or VP1. For example, the peptides may have one or two amino acid insertions or substitutions in the sequence, or a deletion of one or two or more amino acids, for example 1, 2, 3, 4, 5, 6, 7 , 8 or up to 10 amino acids relative to the native sequence, for example to eliminate the occurrence of a disulfide bridge between two cysteines and / or the region between cysteines. In specific examples, the modifications present in the HRV peptides of the present description, in relation to a native HRV sequence are limited to 1 or 2 amino acid insertions, deletions or substitutions, and / or a deletion up to at 10 amino acids contiguous between two residues of cysteine.
When modifications of the HRV sequence are made in the peptides described herein, such modifications may be limited such that a substantial proportion of at least 50% or at least 70% or at least 90% or at least 95% of the amino acids in the peptide correspond to the amino acids in the native HRV VP4 or VP1 sequence.
Alternatively or additionally, any particular HRV peptide may be a chimera of two or three or more HRV peptides as described herein. In the case of any of these modifications of the HRV sequence, the immunogenic character of the HRV sequence is retained. This means that the epitope or HRV epitopes in the peptide that induces the desired immune response are conserved. The object of the modifications may be to improve the properties of the HRV peptide, for example to improve the cross-reactivity with structural proteins of other HRV serotypes.
Nucleic acids encoding RVH peptides, construct containing them and methods for producing chimeric polypeptides
Another aspect of the present disclosure relates to nucleic acid molecules that encode any of the aforementioned peptides and chimeric polypeptides containing the peptides of HRV structural peptides.
In some embodiments, the recombinant nucleic acids that encode the chimeric peptides or polypeptides are codon optimized for expression in a selected prokaryotic or eukaryotic host cell.
To facilitate replication and expression, the nucleic acids that encode the chimeric peptides or polypeptides may be incorporated into a vector such as a prokaryotic or eukaryotic expression vector.
The chimeric peptides and polypeptides described herein can be produced using well established procedures for the expression and purification of recombinant proteins. Sufficient procedures to guide the skilled person can be found in the following references: Sambrooket al.,
Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, NY, 200; and Ausubelet al. Short Protocols in Molecular Biology, 4th ed., John Wiley & Sons, Inc., 999. Additional and specific details are provided below.
Host cells that comprise the nucleic acids encoding the chimeric peptide or polypeptide therefore also constitute one aspect of the present disclosure. Suitable host cells include prokaryotic (i.e., bacterial) host cells such as E. coli, as well as many eukaryotic host cells including fungi (e.g., yeast such as Saccharomyces cerevisiae and Picchia pastoris), insect cells, plant cells and mammalian cells (such as CHO and HEK293 cells). Recombinant nucleic acids that encode the chimeric peptides or polypeptides are introduced (e.g., transduced, transformed, or transfected) into host cells, for example, via a vector such as an expression vector. The vector may be a plasmid, a viral particle, a phage, a baculovirus, etc. Examples of suitable expression hosts include: bacterial cells such as E. coli, Streptomyces, and Salmonella typhimurium; fungi cells such as Saccharomyces cerevisiae, Pichiapastoris, and
Neurosporacrassa; insect cells such as Trichoplusia, Drosophila, Spodopterafrugiperda; mammalian cells such as 3T3, COS, CHO, BHK, HEK 293 or Bowes melanoma; plant cells including algal cells, etc.
Host cells may be cultured in conventional modified nutrient media if appropriate to activate promoters, select transformants, or amplify inserted polynucleotide sequences. Culture conditions such as temperature, pH and the like are those which are used previously with the host cell chosen for expression and will be apparent to those skilled in the art and in the references cited herein, including, for example, Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, Third Edition, Wiley-Liss, New York and references cited therein. In addition to Sambrook, Berger, and Ausubel, details of cell culture can be found in Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sounds, Inc. New York, NY; Gamborg and Phillips (eds.) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Manual, Springer-Verlag (Berlin Heidelberg New York) and. Atlas and Parks (eds.) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, FL.
Immunogenic compositions and methods
Another aspect of the present disclosure is directed to immunogenic compositions that contain picornavirus peptides or constructs of chimeric polypeptides containing them, such as polypeptides that form particles such as VLPs or subviral particles such as capsomers. The immunogenic compositions described herein usually comprise at least one pharmaceutically acceptable diluent, excipient or carrier and optionally, an adjuvant. Pharmaceutically acceptable carriers and excipients are well known and may be selected by those skilled in the art. For example, the vehicle or excipient may favorably include a buffer. Optionally, the vehicle or excipient may also contain at least one component that stabilizes the solubility and / or promotes stability. Examples of solubilizing / stabilizing agents include detergents, for example, lauryl sarcosine and / or tween. Other solubilizing / stabilizing agents include arginine and vitrifying polyols (such as sucrose, trehalose and others). Many pharmaceutically acceptable carriers and / or pharmaceutically acceptable excipients are known in the art and are described for example in Remington's Pharmaceutical Sciences, E. W. Martin, Mack Publishing Co., Easton, PA, 5th Edition (975).
Accordingly, suitable excipients and carriers may be selected by those skilled in the art to produce a formulation suitable for delivery to a subject via a chosen route of administration. Suitable excipients include, but are not limited to: glycerol, polyethylene glycol (PEG), sorbitol, trehalose, N-lauroylsarcosine sodium salt, L-proline, non-detergent sulfobetaine, guanidine urea, trimethylamine oxide, KCl, Ca2 +, Mg2 +, Mn2 +, Zn2 + and other divalent cation-related salts, dithiothreitol, dithioerytrol and β-mercaptoethanol. Other excipients may be detergents (including: Tween 80, Tween 20,
Triton X-00, NP-40, Empigen BB, octylglucoside, lauroylmaltoside, Zwittergent 3-08, Zwittergent 3-0, Zwittergent 3-2, Zwittergent 3-4, Zwittergent 3- 6, CHAPS, sodium deoxycholate, sodium dodecyl sulphate, cetyltrimethylammonium bromide).
Optionally, the immunogenic compositions also comprise an adjuvant. The adjuvant is chosen to be safe and well tolerated in the target population. For example, in the case of an adjuvant chosen for its safety and efficacy in young children, a dose of adjuvant can be chosen which is a dilution (eg, a divided dose) of a dose usually given to a patient. adult subject.
A suitable adjuvant is a nontoxic bacterial lipopolysaccharide derivative. An example of a suitable nontoxic derivative of lipid A is monophosphoryl lipid A or more particularly 3-deacylated monophosphoryl lipid A (3D-MPL). 3D-MPL is marketed under the name of MPL by GlaxoSmithKline Biologicals N.A., and is named throughout this document, MPL or 3D-MPL. See, for example, U.S. Patent Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094.
3D-MPL mainly promotes CD4 + T cell responses with an IFN-γ (Th1) phenotype. 3D-MPL can be produced according to the methods described in GB2220211 A. Chemically, it is a mixture of 3-deacylated monophosphoryl lipid A having 3, 4, 5 or 6 acylated chains. In the compositions of the present invention, small particles of 3D-MPL can be used. The small particle 3D-MPL has a particle size such that it can be sterile filtered in a 0.22 μm filter. These preparations are described in WO94 / 21292.
A lipopolysaccharide, such as 3D-MPL, can be used in amounts between 1 and 50 μg, per human dose of the immunogenic composition. 3D-MPL can be used at a level of about 25 μg, for example between 20 and 30 μg, suitably between 21 and 29 μg or between 22 and 28 μg or between 23 and 27 μg or between 24 and 26 μg. pg or 25 pg. In another embodiment, the human dose of the immunogenic composition comprises 3D-MPL at a level of about 10 μg, for example between 5 and 15 μg, suitably between 6 and 14 μg, for example between 7 μg. and 13 μg or between 8 and 12 μg or between 9 and 11 μg, or 10 μg. In another embodiment, the human dose of the immunogenic composition comprises 3D-MPL at a level of about 5 μg, for example between 1 and 9 μg or between 2 and 8 μg or suitably between 3 and 7 μg. pg or 4 and μg, or 5 μg.
In other embodiments, the lipopolysaccharide may be a disaccharide of β (1-6) glucosamine as described in US Patent No. 6,005,099 and EP Patent No. 0,729,473 B1. can easily produce various lipopolysaccharides, such as 3D-MPL, based on the teachings in these references. Nevertheless, each of these references is incorporated in the present application. In addition to the aforementioned immunostimulants (which are structurally similar to LPS or MPL or 3D-MPL), the acylated monosaccharide and disaccharide derivatives which are a subset of the above MPL structure are also suitable adjuvants. . In other embodiments, the adjuvant is a synthetic derivative of lipid A, some of which are described as TLR-4 antagonists and include, but are not limited to: 1ΌΜ174 (2-deoxy-6-o- [2 deoxy-2 - [(R) -3-dodecanoyloxytetradanoylamino] -4-o-phosphono-β-D-glucopyranosyl] -2 - [(R) -3-hydroxytetrafanoylamino] -α-D-glucopyranosyldihydrogenphosphate), ( WO 95/14026); 299 (3S, 9R) -3 - [(R) -Dodecanoyloxytetradecanoylamino] -4-oxo-5-aza-9 (R) - [(R) -3-hydroxytetradecanoylamino] decane-1,10-diol 1,10-bis (dihydrogenphosphate) (WO 99/64301 and WO 00/0462); and OM 197 MP-Ac DP (3S-, 9R) -3 - [(R) -dodecanoyloxytetradecanoylamino] -4-oxo-5-aza-9 - [(R) -3-hydroxytetradecanoylamino] decan-1.10 diol, 1-dihydrogen phosphate 10- (6-aminohexanoate) (WO 01/46127). Other TLR4 ligands that can be used are alkylglucosaminide (AGP) phosphates such as those described in WO 98/50399 or US Pat. No. 6,303,347 (processes for the preparation of AGP are also described), appropriately RC527 or RC529 or pharmaceutically acceptable AGP salts described in US Pat. No. 6,764,840. Some AGPs are TLR4 agonists and others are TLR4 antagonists. Both are supposed to be useful additives. Other suitable TLR-4 ligands capable of eliciting a TLR-4 signaling response (Sabroe et al, JI 2003 pl630-5) are, for example, gram-negative bacterial lipopolysaccharide and its derivatives or fragments, in particular, a nontoxic derivative of LPS (such as 3D-MPL). Other suitable TLR agonists are: heat shock proteins (HSP) 10, 60, 65, 70, 75 or 90; surfactant A protein, hyaluronan oligosaccharides, heparan sulfate fragments, fibronectin fragments, fibrinogen and b-defensin-2 peptides, and muramyl dipeptide (MDP). In one embodiment, the TLR agonist is HSP 60, 70 or 90. Other suitable TLR-4 ligands are as described in WO 2003/011223 and WO 2003/099195, such as the compound I, the compound II and the compound III described on pages 4 and 5 of the document WO 2003/011223 or on pages 3 and 4 of the document WO 2003/099195 and in particular the compounds described in the document WO 2003/011223 under the names ER803022, ER803058, ER803732, ER804053, ER804057, ER804058, ER804059, ER804442, ER804680 and ER804764. For example, a suitable TLR-4 ligand is ER804057.
Additional TLR agonists are also useful as adjuvants. The term "TLR agonist" refers to an agent that is capable of driving a signaling response by a TLR signaling pathway, either as a direct ligand or indirectly by the generation of an endogenous or exogenous ligand. These natural or synthetic TLR agonists can be used as alternative or additional adjuvants. A brief review of the role of TLRs as adjuvant receptors is provided by Kaisho & Akira, in Biochimica and BiophysicaActa 1589: 1-13, 2002. These potential adjuvants include, but are not limited to, TLR2, TLR3, TLR7, TLR8 and TLR9 agonists. Accordingly, in one embodiment, the adjuvant and the immunogenic composition further comprise an adjuvant that is selected from the group consisting of: a TLR-1 agonist, a TLR-2 agonist, a TLR-3 agonist, a TLR-4 agonist, a TLR-5 agonist, a TLR-6 agonist, a TLR-7 agonist, a TLR-8 agonist, a TLR-9 agonist or a combination thereof.
In one embodiment of the present invention, a TLR agonist is used which is capable of driving a signaling response through TLR-1. Suitably, the TLR agonist capable of driving a signaling response through TLR-1 is selected from: triacylated lipopeptides (LPs); phenol-soluble modulin; Mycobacterium tuberculosis LP; S- (2,3-bis (palmitoyloxy) - (2-RS) -propyl) -N-palmitoyl- (R) -Cys- (S) -Ser- (S) -Lys (4) -OH trihydrochloride (Pam3Cys) LP which mimics the acetylated amino terminus of a bacterial lipoprotein and Borrelia burgdorferi OspA LP.
In another embodiment, a TLR agonist is used which is capable of driving a signaling response through TLR-2. Suitably, the TLR agonist capable of driving a signaling response through TLR-2 is one or more of a lipoprotein, a peptidoglycan, a bacterial lipopeptide of M. tuberculosis, B. burgdorferi or T. pallidum; peptidoglycans from species including Staphylococcus aureus; lipoteichoic acids, mannuronic acids, Neisseria porins, bacterial fimbriae, Yersina virulence factors, CMV virions, measles virus haemagglutinin and yeast zymosan.
In another embodiment, a TLR agonist is used which is capable of driving a signaling response through TLR-3. Suitably, the TLR agonist capable of driving a signaling response through TLR-3 is double-stranded RNA (dsRNA) or polyinosinic-polycytidylic acid (Poly IC), a nucleic acid configuration. associated with a viral infection.
In another embodiment, a TLR agonist is used which is capable of driving a signaling response through TLR-5. Suitably, the TLR agonist capable of driving a signaling response through TLR-5 is a bacterial flagellin.
In another embodiment, a TLR agonist is used which is capable of driving a signaling response through TLR-6. Suitably, the TLR agonist capable of driving a signaling response through TLR-6 is a mycobacterial lipoprotein, a di-acylated LP and a phenol-soluble modulin. Additional TLR6 agonists are described in WO 2003/043572.
In another embodiment, a TLR agonist is used which is capable of driving a signaling response through TLR-7. Suitably, the TLR agonist capable of driving a signaling response through TLR-7 is a single-stranded RNA (sRNA), loxoribine, a guanosine analog at the N7 and C8 positions or a compound imidazoquinoline or derivatives thereof. In one embodiment, the TLR agonist is imiquimod. Other TLR7 agonists are described in WO 2002/085905.
In another embodiment, a TLR agonist is used, which is capable of driving a signaling response through TLR-8. Suitably, the TLR agonist capable of driving a TLR-8 signaling response is a single-stranded RNA (ARNbs), an imidazoquinoline molecule having antiviral activity, for example resiquimod (R848); ResiMimod can also be recognized by TLR-7. Other TLR-8 agonists that can be used include those described in WO 2004/071459.
In another embodiment, a TLR agonist is used that is capable of driving a signaling response through TLR-9. In one embodiment, the TLR agonist capable of driving a signaling response through TLR-9 and HSP90. Alternatively, the TLR agonist capable of driving a signaling response through TLR-9 is bacterial or viral DNA, DNA containing unmethylated CpG nucleotides, particularly particular sequence contexts known under the CpG pattern name. Oligonucleotides containing CpG mainly induce a Th1 response. These oligonucleotides are well known and are described, for example, in WO 96/02555, WO 99/33488 and US Pat. Nos. 6,008,200 and 5,856,462. Suitably, CpG nucleotides are CpG oligonucleotides. . Oligonucleotides suitable for use in the immunogenic compositions of the present invention are oligonucleotides containing CpG, optionally containing two CpG units of dinucleotides separated by at least three, suitably at least six or more nucleotides. A CpG motif is a cytosine nucleotide followed by a guanine nucleotide. The CpG oligonucleotides of the present invention are usually deoxynucleotides. In a specific embodiment, the internucleotide in the oligonucleotide is phosphorodithioate or, suitably, a phosphorothioate linkage although phosphodiester bonds and other internucleotide linkages fall within the scope of the invention. In the context of the invention are also included oligonucleotides comprising mixed internucleotide linkages. Methods for producing phosphorothioate or phosphorodithioate oligonucleotides are disclosed in US Patent Nos. 5,666,153, 5,278,302 and WO 95/26204. Other adjuvants that may be used in immunogenic compositions with picornavirus peptides or chimeric polypeptide constructs, for example, of themselves or in combination with 3D-MPL, or other adjuvant described herein, are saponins, such as QS21.
Saponins are described by Lacaille-Dubois, M. and Wagner H. (1996. A review of the biological and pharmacological activities of saponins, Phytomedicinevol 2 pp. 363-386). Saponins are steroids or triterpene glycosides widely distributed in the plant kingdom and marine animal. Saponins are known to form colloidal solutions in water, which foam when agitated and to precipitate cholesterol. When the saponins are close to the cell membranes, they create porous-type structures in the membrane, which causes the membrane to burst. Haemolysis of erythrocytes is an example of this phenomenon which is a property of some saponins but not all.
Saponins are known as adjuvants in vaccines for systemic administration. Adjuvant and haemolytic activity of individual saponins have been widely studied in art (Lacaille-Dubois and Wagner, supra). For example, Quil A (derived from the bark of the South American tree Quillaja Saponaria Molina), and fractions thereof are described in US 5,057,540 and in "Saponins as vaccine adjuvants". , Kensil, CR, Crit Rev. Ther Drug Carrier Syst, 1996, 12 (1-2): 1-55; and EP 0 362 279 B1. Particle structures termed immune-stimulating complexes (ISCOMS) comprising Quil A moieties are hemolytic and have been used in the production of vaccines (Morein, B., EP 0 109 942 B1, WO 96/11711; WO 96/33739). The hemolytic saponins QS21 and QS17 (HPLC purified fractions of Quil A) have been described as potent systemic adjuvants and the method for their production is described in US Patent No. 5,057,540 and EP 0 362 279 B1 which are included. attached for reference. Other saponins that have been used in systemic vaccination studies include those derived from other plant species such as gypsophila and saponaria (Bomford et al., Vaccine, 10 (9): 572-577, 1992).
QS21 is a purified non-toxic Hplc fraction derived from Quillaja Saponaria Molina bark. A process for producing QS21 is described in US Patent No. 5,057,540. Non-reactogenic adjuvant formulations containing QS21 are described in WO 96/33739. The references mentioned above are incorporated in the appendix to this document. Said immunologically active saponin such as QS21 can be used in amounts of between 1 and 50 μg per human dose of the immunogenic composition. Advantageously, QS21 is used at the level of about 25 μg, for example between 20 and 30 μg, suitably between 21 and 29 μg or between 22 and 28 μg or between 23 and 27 μg or between 24 and 26 μg, or 25 pg. In another embodiment, the human dose of the immunogenic composition comprises QS21 at a level of about 10 μg, for example between 5 and 15 μg, suitably between 6 and 14 μg, for example, between 7 and 13 μg. pg or between 8 and 12 pg or between 9 and 11 pg, or 10 pg. In another embodiment, the human dose of the immunogenic composition comprises QS21 at a level of about 5 μg, for example between 1 and 9 μg, or between 2 and 8 μg or suitably between 3 and 7 μg or 4-6 μg, or 5 μg. These formulations comprising QS21 and cholesterol have been found to be effective Th1 stimulating adjuvants when formulated together with an antigen. Therefore, for example, picornavirus peptides and chimeric polypeptide constructs can be favorably used in immunogenic compositions with an adjuvant comprising a combination of QS21 and cholesterol.
Optionally, the adjuvant may also include inorganic salts such as aluminum or calcium salts, in particular aluminum hydroxide, aluminum phosphate and calcium phosphate. For example, an adjuvant containing 3D-MPL in combination with an aluminum salt (eg, aluminum hydroxide or "alum") is suitable for formulation into an immunogenic composition containing picornavirus peptides or construct of chimeric polypeptide for administration to a human subject.
Another class of Thl-based adjuvants for use in formulations with picornavirus peptides and chimeric polypeptide constructs include OMP-based immunostimulatory compositions. OMP immunostimulatory compositions are particularly suitable as mucosal adjuvants, for example, for intranasal administration. OMP-based immunostimulatory compositions are a kind of outer membrane protein preparations (OMP including certain porins) of gram-negative bacteria such as, but not limited to, Neisseria species (see, e.g., Lowell et al., J. 167: 658, 1988; Lowell et al., Science 240: 800, 1988; Lynch et al., Biophys. J. 45: 104, 1984; Lowell, in "New Generation Vaccines" 2nd ed., Marcel Dekker, Inc., New York, Basil, Hong Kong, page 193, 1997; US Patent No. 5,726,292; US Patent No. 4,707,543) which are useful as vehicles or in compositions for immunogens such as bacterial or viral antigens. Some immunostimulatory OMP-based compositions may be termed "proteosomes", which are hydrophobic and harmless to humans. The proteosomes have the ability to self-assemble into vesicles or OMPs of vesicle type from about 20 nm to about 800 nm and to incorporate, coordinate, associate non-covalently, ( for example, electrostatically or hydrophobically), or otherwise cooperate with protein (Ag) antigens, particularly antigens that have a hydrophobic moiety. Any method of preparation which provides the vesicle-like or vesicle-like outer membrane protein component, comprising multimolecular membrane structures or molten globular OMP compositions of one or more OMPs, is included in the definition of proteosome. Proteosomes can be prepared, for example, as described in the art (see for example, U.S. Patent No. 5,726,292 or U.S. No. 5,985,284). The proteosomes may also contain a lipopolysaccharide, an endogenous lipooligosaccharide (LPS or LOS, respectively) from the bacteria used to produce OMP porins (eg, Neisseria species) which will generally constitute less than 2% of the Total OMP.
Proteosomes consist mainly of chemically extracted outer membrane (OMP) proteins of Neisseria menigitidis (mainly porins A and B as well as class 4 OMPs), maintained in detergent solution (Lowell GH, Proteosomes for Improved Nasal). , Oral, Injectable Gold Vaccines, in: MM Levine, Woodrow GC, Kaper JB, Cobon GS, Ed, New Generation Vaccines, New York: Marcel Dekker, Inc. 1997; 193-206). The proteosomes can be formulated with various antigens such as purified or recombinant proteins derived from viral sources, including picornavirus peptides and chimeric polypeptide constructs described herein, for example by diafiltration or conventional dialysis methods. The gradual removal of detergent allows the formation of particulate hydrophobic complexes of about 100 to 200 nm in diameter (Lowell GH Proteosomes for Improved Nasal, Oral, or Injectable Vaccines in: MM Levine, Woodrow GC, Kaper JB, Cobon GS , ed., New Generation Vaccines, New York: Marcel Dekker, Inc. 1997, 193-206).
The terms "proteosome: LPS or protolline" as used herein refer to mixed proteosome preparations, for example, by exogenous addition, with at least one kind of lipopolysaccharide to give OMP-LPS composition (which can be immunostimulatory composition effect). Therefore, the OMP-LPS composition can be comprised of two of the basic components of protollin, which comprises (1) a proteosome outer membrane protein preparation (e.g., Projuvant) prepared from gram negative bacteria such as Neisseria meningitidis, and (2) a preparation of one or more liposaccharides. A lipo-oligosaccharide may be endogenous (e.g., naturally occurring with the OMP proteosome preparation), may be mixed with or combined with an OMP preparation of an exogenously prepared lipo-oligosaccharide (eg for example, prepared from a different microorganism culture than the OMP preparation), or can be a combination thereof. This exogenously added LPS may be from the same gram negative bacterium from which the OMP preparation was produced or from a different gram-negative bacterium. Protolline should also be understood as possibly including lipids, glycolipids, glycoproteins, small molecules or the like, and combinations thereof. The protolline can be prepared, for example, as described in the publication of the US patent application. No. 2003/0044425.
Combinations of different adjuvants such as those mentioned above, can also be used in compositions with picornavirus peptides and chimeric polypeptide constructs. For example, as already indicated, QS21 can be formulated in conjunction with 3D-MPL. The ratios of QS21: 3D-MPL will usually be in the range of 1:10 to 10: 1; such as 1: 5 to 5: 1, and often, substantially, 1: 1. Usually, the ratio is of the order of 2.5: there 1: 1 of 3D-MPL: QS21. Another combined adjuvant formulation comprises 3D-MPL and an aluminum salt such as aluminum hydroxide. When combined, this combination may enhance an antigen-specific Th1 immune response.
In some cases, the adjuvant formulation comprises a mineral salt such as a calcium or aluminum salt (alum), for example, calcium phosphate, aluminum phosphate or aluminum hydroxide. In the presence of alum, for example in combination with 3D-MPL, the amount is usually between about 100 Hg and 1 mg, for example, about 100 μρ or about 200 μρ to about 750 μg, for example about 500 μl per dose.
In some embodiments, the adjuvant comprises an emulsion of oil and water, for example, an oil-in-water emulsion. An example of an oil-in-water emulsion includes a metabolizable oil such as squalene, a tocol such as tocopherol, e.g. alpha-tocopherol and a surfactant such as sorbitan trioleate (Span 85 ™) or mono-oleate of polyoxyethylene sorbitan (Tween 80 ™) in an aqueous vehicle. In some embodiments, the oil-in-water emulsion does not contain any additional immunostimulants, (in particular, it does not contain a non-toxic lipid A derivative such as 3D-MPL or a saponin, such as QS21). The aqueous vehicle may be, for example, a phosphate buffered saline solution. In addition, the oil-in-water emulsion may contain span 85 and / or lecithin and / or tricapryline.
In another embodiment of the invention there is provided a vaccine composition comprising an antigen or antigenic composition and an adjuvant composition comprising an oil-in-water emulsion and optionally one or more other immunostimulants, wherein said oil-in-water emulsion comprises 0.5 to 10 mg of metabolizable oil (suitably squalene), 0.5 to 11 mg of tocol (suitably a tocopherol, such as alpha-tocopherol) and 0.4 to 4 mg of emulsifier.
In a specific embodiment, the adjuvant formulation comprises 3D-MPL prepared as an emulsion, such as an oil-in-water emulsion. In some cases, the emulsion has a small particle size of less than 0.2 μιη in diameter as described in WO 94/21292. For example, the 3D-MPL particles may be small enough to be sterile filtered in a 0.22 micron membrane (as described in European Patent No. 0 689 454). Alternatively, 3D-MPL can be prepared in a liposomal formulation. Optionally, the adjuvant containing 3D-MPL (or a derivative thereof) also includes an additional immunostimulatory component.
It should be noted that regardless of the adjuvant chosen, the concentration in the final formulation is calculated to be safe and effective in the target population. For example, immunogenic compositions may be intended to induce an immune response against a picornavirus such as HRV in human infants (e.g., infants between birth and 1 year, e.g., 0 to 6 months of age, at initial administration). In another example, the immunogenic compositions may be intended to induce an immune response against a picornavirus such as HRV in elderly humans. The immunogenic composition may also be for administration to adults or children. It will be understood that the choice of an adjuvant may be different in these different applications and the optimal adjuvant and concentration for each situation can be determined empirically by those skilled in the art.
Chimeric polypeptide constructs in the form of particles for use as described herein can be adsorbed on aluminum-containing adjuvants. In the case of several different chimeric polypeptide constructs, for example, a particle such as VLP, the adjuvant can be added to the different constructs or particles or VLPs to pre-adsorb them before mixing the different constructs or particles or VLPs to form the final immunogenic composition.
The immunogenic composition may also comprise aluminum or an aluminum compound such as a stabilizer and the present disclosure also relates to a stabilized composition in which chimeric polypeptide constructs such as VLPs are adsorbed on an aluminum salt. Suitably, the VLPs are more stable in time after adsorption on an aluminum salt than in the absence of aluminum.
The immunogenic compositions described herein may be administered in the form of vaccines by any of the various routes such as the oral, topical, subcutaneous, mucosal, intravenous, intramuscular, intranasal, sublingual, intradermal and suppository routes. Intramuscular, sublingual and intradermal distributions are preferred.
The assay of chimeric peptides or polypeptide constructs such as VLPs may vary depending on the health status, gender, age and weight of the individual and the route of administration of the vaccine. The amount may also vary depending on the number of peptides or different chimeric constructs.
An immunogenic composition usually contains an immunoprotective amount (or a fractional dose thereof) of the antigen and can be prepared by conventional techniques. A preparation of immunogenic compositions, including those intended for administration to human subjects, is generally described in Pharmaceutical Biotechnology, vol. 61 Vaccine Design-the subunit and adjuvant approach, edited by Powell and Newman, Plenum Press, 1995. New Trends and Developments in Vaccines, edited by Voiler et al., University Park Press, Baltimore, Maryland, USA 1978. Encapsulation in liposomes are described, for example, by Fullerton, US Pat. No. 4,235,877. The conjugation of proteins to macromolecules is described, for example, by Likhite, US Pat. No. 4,372,945 and Armoret et al., US Pat. No. 4,474,757.
Usually, the amount of protein in each dose of immunogenic composition is selected as an amount that induces an immunoprotective response without causing significant undesirable side effects in the human subject. The term "immunoprotective" in this context does not necessarily mean "completely protective" of an infection; it means protection against symptoms or diseases, in particular, serious diseases associated with the virus. The amount of antigen may vary depending on the specific immunogen that is used. Generally, it is expected that each human dose will contain 1 to 1000 μg of protein. Suitably, each vaccine dose comprises 1 to 100 μl of each chimeric polypeptide peptide or construct conjugate, suitably at least 5 μρ, or at least 10 μg, for example, between 5 and 50 μρ of each conjugate of peptide or chimeric polypeptide construct, most suitably 10 to 50 μρ each, for example, 10 μρ, 15 μρ, 20 μρ, 40 μg or 50 μg. For example, one dose of vaccine may contain 10 or 15 or 20 or 30 or 40 μg of each peptide conjugate or construct of chimeric polypeptides. The amount used in an immunogenic composition is chosen on the basis of the target population (e.g., infants or elderly). An optimal amount for a particular composition can be provided by standard studies including observation of antibody titers and other responses in subjects. After an initial vaccination, subjects may be recalled approximately 4 weeks later.
The immunogenic compositions described herein suitably generate an immune response in a human or animal subject against at least two different picornaviruses or two different serotypes of a picornavirus such as two different HRV serotypes, suitably 2 or more, 3 or more, 4 or more, 5 or more, or 10 or more different serotypes.
The HRV compositions described herein suitably provide protection against infection and / or disease of at least 2 different HRV serotypes, suitably 2 or more, 3 or more, 4 or more, 5 or more. more or 10 different serotypes or more.
In addition, the compositions described herein which comprise a carrier protein or a chimeric polypeptide such as VLP will also generate an immune response against the carrier protein or the VLP itself. It can be a protective response. Therefore, the immunogenic compositions can provide protection against infection or disease caused by the native virus corresponding to the VLP of the immunogenic composition. For example, a chimeric particle of HBsAg or VLP containing one or more peptides of a picornavirus such as HRV may protect against infection or disease caused by HBsAg as well as against picornavirus infection. Likewise, a non-structural chimeric rhinovirus protein particle or a VLP containing one or more peptides of a picornavirus such as HRV may confer another beneficial immune response against the non-structural picornavirus protein.
Optionally, the immunogenic composition of HRV or the vaccine may also be formulated or coadministered with other antigens such as antigens of other respiratory viruses such as influenza virus or RSV, or other causes of COPD such as nontypeable Haemophilus influenzae, Moraxella catharralis and Streptococcus pneumoniae.
For all vaccines described herein, the vaccine is appropriately used for vaccination in all age groups, particularly for vaccination of children and elderly populations.
Suitably, the vaccine is dispensed according to a schedule of 2 or 3 doses, for example according to a schedule of 0, 1 or 0, 2 or 0, 3 or 0, 4 or 0, 5 or 0, 6 or 0, 12 months or a schedule of 0, 1, 6 or 0, 2, 6 or 0, 6, 12 months, respectively.
Suitably, the vaccine is a liquid vaccine formulation, although the vaccine can be lyophilized or reconstituted prior to administration.
Examples
Example 1
Immunogenicity of peptides and full-length proteins associated with human rhinovirus
Goals
In this experiment, the following points were evaluated: (1) the immunogenicity of KLH-conjugated VP1 / VP4-associated peptides or full-length proteins and (2) the performance of HRV / antigen peptide chimeric polypeptide constructs Hepatitis B surface area
Peptides were selected on the basis of bioinformatic predictions and compared to published data showing the ability of various peptides to induce neutralizing (cross) antibodies (McCray & Werner, 1987, 1989; Katpally et al. 2009, Miao et al., 2009, Edlmayr et al., 2011). In parallel with the peptides, concatemers of full length proteins of clade B VP4 were produced and purified. These concatemers were designed on the basis of amino acid sequence analysis to cover the entire panel of existing sequences in frame B.
The specific reactivity and cross-reactivity of rabbit sera were measured using a peptide-based ELISA and cross-neutralization assays.
Materials and methods
Study Design The immunogenicity of HRV-related peptides and full-length proteins was studied in four similarly designed experiments.
Groups of NZW rabbits (N = 2-3 / group) were immunized intramuscularly, (in tibial muscle) on days 0, 14, 42 and 70, with 20 to 100 μg of antigen formulated with adjuvant water in oil (Specol, Leonards et al, 1994). A blood sample was taken 14 days after the 2nd, 3rd and 4th injections. Unless otherwise indicated, the humoral response was measured 14 days after the 4th injection by ELISA and neutralization tests.
Table 2 below shows the peptides that were injected and also the two full-length 5x VP4 concatamer constructs that were used. HBsAg chimeric polypeptide constructs with rhinovirus VP4 peptides were prepared as described in Example 2.
Table 2
peptides
HRV peptides were produced by
Polypeptide Laboratories. The peptides were conjugated to KLH using N-hydroxysuccinimide-mimetimidobenzoic acid ester (MBS) after adding cysteine to the N-terminal region and amidation of the C-terminal region.
Cloning, expression and purification of clade A (Rhi002), clade B (Rhi004), clade C (Rhi006) and serotype 5x HRV14 clade B (Rhi008) VP4 proteins
Expression plasmid and recombinant strain
Genes encoding full-length proteins of VP4 polyproteins (RhiO 2 O / RhiO 4 / RhiOl / RhiO) and a His tag were cloned into the pET24b (+) expression vector (Novagen) using the NdeI / XhoI restriction sites. according to standard procedures. Final constructs were generated by E. strain transformation. coli C43 (DE3) (RhiO4 / RhiO6 / RhiO8) or Rosetta2 (DE3) (RhiO2) with the recombinant expression vector according to a standard method with CaCl2-treated cells (Hanahan D. Plasmid transformation by Simanis, at Glover, DM (eds.), DNA cloning, IRL Press London (1985): pp. 109-135.
Host strain: C43 (DE3) is a derivative of strain C41 which is a derivative of BL21. Strains with the designation (DE3) are lysogenic for a prophage which contains an IPTG inducible T7 RNA polymerase. DE3 lysogens are designed for expression of the protein from pET vectors. This strain is also deficient in proteases Ion and ompT. Strains C43 contain phenotypically selected genetic mutations, so as to confer tolerance to toxic proteins. This strain has at least one mutation that prevents cell death associated with the expression of many recombinant toxic proteins and is selected for its resistance to a different toxic protein and can express a different group of toxic proteins. Genotype: E. coli strain C43 :: DE3, F ~ ompThsdSB (rB_me)) gal dem (DE3) #
Rosetta2 (DE3) is a BL21 derivative designed to enhance the expression of eukaryotic proteins that contain codons rarely used in E. coli. These strains provide tRNAs for 7 rare codons (AGA, AGG, AUA, CUA, GGA, CCC and CGG). Strains with the designation (DE3) are lysogenic for a prophage which contains an IPTG inducible T7 RNA polymerase. DE3 lysogens are designed for expression of the protein from pET vectors. This strain is also deficient in proteases Ion and ompT. Genotype: E. coli, strain Rosetta2 :: DE3, F-ompThsdSB (rB "mB_) gal dem (DE3) pRARE2 (CamR).
Expression of recombinant proteins:
Transformants of E. coli are taken from agar plates and used to inoculate 200 ml of LBT broth ± 1% (w / v) glucose + kanamycin (50 μg / ml) to obtain an O.D.6oonm between 0.1 and 0.2. The cultures are incubated overnight at 37 ° C at 250 rpm.
These overnight cultures were diluted 1: 20 in 500 ml LBT medium containing kanamycin (50 μg / ml) and cultured at 37 ° C at a stirring speed of 250 rpm. to achieve an OD620 value of 0.5 / 0.6. At an OD600 of around 0.6, cultures were induced to determine the expression of recombinant proteins by the addition of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG, EMD Chemicals Inc. catalog: 5815) and incubated overnight at 37 ° C, 250 t / m for strain C43 (DE3) (RhiO4 / RhiO6 / RhiO8) or for 3 h at 37 ° C, 250 t / m for Rosetta strain 2 (DE3) (RhiO2).
After induction overnight (about 16 hours) or 3 hours, the OD0oonm value was evaluated and the cultures were centrifuged at 14,000 rpm for 15 minutes and the agglomerates were frozen separately at -20 ° C. .
Purification of RhiO 2: The bacterial agglomerate was suspended in PBS (pH 7.4). The bacteria were lysed using a French Press 3 X 20 000 PSI system. Soluble (supernatant) and insoluble (agglomerate) components were separated by centrifugation at 20,000 xg for 30 min at 4 ° C.
The 6-His-tagged protein was purified under denaturing conditions on IMAC. The insoluble components were solubilized in 50 mM bicine buffer, pH 8.0, containing 6 M guanidine, 500 mM NaCl. The solubilized component was loaded onto a 5 ml GE Histrap (GE) column pre-equilibrated with the same buffer as that used for solubilization of the agglomerate. After loading the column, the column was washed with 50 mM bicine buffer, pH 8.0, containing 6 M urea and 500 mM NaCl. Elution was performed using 50mM bicine buffer, pH 8.0, containing 6M urea, 500mM NaCl, and imidazole (250mM).
After gel analysis, the elution of IMAC containing the RhiO 2 2 fragment was dialyzed against a bicine buffer (25 mM bicine, 4 M urea, 500 mM NaCl, 0.1% pluronic acid - 5 mM EDTA, 1% sucrose pH 9.5). The dialyzed fraction was loaded onto a SEC chromatography column for a further purification step.
After SEC chromatography, purer fractions were selected and dialysed against PBS, pH 7.4 containing 1% of interferon.
Protein concentration was determined using a BioRad Lowry RC / DC protein assay. The proteins were thus pooled, sterile filtered through a 0.22 μm filter, stored at -80 ° C.
Purification of RhiO.sub.4: The bacterial agglomerate was resuspended in 20mM bicine buffer, pH 8.3 containing 500mM NaCl-benzonase and a protease inhibitor cocktail without EDTA (Roche). The bacteria were lysed using a French Press 2 X 20 000 PSI system. Soluble (supernatant) and insoluble (agglomerate) components were separated by centrifugation at 20,000 xg for 30 min at 4 ° C.
The 6-His tagged protein was purified under native conditions on IMAC. Soluble components (supernatant) were loaded onto a GE Histrap (GE) 5 ml column pre-equilibrated with the same buffer used to lyse the cells, without benzonase, and a protease inhibitor cocktail without EDTA (Roche). After loading the column, the column was washed with 20 mM bicine buffer, pH 8.3 containing 500 mM NaCl. Elution was performed using a 20 mM bicine buffer, pH 8.3 containing 500 mM NaCl and imidazole (500 mM gradient). After gel analysis, purer fractions were selected, concentrated and loaded onto a SEC superdex 75 chromatography column for a further purification step.
After SEC chromatography in 20 mM bicine, pH 8.3 containing 150 mM NaCl, 5 mM EDTA, purer fractions were selected for a further purification step. The purer fractions were pooled and loaded onto a SEC G25 chromatography column in 20 mM bicine, pH 8.3 containing 500 mM NaCl.
After the gel analysis, purer fractions were selected and loaded onto a 5 ml GE Histrap (GE) column pre-equilibrated with 20 mM bicine buffer, pH 8.3 containing 500 mM NaCl. After loading the column, the column was washed with 20 mM bicine buffer, pH 8.3 containing 500 mM NaCl. Elution was performed using a 20 mM bicine buffer, pH 8.3, containing 500 mM NaCl and imidazole (500 mM gradient). After gel analysis, purer fractions were selected, pooled and dialyzed against 20 mM bicine buffer containing 150 mM NaCl and 5 mM EDTA.
The protein concentration was determined from a BioRad Lowry DC protein assay. The proteins were thus pooled, sterile filtered through a 0.22 μm filter, stored at -80 ° C.
Purification of RhiO.sub.6: The bacterial agglomerate was suspended in PBS (pH 7.4). The bacteria were lysed using a French Press 1 X 20 000 PSI system. Soluble (supernatant) and insoluble (agglomerate) components were separated by centrifugation at 20,000 xg for 30 min at 4 ° C.
The 6-His-tagged protein was purified under denaturing conditions on IMAC. The insoluble components were solubilized in 50 mM bicine buffer, pH 8.0, containing 6 M guanidine, 500 mM NaCl, a complete protease inhibitor cocktail without EDTA (Roche). The solubilized component was loaded onto a 5 ml GE Histrap (GE) column pre-equilibrated with the same buffer as that used for solubilization of the agglomerate. After loading the column, the column was washed with 50 mM bicine buffer, pH 8.0, containing 6 M urea and 500 mM NaCl. Elution was performed using 50mM bicine buffer, pH 8.0, containing 6M urea, 500mM NaCl, and imidazole (250mM).
After gel analysis, the elution of IMAC containing the RhiO6 fragment was dialyzed against PBS, pH 8 containing 4 M urea. The dialyzed fraction was loaded onto a SEC chromatography column for a further purification step. After SEC chromatography, purer fractions were selected and dialysed against PBS, pH 8 containing 1 M urea and 5 mM EDTA.
Protein concentration was determined using a BioRad Lowry RC / DC protein assay. The proteins were thus pooled, sterile filtered through a 0.22 μm filter, stored at -80 ° C.
Purification of RhiO.sub.8: The bacterial agglomerate was resuspended in PBS (pH 7.4) containing a complete protease inhibitor cocktail without EDTA (Roche). The bacteria were lysed using a French Press 2 X 20 000 PSI system. Soluble (supernatant) and insoluble (agglomerate) components were separated by centrifugation at 20,000 xg for 30 min at 4 ° C.
The 6-His-tagged protein was purified under denaturing conditions on IMAC. The insoluble components were solubilized in 50 mM bicine buffer, pH 8.3, containing 8 M urea, 500 mM NaCl, a complete protease inhibitor cocktail without EDTA (Roche). The solubilized component was loaded onto a 10 ml column of NiNTA resin pre-equilibrated with the same buffer as that used for the solubilization of the agglomerate. After loading the column, the column was washed with 50mM bicine buffer, pH 8.3, containing 8M urea and 500mM NaCl. Elution was performed using 50mM bicine buffer, pH 8.0, containing 6M urea, 500mM NaCl and imidazole (500mM).
After gel analysis, the elution of IMAC containing the RhiO8 fragment was dialyzed stepwise against a 25 mM bicine buffer, pH 8.3 containing 4 M urea and 250 mM NaCl, and then in a second step dialysis, against PBS, pH 7.4.
Protein concentration was determined using a BioRad Lowry RC / DC protein assay. The proteins were thus pooled, sterile filtered through a 0.22 μm filter, stored at -80 ° C.
HRV-HBsAq chimeric polypeptide constructs are described in Example 2.
ELISA assay for the detection of antibodies against proteins or peptides
Quantification of antibodies against anti-VP1 / VP4-associated peptides or proteins was performed by ELISA using peptides or concatamers specific for full-length proteins as coating antigen. Antigens were diluted to a final concentration of 2 μg / ml in PBS and adsorbed overnight at 4 ° C in wells of 96-well microtiter plates (Maxisorplmmuno-plate, Nunc, Denmark). The plates were then incubated for 1 h at 37 ° C with PBS + 0.1% Tween 20 + 1% BSA (saturation buffer). Sera diluted in the saturation buffer were added to the plates and incubated for 1 hr 30 min at 37 ° C. The plates were washed four times with 0.1% Tween 20 PBS and anti-rabbit Ig. Biotin conjugate (Amersham Biosciences, UK) diluted in saturation buffer was added to each well and incubated for 1 h at 37 ° C. After the plates were washed four times with 0.1 PBS. % Tween 20, streptavidin-horseradish peroxidase (Roche), diluted in saturation buffer, was added for another 30 min at 37 ° C. The plates were washed 4 times with 0.1% PBS. Tween 20 again, and incubated for 20 min at room temperature with a solution of 0.04% o-phenylenediamine (Sigma) at 0.03% H2O2 in 0.1% Tween 20, 0.05M. citrate buffer, pH 4.5. The reaction was quenched with 2NH2SO4 and the result read at 492/620 nm. The ELISA titers were calculated from a reference by SoftMaxPro (with a four-parameter equation) and expressed in EU / ml.
Rhinovirus production
Strains of HRV2, 8, 10, 14, 39 and 61 were purchased from ATCC and amplified on Hela-H1 cells grown in infection medium (MEM containing 2% FCS, 30 mM MgCl 2 and 1mM glutamine). For this purpose, 5 million Hela-H1 cells were seeded in 75 cm 2 plates and grown overnight at 34 ° C. The plates were then infected at a multiplicity of infection of 15 and then incubated until complete lysis of cell monolayers (24 h to 48 h depending on the strain). The supernatants were collected and the debris removed by centrifugation (1000 g-10 min). The purified supernatants were aliquoted, stored at -80 ° C and drawn on Hela-H1.
Titration of rhinovirus production
Hela-H1 cells were seeded into 96-well plates (5,000 cells / well) and grown overnight at 34 ° C with 5% CO2. The plates were then infected with double serial dilutions (starting dilution = 1/1000) of rhinovirus suspensions tested in 8 copies. Three to five days after infection, the presence of HRV-mediated cytopathic effects was revealed by measuring cell viability. For this purpose, the cells were incubated with the WST-1 reagent according to the manufacturer's instructions (Promega). A threshold value was then defined for each plate as average -3 standard deviations of the optical densities (O.D.) of wells containing uninfected cells. Wells with an O.D. below this threshold value were rated positive at EPC. The viral concentration was calculated according to the formula of Reed and Muench.
Neutralization test
Hela-H1 cells were plated on 96-well plates (5,000 cells / well) and incubated overnight at 37 ° C with 5% CO2. The sera were inactivated at 56 ° C for 30 min. 100 TCID50 of HRV (relevant serotype, depending on the neutralization or cross-neutralization assay) were placed in the presence of serial dilutions (double dilutions from dilution = 1/2) of 37 ° rabbit serum. C for 2 h. The mixture was then placed on cell monolayers and the plates were further incubated at 37 ° C for 3-5 days until complete lysis of control cell monolayers was performed. Viability of the cells was then measured using a WST-1 reagent according to the manufacturer's instructions (Promega). The O.D. was then converted to a percentage of cytopathic and reciprocal effect of the dilution giving a 50% reduction in cytopathic effect (CPE) compared with the control cells. The CPE was then extrapolated using non-linear regression with the GraphpadPrism software. Results
Peptide immunogenicity - specific peptide response
The specific peptide response in rabbits that received KLH or peptides conjugated to HB-S was measured 14 days after the 4th immunization using the peptide-based ELISA described herein.
The results were as follows and are shown in Figures 2 and 3. No response was detected in the NaCl control group.
VP1 - KLH conjugates (Figure 2)
High levels of VP1-specific antibodies were detected in rabbits that received - HRV14 VP1 147-162, - HRV14 VP1 1-30 and - HRV14 VP1 32-45
Low levels of antibodies were induced by the HRV8, HRV25 or HRV_C_026VP1 32-45 peptides, although the increase in the antigen dose positively affected the antibody titers.
VP4 - KLH Conjugates (Figure 3)
Overall, VP4-associated peptides induced lower levels of antibodies than VP1 peptides. However, high titres of VP4 peptide-specific antibodies were measured in rabbits that received HRV14 VP4 39-68 or HRV14, HRVIOO and HRVC VP4 1-16.
Unlike the article published by Katpally et al. (2009), HRV14 VP4 1-31 was weakly immunogenic. However, this can be explained by the aggregation and low solubility of this peptide observed after KLH conjugation.
Chimeric constructs of VP4 - HbsAg (Figure 3) The insertion of HRV14 VP4 1-31 in loop A but not in the N-terminus of HBsAg induces high levels of peptide-specific antibodies in 2 out of 3 rabbits. shown in Figure 3.
Immunogenicity of Full-Length Proteins - Specific Protein Response The immunogenicity of full-length proteins in the form of concatemers was measured 14 days after the 4th immunization with ELISA. The results are shown in Figure 4. - No response was detected in the group
NaCl - High levels of antibodies against VP4 were detected in rabbits that received clade B concatemers or HRV14 VP4 proteins.
Comparison of Neutralizing Antibody Titers Induced by Peptides and Full Length Proteins
The levels of neutralizing antibodies induced by peptides and proteins associated with VP1 / VP4 were measured 14 days after the 4th vaccination using an HRV strain panel (i.e., HRV2, 8, 10, 14, 39 and 61). The results are shown in Figure 5 and Figure 6. - No neutralizing antibodies were detected in the control NaCl group - Specific and / or cross-neutralizing antibodies were detected in all groups that received the associated peptides at VP1. Importantly, the HRV14 VP1 32-45 peptide induced the broadest cross-reactivity because this particular peptide could neutralize all the strains tested. Significant levels of neutralizing and cross-neutralizing antibodies were detected in rabbits immunized with the peptides. HRV14 VP4 39-68, VP4 1-16 and HRV100 VP4 1-16 whereas a contradictory response was observed in other groups vaccinated with KLH-conjugated peptides or full-length proteins. In particular, the HRV100 VP4 1-16 peptide induced the broadest cross-reactivity because this particular peptide was able to neutralize all 6 strains tested. Therefore, in VP4 1-16, a shorter peptide than VP4 1-31 has been identified, which is more conserved and can introduce a broader cross-neutralizing activity.
Complete VP4 proteins induce low levels of antibodies specific to regions 1 to 16
Data collected in neutralization assays suggested that a neutralizing epitope is in the VP4 1-16 regions and that immunization with more peptide sequences. Long (1-31) or full-length VP4 protein could give the wrong immune response against non-neutralizing epitopes. A similar mechanism, contributing to the immune evasion of HRV, has been described for the VP1 protein (Niespodziana et al 2012).
Indeed, most of the antibodies were directed against region 1 to 30 of VP1 after immunization with the full length VP1 protein and this region is well known to induce low neutralization (cross) antibodies (Niespodziana et al 2012) ( and as observed in these experiments). It was therefore verified whether the full-length protein induced antibodies directed against the VP4 1-16 region. Rabbit sera were tested for the presence of VP4 1-16 specific antibodies by ELISA and the relative titers (relative to the HRV14 VP4 1-16 vaccinated group) were calculated. Very low levels of VP4 1-16 antibodies were detected in rabbits that received VP4 1-31 in the HBsAg loop or the VP4 full-length protein. The results are shown in Figure 7. Overall, these results suggest that immunization with full-length VP4 protein (or VP1) resulted in the poor immune response against non-neutralizing epitopes.
Table 3
Titers of anti-HRV14 VP4 1-16 peptide antibodies induced by peptide HRV14 VP4 1-16, peptide HRV14 VP4 1-31 and full length VP4 protein. HRV14 VP4 1-16 17561.5 HRV14 VP4 1-31 in HBS (one 2163 loop) 5x HRV14 complete 427
NaCl <25
Full length VP4 protein
Rabbits: the immunogenicity of full-length VP4 proteins of clades Ä and C and HRV14 VP1 protein
In another experiment, ELISA and neutralization tests were performed 14 days post-IV. As shown in Table 4 below, high levels of cross-reactive anti-VP4 IgG were induced by concatamers of VP4 proteins and to a lesser extent by the chimeric VP4-HBs construct. However, even if some neutralization of strain HRV14 was observed, none of these constructs could induce antibodies that neutralize HRV39. These data support previous findings suggesting that full-length HRV VP4 proteins induce high levels of non-neutralizing antibodies. Similarly, the HRV14 VP1 protein was highly immunogenic but did not induce functional antibodies.
Table 4
conclusions
This study demonstrates that HRV14 VP1 32-45 and HRV100 VP4 1-16 are immunogenic and induce broad cross-reactive antibodies. In contrast, full-length VP4 proteins induced high levels of antibodies that were not found to be functional. The data suggest that immunization with full-length VP4 proteins results in a poor immune response against non-neutralizing epitopes. This has been confirmed by the fact that full-length VP4 proteins do not induce antibodies against the VP4 1-16 region. This mechanism has also been demonstrated for the HRV14 VP1 protein and further confirms the need for peptide vaccination to direct the immune response against well-preserved cross-neutralizing antibodies.
Example 2. Construction of Mixed Particles of VP4-S, S Expressing the Pichiapastoris Strain
Introduction
A construct encoding the VP4i-3i peptide (serotype HRV14) genetically fused to the N-terminus of the Hepatitis B virus antigen S (HBsAg) was generated. This fusion protein (VP4-S) was co-expressed in the yeast Pichiapastoris, with a wild-type HBsAg fragment 230aa (S). The strain obtained synthesizes two polypeptides, S and the VP4-S fusion protein, which spontaneously co-assemble into mixed lipoprotein particles (VP4-S, S).
The Pichiapastoris strain used for the production of these mixed particles carries separate expression cassettes for each protein. These cassettes were stably integrated into the Pichia genome using linear integration vectors.
Construction of the recombinant peptide plasmid VP4-pMK
A synthetic DNA fragment encoding the VP4 peptide (31aa) was generated by Geneart. The fragment was cloned into the pMK vector using the cloning sites Pad and Ascl (plasmid property of Geneart). The nucleotide sequence (codon optimized for expression in Pichia) and the corresponding amino acid sequence are shown in Figure 16-A. The VP4-pMK peptide plasmid map is shown in Figure 16-B.
Construction of the PHIL-D2mod / VP4-S Integration Vector The VP4 synthetic DNA fragment was amplified by PCR using the VP4-pMK peptide plasmid as template and the following primer pair: VP4-Fw: CTCACTATAGGGCGAATTGAAGGAAGG VP4-Rv: TTTGAATAGTATCCCGGGGTAGTTGATAAC The PCR product was cleaved with Ncoland Smal, gel purified and cloned into the PHIL-D2mod vector which already carried the S gene of hepatitis B virus (PHIL-D2mod / S fusion vector) . The resulting recombinant plasmid carries the VP4-S fusion gene and was named PHIL-D2mod / VP4-S vector. The VP4-S fusion gene and the corresponding amino acid sequence are detailed in Figure 17.
The map of the PHIL-D2mod / VP4-S vector is illustrated in FIG. 20. In this expression vector, the recombinant gene is under the control of the strong, tightly regulated, methanol-inducible AOX1 promoter.
The PHIL-D2-mod backbone vector is a derivative of the commercially available PHIL-D2 vector (Invitrogen). The commercial PHIL-D2 vector has been modified so that the expression of a heterologous protein begins at the native ATG start codon of the Pichia AOX1 gene and will potentially produce a recombinant protein with an exterminal histidine tag. The vector PHIL-D2-mod is illustrated in FIG.
Construction of the integration vector PHIL-D2mod / S
The PHIL-D2mod / S vector was designed to allow the production of S antigen alone (without fusion partner).
A synthetic DNA fragment encoding the S antigen (227aa) was generated by Geneart. The synthetic gene was optimized by a codon for expression in Pichiapastoris (named Sco gene). This synthetic DNA fragment was cloned into the PHIL-D2mod vector between the Ncol and EcoRI sites. The recombinant plasmid containing the Sco gene was named PHIL-D2mod / S. The map of the PHIL-D2mod / S vector is illustrated in FIG. 21. In this expression vector, the recombinant gene is under the control of the strong, tightly regulated, methanol-inducible AOX1 promoter.
Generation of the P.pastoris strain co-expressing the VP4-S and S proteins
The expression vectors PHIL-D2mod / VP4-S and PHIL-D2mod / S were used to transform the strain Pichiapastoris GS115 (his4). Prior to transformation, the vectors were digested with the NotI enzyme to release a DNA fragment containing the expression cassette (VP4-S or S) plus the HIS4 gene (to complete his4 in the host genome ).
Since both ends of the NotI DNA integration fragment are homologous to the A0X1 region of the Pichia genome, it can integrate into the A0X1 locus by homologous recombination. In total, 100 His + transformants were obtained and "multicopy" integration clones were selected by semi-quantitative dot-blot DNA analysis. Some "high copy number" candidates were selected, induced by methanol and their production of recombinant protein was analyzed on Coomassie blue stained gel and Western blot. Finally, the clone transform No. 49 was selected for a new analysis.
Evaluation of particle formation
In order to determine whether the VP4-S and S proteins produced in the recombinant Pichia clone # 49 assemble into particulate structures, the soluble extract was prepared (after methanol induction) and analyzed by CsCl density gradient centrifugation. The soluble extract (15 mg of total protein) was loaded on a gradient of 10 ml, 1.5 M, CsCl (72 hours at 40,000 rpm, + 8 ° C in a Beckman 50 Ti rotor). Fractions (0.5 ml) were collected and run on 12% SDS-PAGE, transferred to nitrocellulose membrane and analyzed using an anti-S monoclonal antibody (Group A). As shown in FIG. 22, the Western blot peaks corresponding to the VP4-S fusion protein and the S protein appear on the same fraction of the gradient corresponding to a rho density of rho = 1.20, suggesting that particles mixtures containing both the VP4-S and S monomers are formed in this strain.
The density (rho) of the peak fraction was calculated from a measurement of its refractive index (group A).
Group B: Two peak fractions were analyzed by silver staining (left) and Coomassie blue (right).
Purification of mixed particles VP4-S, S
The following method was used for the VP4-S, S mixed particles from the soluble fraction of the Pichia recombinant clone # 49.
The purification process comprises the following steps: homogenization of the French Press pulp - clarification by centrifugation
- 2 successive gradients of CsCl 1.5 M - 1 gel filtration step (HR300) - Concentration (Amicon) - Sterile filtration (0.22 μm) For example, the mass of purified BMP201 is illustrated on the figure 23.
Analysis by ME
An electron microscopy analysis was performed on the purified mass of BMP201. The particles were visualized after negative staining with phosphotungstic acid. The scale is equivalent to 100 nm (Figure 24). Many particles (20 to 40 nm) could clearly be identified.
List of references
Arnold E, Rossmann MG (1990). Analysis of the structure of a common cold virus, human rhinovirus 14, refined at a resolution of 3.0 A. J Mol Biol. 211 (4): 763-801.
Bartlett NW et al (2008) Mouse models of rhinovirus-induced disease and exacerbation of allergic airway inflammation. Nat Med. Feb. 14 (2): 199-204. doi: 10.1038 / nml713. Epub 2008 Feb 3.
Chen et al (2000). Mol. Cell 5, 557-567.
Cheng-Yu Chung et al (2010). Vaccine 28 (2010) 6951-6957.
Edlmayr et al (2011). Antibodies induced with recombinant VPl from human rhinovirus exhibitcross-neutralization. Eur. Respir. J. 37: 44-52.
Katpally U, Fu TM, DC Freed, Casimiro DR, Smith TJ (2009). Antibodies to the buried N terminus of rhinovirus VP4 exhibit cross-serotypic neutralization.J Virol. 83 (14): 7040-8.
Leenaars PP, Hendriksei CF, Angulo AF, Koedam MA, Claassen E. (1994) Evaluation of several adjuvants as alternatives to the use of Freund's adjuvant in rabbits. Vet Immunol Immunopathol. Mar. 40 (3): 225-41.
Lewis JK, Bothner B, Smith TJ, Siuzdak G (1998). Antiviral agent blocks breathing of the common cold virus. Proc Natl Acad Sci USA. 95 (12): 6774-8.
Lewis-Rogers N, ML Bendall, Crandall KA (2009). Phylogenetic Relationships and Molecular Adaptation Dynamics of Human Rhinoviruses. Mol Biol. Evol. 26 (5): 969-981.
Miao LY et al (2009). Monoclonal Antibodies to VP1 Recognize a Broad Range of Enteroviruses. J. Clin. Micorbiol. Vol 47, No. 10, 3108-3113.
McCray J, Werner G (1987). Different rhinovirus serotypes neutralized by antipeptide antibodies. Nature. Oct 22-28; 329 (6141): 736-8.
Niespodziana K et al (2012). Misdirected antibody responses against an N-terminal epitope on human rhinovirus VP1 as explained for recurrent RV infections. The FASEB Journal. Vol 26, 1001-1008.
Palmenberg AC, Rathe JA, Liggett SB (2010). Analysis of the complete genome sequence of human rhinovirus. J. Allergy Clin. Immunol. Vol 125, No 6, 1190-1199.
Phillips T, Jenkinson L, McCrae C, Thong B, Unitt J. (2011). Development of a high-throughput human rhinovirus-based cell-based assay for identifying antiviral compounds. J Virol Methods. May. 173 (2): 182-8.
Rollinger JM and Schmidtke M (2009). The Human Rhinovirus: Human-Pathological Impact, Mechanisms of Antirhinoviral Agents, and Strategies for Their Discovery. Medicinal Research Reviews, 31, No. 1, 42-92.

权利要求:
Claims (30)
[1]
An immunogenic composition comprising a first and a second peptide derived from a structural protein of a picornavirus, said peptides being capable of inducing a cross-neutralizing immune response against two or more picornaviruses or serotypes of picornavirus, and a diluent, excipient or a pharmaceutically acceptable carrier, wherein said picornavirus peptides are from the N-terminal region of picornavirus structural proteins.
[2]
The immunogenic composition of claim 1, wherein the first and second peptides are derived from different picornavirus proteins.
[3]
An immunogenic composition according to claim 1 or claim 2, wherein the first and second peptides are derived from the picomavirus VP1 and VP4 structural proteins.
[4]
The immunogenic composition according to any one of claims 1 to 3, wherein one or both of the first and second picornavirus peptides consist of less than 20 amino acids of the picornavirus structural protein.
[5]
An immunogenic composition according to any one of claims 1 to 4, wherein one or both of the first and second peptides are from a human enterovirus and the enterovirus peptides are capable of inducing an immune neutralization response. crossed against two or more enteroviruses.
[6]
An immunogenic composition according to claim 5, wherein one or both of the first and second peptides are from a human rhinovirus and the rhinovirus peptides are capable of inducing a cross-neutralizing immune response against two or more rhinoviruses.
[7]
An immunogenic composition according to any one of claims 1 to 6, wherein the first peptide consists of amino acids 32 to 45 of VP1 or a variant of amino acids 32 to 45 of VP1 having 1 to 4 additions or deletions of amino acids at either end and / or 1 to 2 substitutions or additions or deletions of amino acids in the peptide sequence.
[8]
The immunogenic composition according to claim 7, wherein the peptide is selected from: HRV14 (B): 32-PILTANETGATMPV-45 [SEQ ID NO: 1] HRV8 (AM): 32-PALDAAETGHTSSV-45 [SEQ ID NO. : HRV25 (Am): 32-PILDAAETGHTSNV-45 [SEQ ID NO: 3] HRV_C_026: 32-QALGAVEIGATADV-45 [SEQ ID NO: 4] or a variant thereof having 1 to 4 additions or deletions of amino acids at either end and / or 1 to 2 substitutions or additions or deletions of amino acids in the peptide sequence.
[9]
The immunogenic composition according to any one of claims 1 to 8, wherein the second peptide consists of amino acids 1 to 16 of VP4 or a variant of amino acids 1 to 16 of VP4 having 1 to 4 additions or deletions of amino acids at either or both ends and / or 1 to 2 substitutions or additions or deletions of amino acids with the peptide sequence.
[10]
The immunogenic composition according to claim 9, wherein the peptide is selected from: HRV14 (B): 1-GAQVSTQKSGSHENQN-16 [SEQ ID NO: 5] HRVIOO (AM): 1-GAQVSRQNVGTHSTQN-16 [SEQ ID NO. : 6]. HRV_C_026: 1-GAQVSRQSVGSHETMI-16 [SEQ ID NO: 7] or a variant thereof having 1 to 4 amino acid deletions or deletions at either or both ends and / or 1 to 2 substitutions or amino acid additions or deletions in the peptide sequence.
[11]
Immunogenic composition according to any one of claims 1 to 10, wherein the first and second peptides are coupled to carrier proteins.
[12]
The immunogenic composition of claim 11, wherein the first and second peptides are coupled to CRM197.
[13]
An immunogenic composition according to any one of claims 1 to 10, comprising at least one chimeric polypeptide construct comprising the first and second peptides.
[14]
The immunogenic composition of claim 13, wherein the first and second peptides are in separate chimeric constructs.
[15]
The immunogenic composition of claim 13, wherein the first and second peptides are in the same construct of chimeric polypeptides.
[16]
An immunogenic composition according to any one of claims 13 to 15, wherein the chimeric polypeptide construct is in the form of a particle.
[17]
An immunogenic composition according to any one of claims 13 to 16, wherein the backbone polypeptide is derived from a human papillomavirus (HPV), a rhinovirus, the hepatitis B virus surface antigen ( HBsAg), EV-71, influenza virus, norovirus.
[18]
18. An immunogenic composition according to claim 16 or 17, wherein the particle is a virus-like particle (VLP).
[19]
19. An immunogenic composition according to any one of claims 1 to 18, further comprising an adjuvant.
[20]
The immunogenic composition of claim 19, wherein the adjuvant comprises an aluminum salt.
[21]
21. The immunogenic composition of claim 20, wherein the adjuvant comprises aluminum hydroxide.
[22]
22. An immunogenic composition according to any one of claims 19 to 21, wherein the adjuvant comprises 3-O-deacylated monophosphoryl lipid A (3D-MPL).
[23]
23. An immunogenic composition according to any one of claims 19 to 22, wherein the adjuvant comprises QS21.
[24]
The immunogenic composition of claim 22 or 23, wherein the adjuvant comprises 3D-MPL and QS21 in a liposome formulation.
[25]
25. Use of an immunogenic composition according to any one of claims 1 to 24 in the prevention or treatment of a picornavirus infection such as an HRV infection.
[26]
26. Use of an immunogenic composition according to any one of claims 1 to 24 in the manufacture of a medicament for the prevention or treatment of a picornavirus infection such as an HRV infection.
[27]
27. A method of inducing neutralizing antibodies against a picornavirus such as HRV in humans comprising administering to an human an immunogenic composition according to any one of claims 1 to 24.
[28]
28. A method of inducing cross-neutralizing antibodies against a picornavirus such as HRV in humans, comprising administering to an human an immunogenic composition according to any one of claims 1 to 24.
[29]
29. A method of preventing picornavirus infection or picornavirus infection associated with picornavirus infection such as HRV infection or HRV disease associated with HRV infection, said method comprising administering to a of an immunogenic composition according to any one of claims 1 to 24.
[30]
A process for preparing an immunogenic composition, said method comprising combining (i) two or more picornavirus peptides from the N-terminal region of the picornavirus structural proteins, said peptides being capable of inducing an immune response cross neutralization against two or more picornaviruses or picornavirus serotypes and (ii) a pharmaceutically acceptable diluent, excipient or vehicle.

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同族专利:

---

公开号 | 公开日

---

WO2014140166A2|2014-09-18|

---

TW201502136A|2015-01-16|

---

US10493145B2|2019-12-03|

---

US10058603B2|2018-08-28|

---

EP3608332A1|2020-02-12|

---

EP2968521A2|2016-01-20|

---

WO2014140166A3|2014-11-20|

---

AR095425A1|2015-10-14|

---

US20160022803A1|2016-01-28|

---

UY35418A|2014-10-31|

---

US20190076519A1|2019-03-14|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

EP0358485A2|1988-09-08|1990-03-14|The Wellcome Foundation Limited|Human rhinovirus peptides|

---

WO1992003475A1|1990-08-16|1992-03-05|Replico Medical Aktiebolag|Enterovirus peptides|

---

WO2006029887A2|2004-09-16|2006-03-23|Glaxosmithkline Biologicals Sa|Vaccines comprising plasmodium antigens|

---

WO2008057158A2|2006-09-29|2008-05-15|Sanofi Pasteur Biologics Co.|Novel neutralizing immunogen of rhinovirus and its use for vaccine applications|

---

US4372945A|1979-11-13|1983-02-08|Likhite Vilas V|Antigen compounds|

---

US4235877A|1979-06-27|1980-11-25|Merck & Co., Inc.|Liposome particle containing viral or bacterial antigenic subunit|

---

IL61904A|1981-01-13|1985-07-31|Yeda Res & Dev|Synthetic vaccine against influenza virus infections comprising a synthetic peptide and process for producing same|

---

US4866034A|1982-05-26|1989-09-12|Ribi Immunochem Research Inc.|Refined detoxified endotoxin|

---

US4436727A|1982-05-26|1984-03-13|Ribi Immunochem Research, Inc.|Refined detoxified endotoxin product|

---

SE8205892D0|1982-10-18|1982-10-18|Bror Morein|IMMUNOGENT MEMBRANE PROTEIN COMPLEX, SET FOR PREPARATION AND USE THEREOF|

---

US4707543A|1985-09-17|1987-11-17|The United States Of America As Represented By The Secretary Of The Army|Process for the preparation of detoxified polysaccharide-outer membrane protein complexes, and their use as antibacterial vaccines|

---

US4877611A|1986-04-15|1989-10-31|Ribi Immunochem Research Inc.|Vaccine containing tumor antigens and adjuvants|

---

US5057540A|1987-05-29|1991-10-15|Cambridge Biotech Corporation|Saponin adjuvant|

---

CA1331443C|1987-05-29|1994-08-16|Charlotte A. Kensil|Saponin adjuvant|

---

US5726292A|1987-06-23|1998-03-10|Lowell; George H.|Immuno-potentiating systems for preparation of immunogenic materials|

---

US5278302A|1988-05-26|1994-01-11|University Patents, Inc.|Polynucleotide phosphorodithioates|

---

US4912094B1|1988-06-29|1994-02-15|Ribi Immunochem Research Inc.|Modified lipopolysaccharides and process of preparation|

---

JP3954643B2|1991-11-16|2007-08-08|グラクソスミスクライン・バイオロジカルス・ソシエテ・アノニム|Hybrid protein between CS and HBsAG derived from Plasmodium|

---

EP0812593B8|1993-03-23|2010-11-10|SmithKline Beecham Biologicals S.A.|Vaccine compositions containing 3-0 deacylated monophosphoryl lipid a|

---

US5961970A|1993-10-29|1999-10-05|Pharmos Corporation|Submicron emulsions as vaccine adjuvants|

---

CA2175375C|1993-11-17|2010-01-05|John Gwynfor Davies|Glucosamine disaccharides, method for their preparation, pharmaceutical composition comprising same, and their use|

---

WO1995026204A1|1994-03-25|1995-10-05|Isis Pharmaceuticals, Inc.|Immune stimulation by phosphorothioate oligonucleotide analogs|

---

DK0772619T4|1994-07-15|2011-02-21|Univ Iowa Res Found|Immunomodulatory oligonucleotides|

---

AUPM873294A0|1994-10-12|1994-11-03|Csl Limited|Saponin preparations and use thereof in iscoms|

---

DE69637254T2|1995-04-25|2008-06-19|Glaxosmithkline Biologicals S.A.|Vaccines containing a saponin and a sterol|

---

US5666153A|1995-10-03|1997-09-09|Virtual Shopping, Inc.|Retractable teleconferencing apparatus|

---

US5856462A|1996-09-10|1999-01-05|Hybridon Incorporated|Oligonucleotides having modified CpG dinucleosides|

---

US6764840B2|1997-05-08|2004-07-20|Corixa Corporation|Aminoalkyl glucosaminide phosphate compounds and their use as adjuvants and immunoeffectors|

---

US6303347B1|1997-05-08|2001-10-16|Corixa Corporation|Aminoalkyl glucosaminide phosphate compounds and their use as adjuvants and immunoeffectors|

---

US6113918A|1997-05-08|2000-09-05|Ribi Immunochem Research, Inc.|Aminoalkyl glucosamine phosphate compounds and their use as adjuvants and immunoeffectors|

---

GB9727262D0|1997-12-24|1998-02-25|Smithkline Beecham Biolog|Vaccine|

---

HU0102332A3|1998-06-08|2002-11-28|Sca Emballage France|Fast flattening packaging|

---

CZ302062B6|1998-06-30|2010-09-22|Om Pharma|Acyl dipeptide derivative process for its preparation, intermediates for the preparation thereof and pharmaceutical composition in which the derivative is comprised|

---

US6551600B2|1999-02-01|2003-04-22|Eisai Co., Ltd.|Immunological adjuvant compounds compositions and methods of use thereof|

---

US20040006242A1|1999-02-01|2004-01-08|Hawkins Lynn D.|Immunomodulatory compounds and method of use thereof|

---

WO2001046127A1|1999-12-22|2001-06-28|Om Pharma|Acyl pseudopeptides bearing a functionalised auxiliary spacer|

---

US7524509B2|2001-03-09|2009-04-28|Id Biomedical Corporation Of Quebec|Proteosome-liposaccharide vaccine adjuvant|

---

AT404561T|2001-04-17|2008-08-15|Dainippon Sumitomo Pharma Co|NEW ADENINE DERIVATIVES|

---

DE60230340D1|2001-11-16|2009-01-22|3M Innovative Properties Co|N-Ä4- -butyl-methanesulfonamide, pharmaceutical composition containing the same and their use|

---

EP1592302A4|2003-02-13|2007-04-25|3M Innovative Properties Co|Methods and compositions related to irm compounds and toll-like receptor 8|

---

US8153760B2|2005-01-19|2012-04-10|Donald Danforth Plant Science Center|Rhinovirus vaccines|

---

GB0505996D0|2005-03-23|2005-04-27|Glaxosmithkline Biolog Sa|Fermentation process|

---

WO2009127081A1|2008-04-17|2009-10-22|Biomerieux|A newly identified human rhinovirus of hrv-c and methods and kits for detecting hrv-cs|

---

GB0815872D0|2008-09-01|2008-10-08|Pasteur Institut|Novel method and compositions|

---

EP2316481A1|2009-10-30|2011-05-04|Biomay Ag|Pharmaceutical composition for the treatment and prevention of a rhinovirus infection|

---

EP2694102A4|2011-04-04|2015-03-04|Univ Iowa Res Found|Methods of improving vaccine immunogenicity|

---

KR101839496B1|2011-06-01|2018-03-19|시아먼 유니버시티|Fusion protein comprising diphtheria toxin non-toxic mutant crm197 or fragment thereof|

---

US20140236070A1|2011-07-29|2014-08-21|Kate Broderick|Linear expression cassettes and uses thereof|WO2016134288A1|2015-02-19|2016-08-25|University Of Virginia Patent Foundation|Compositions and methods for preventing and treating rhinovirus infections|

---

RU2617043C1|2016-03-24|2017-04-19|Федеральное государственное бюджетное научное учреждение "Всероссийский научно-исследовательский и технологический институт биологической промышленности" |Vaccination against foot and mouth disease and method for its production and use|

---

US20190328900A1|2016-07-01|2019-10-31|Glaxosmithkline Intellectual Property Development Limited|Antibody-drug conjugates and therapeutic methods using the same|

---

GB201616904D0|2016-10-05|2016-11-16|Glaxosmithkline Biologicals Sa|Vaccine|

法律状态:
2019-12-05| MM| Lapsed because of non-payment of the annual fee|Effective date: 20190331 |

优先权:

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申请号 | 申请日 | 专利标题

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US201361786765P| true| 2013-03-15|2013-03-15|

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US61/786765|2013-03-15|

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