WO2007010291A1

WO2007010291A1 - Vaccine

Info

Publication number: WO2007010291A1
Application number: PCT/GB2006/002762
Authority: WO
Inventors: Douglas Campbell Thomson; Joseph David Santangelo
Original assignee: Singvax Pte Ltd.
Priority date: 2005-07-22
Filing date: 2006-07-24
Publication date: 2007-01-25
Also published as: GB0515135D0

Abstract

The present invention relates to a peptide comprising the amino acid sequence identified herein as SEQ ID NO: 1 or a homologue thereof with at least 60% sequence similarity, or a fragment thereof capable of eliciting an immunogenic reaction. The peptides of the invention are useful in the treatment of hand, foot and mouth disease.

Description

VACCINE

Field of the Invention

The present invention relates to peptides for use in the treatment of hand, foot and mouth disease, in particular to the preparation of vaccines for use against infection by Enterovirus 71. Background to the Invention

Enterovirus 71 (EV71) is the major causative agent of hand, foot and mouth disease (HFMD), resulting in major neurological complications including encephalitis, meningitis, neurogenic pulmonary oedema and polio-like paralysis. The disease results in significant mortality in children and infants.

Since 1997, there has been a significant increase in EV71 epidemic activity throughout the Asia-Pacific region, which has been associated with higher rates of severe neurological sequelae. In an outbreak in Malaysia at least 35 deaths were reported and, in 1998, 78 deaths were reported in Taiwan during an epidemic that spread across the whole island. Further epidemics have been reported in 2000 again in Taiwan, Korea in 2003 and in Australia.

EV71 is a picornavirus of the genus enterovirus. Picornaviruses are small, non-enveloped RNA viruses characterised by an isocahedral capsid, composed of 60 copies each of four structural proteins VP1-4. The viral capsid is known to play a significant role in contributing to the pathogenicity of the enteroviruses.

Ranganathan et al, Applied Bioinformatics, 2002; 1(1): 43-53 discloses a method for identifying antigenic sites of EV71 using computer modelling. A consensus structural model for EV71 coat protein was obtained and functional regions from closely related viruses were mapped onto the surface of the predicted structure. The method revealed two putative immunogenic regions, one of which was unique to EV71. No experiments were carried out to determine whether the putative immunogenic regions provided a sufficient immunogenic response to make the epitopes viable vaccine candidates.

Ooi et al, Emerging Infectious Diseases, 2002; 8(9): 995-997 and Cardosa ef a/, Emerging Infectious Diseases, 2003; 9(4): 461-468, each relate to epidemiology studies to identify the phylogenetic relationships between the HEV71 (Human EV71) strains circulating in recent outbreaks.

Singh ef a/, Microbiol. Immunol., 2002; 46(1 1): 801-808 describes the sequencing of two EV71 strains; one fatal and one not fatal. The publication discloses homology levels between the two strains and states that the VP1 BC loop region is known to influence neurovirulence. Interestingly, the publication states that attempts to elucidate neurovirulence factors have so far been unsuccessful.

There is still a need for development of a vaccine for immunisation against infection by EV71. Summary of the Invention

The present invention is based on the surprising identification of a consensus VP1 protein which may be used in viral neutralisation.

According to a first aspect of the invention, a peptide of the invention comprises the amino acid sequence identified herein as SEQ ID NO: 1 , or a homologue thereof with at least 60% sequence similarity, or a fragment thereof capable of eliciting an immunogenic reaction.

According to a second aspect of the invention, an immunogenic composition comprises a peptide as defined above and a pharmaceutically acceptable excipient, diluent or adjuvant.

According to a third aspect of the present invention, a vaccine composition comprises one or more of the peptides defined above, and an adjuvant.

According to a fourth aspect of the present invention, a peptide defined above may be used in the manufacture of a medicament for the treatment of hand, foot and mouth disease.

According to a fifth aspect of the present invention, an antibody is raised against a peptide as defined above.

According to a sixth aspect of the present invention, a cell comprises a peptide as defined above.

According to a seventh aspect of the present invention, a recombinant vector encodes a peptide comprising two or more of the peptides defined above. 8. According to an eighth aspect of the invention, an assay for screening potential antiviral molecules comprises the steps of:

(i) contacting a molecule with a peptide as defined above;

(ii) detecting the interaction between the molecule and the peptide; and

(Hi) selecting those molecules that bind to the peptide. Description of the Invention

The present invention is based on an appreciation that a specific EV71 VP1 consensus protein may be used in the preparation of vaccine compositions to prevent infection by EV71. The consensus protein identified herein as SEQ ID NO. 1 is useful in therapy. It should be understood that reference to therapy includes preventative treatments, e.g. vaccination. SEQ ID No. 1 shows the Enterovirus 71 (EV71) VP1 consensus sequence according to the invention. There are 9 positions of variability, namely positions 43, 58, 145, 164, 184, 240, 249, 289 and 292. These positions of variability are represented by an "Xaa" in the sequence, with the variable amino acids identified in the <223> fields of the sequence listing. There are two possible amino acids in 8 out of the 9 variable positions, whereas in position 145 there are three possible amino acid residues.

Preferably, the peptides that may be useful in the various aspects of the invention include those having greater than 60% similarity or identity with the peptide identified herein as SEQ ID No: 1 , or a fragment thereof of at least 5 amino acids of SEQ ID NO: 1 , preferably at least 10, 15, 20, 25, 30 amino acids. More preferably, the peptides have greater than 70%, 80%, 90% or 95% sequence similarity or identity. Most preferably, the peptides have greater than 98% sequence similarity or identity, e.g. 99% similarity or identity.

The term "similarity" is known in the art and refers to a comparison between amino acid sequences, and takes into account not only identical amino acid sequences at corresponding positions, but also functionally similar amino acids in corresponding positions. Thus similarity between peptide sequences indicates functional similarity, in addition to sequence similarity. Although peptides within the scope of the invention may not retain the biological activity of the VP1 full length protein, they will function to elicit an immunogenic response.

Levels of similarity between amino acid sequences can be calculated using known methods. In relation to the present invention, publicly available computer based methods for determining the similarity include the BLASTP, BLASTN and FASTA (Atschul ef a/, J. Molec. Biol., 1990; 215: 403-410), the BLASTX programme available from NCBI, and the Gap programme from Genetics Computer Group, Madison Wl. The levels of similarity provided herein are calculated using the Gap programme with a gap penalty of 12 and a gap length penalty of 4 for determining the amino acid sequence comparisons.

The term "peptide" is used herein to refer to peptides and proteins. For the avoidance of doubt, the term "peptide" refers to a chain of two or more amino acid residues linked by the peptide linkage.

The peptides of the invention are defined with reference to the amino acid sequence specified herein as SEQ ID NO: 1. The amino acid sequence of SEQ ID NO: 1 indicates that there are several positions where alternative amino acid residues may be present. The alternative residues are indicated. *** Peptides according to the invention may be prepared by methods known in the art. In particular, knowledge of the gene sequence allows the skilled person to use recombinant techniques to express the gene sequence in a suitable host. Methods for the production and isolation of the peptides using gene expression in a suitable host organism will be apparent to the skilled person. Preferably, the host is a cell that is isolated in vitro, such as a mammalian (human or non-human) cell-line, insect cell- line, bacterial or yeast cell culture. A recombinant vector containing a polynucleotide encoding the peptide of the current invention is within the scope of the invention. Preferably, the vector is an "expression vector", examples of which will be apparent to the skilled person. Alternatively, the peptides may be made using synthetic peptide chemistry, as will be appreciated by the skilled person.

In a preferred embodiment, a peptide of the invention comprises an affinity tag. The skilled person will realise that the term "affinity tag" refers to a tag that simplifies purification of the protein of interest by use of an interaction between the affinity tag and its binding partner, in affinity chromatography. Preferably, the affinity tag is an amino acid sequence that is expressed as a fusion polypeptide with the peptide of the invention. Preferred affinity tags include a histidine tag (for example six to ten histidine residues), for purification on a nickel column, and a glutathione-S- transferase tag for purification on a glutathione column.

Active fragments and homologues of the peptides can be identified for use in therapy. As used herein, the term "active fragment" refers to a fragment of the consensus VP1 sequence that retains the ability to elicit an immunogenic response. Preferably, the immunogenic response generated by the fragment is similar to the response generated by the full-length VP1 consensus sequence. An antibody that binds to an active fragment will also bind to the full-length sequence (in the region that corresponds to the fragment). Preferably, antibodies raised in an immunogenic response against an active fragment will bind specifically to the full length VP 1 protein. As used herein, reference to "specific binding" refers to an antibody binding selectively to a specific antigen or epitope and not to other (non-related) antigens or epitopes. Preferably, the binding affinity between a peptide of the invention and an antibody is in the micromolar range, for example 50μM or less, more preferably 10μM or less.

The preparation of vaccines incorporating one or more peptides of the invention will be known to those skilled in the art. Vaccine compositions can be formulated with suitable carriers or adjuvants, e.g. alum, as necessary or desired, to provide effective immunisation against infection. Suitable adjuvants include, but are not limited to aluminium salts, squalene mixtures, aquiline mixtures, saponin derivatives, immunostimulating complexes (ISCONs), non-ionic block copolymer surfactants and β-glucan.

More generally, and as is well known to those skilled in the art, a suitable amount of an active component of the invention can be selected, for therapeutic use, as can suitable carriers or excipients and routes of administration. These factors are chosen or determined according to known criteria such as the nature/severity of the condition to be treated, the type of health of the subject etc.

The peptides of the invention may also be prepared with suitable protective agents which stabilise the peptides on storage. Suitable stabilising agents include carbohydrates, e.g. trehalose, sucrose and mannitol. Such stabilising formulations may be prepared with the peptides of the invention incorporated into an amorphous or crystalline carbohydrate structure, for example by spray drying the peptides of the invention together with a stabilising carbohydrate, as disclosed in WO-A-005/053717. Alternatively, compositions of the invention comprising the peptides may be lyophilised after adding a stabiliser.

Compositions intended for immunisation may be prepared with one of the peptides of the invention or may comprise a combination of two or more for example 3, 4, 5 or more peptides of the invention. If two or more peptides are to be used, these may be presented individually in the immunogenic composition or may be comprised in a single recombinant fusion peptide. For the avoidance of doubt combinations of each peptide, homologue or active fragment described herein are within the scope of the invention.

Compositions intended for immunisation will be prepared with one or more peptides of the invention or may comprise a combination of one or more peptides of the invention and one or more additional peptides. Preferably, the one or more additional peptides are useful in generating an immune response to EV71. However, a composition that is within the scope of the current invention must include the peptide of the invention, as defined above.

Methods of making fusion peptides are well known in the art and will be apparent to the skilled person. For the avoidance of doubt, a preferred method involves creating a recombinant DNA molecule, preferably an expression vector, comprising the DNA that encodes the peptides to be included in the fusion. The fusion peptide can be expressed from this vector in a cell, preferably an isolated cell in vitro, such as a bacterial, yeast, insect or mammalian cell, or in a cell-free expression system. If two or more peptides are to be used, these may be presented individually in the immunogenic composition or may be comprised in a single recombinant fusion peptide. In a preferred embodiment, multiple copies of the same peptide are included in a fusion peptide, such that the peptide is repeated. In a further preferred embodiment, two or more different peptides are repeated in a fusion peptide. For example, multiple copies of one peptide can be followed by a multiple copies of a second peptide, or alternatively, two peptides can alternate in the fusion peptide so that the combination of the two peptides is repeated. Other suitable combinations and arrangements will be apparent to the skilled person.

A peptide of the invention can be linked or conjugated, covalently or non- covalently, to a different molecule, referred to herein as a "heterologous" molecule. Preferably, the heterologous molecule is an immunomodulating entity. Suitable immunomodulating entities include, but are not limited to, Tetanus Toxoid and Keyhole Cymphocyte Hemocyanin.

In a preferred embodiment, the peptide defined by SEQ ID No.1 is included in a composition intended for immunisation. In a further preferred embodiment, the peptide of SEQ ID No.1 and a further peptide of the invention are included in a composition intended to raise an immunogenic response, eg. for immunisation. As used herein, the term "immunisation" refers to the standard meaning of the term in the art, i.e. the technique of generating a protective immune response in a subject, to provide the subject with resistance against a specific disease. The subject can be a human or a (non-human) animal, such as a (non-human) mammal. Both passive immunisation and active immunisation are within the scope of the invention.

Passive immunisation involves producing, outside of the subject of immunisation, protective antibodies that bind to the peptide of the invention. These antibodies are then administered to the subject to provide the protective effect. The protective antibodies can be produced synthetically or, preferably, by an animal such as a rabbit. Methods of generating antibodies to the epitope of the invention, that are suitable for use in passive immunisation, will be apparent to one skilled in the art.

Active immunisation involves administering a peptide of the invention to the subject and allowing the subject's own immune system to produce protective antibodies.

As used herein, the term "antibody" refers to an immunoglobulin-based molecule that binds to an antigen. The term antibody encompasses all five classes of immunoglobulins (IgG, IgE, IgA, IgM and IgD). Antibody fragments and variations such as Fab and scFv fragments are within the scope of the invention. The skilled person will realise that when an antibody fragment is intended for use in passive immunisation, the Fc region is preferably present. A monoclonal antibody that binds to a peptide of the invention is within the scope of the invention; the use of the peptide of SEQ ID No. 1 to create monoclonal antibodies that bind to the peptide of SEQ ID No. 1 is also within the scope of the invention. Humanised antibodies are also within the scope of the invention.

An antibody within the scope of the invention binds to a peptide of the invention. Preferably, an antibody binds to a peptide of the invention with a K_A (affinity constant) in the micromolar range. In a preferred embodiment, the antibody binds to a peptide of the invention specifically, so that it binds only to a peptide of the invention and does not bind to other peptides.

In an alternative embodiment, the antibody is cross-reactive, i.e. it binds to a peptide of the invention and to a heterologous peptide. Preferably, the heterologous peptide is an epitope from a different enterovirus, i.e. an enterovirus that is not EV71. A preferred heterologous peptide is an epitope from Coxsackie virus 16 (CA16); a preferred antibody therefore binds to a peptide of the invention and a CA16 epitope. For the avoidance of doubt, in a preferred embodiment, an antibody within the scope of the invention is cross-reactive (i.e. binds) to a plurality of enteroviruses. The skilled person will understand that an antibody that binds to a plurality of enteroviruses is useful in immunising against a plurality of enteroviruses, and may be used in diagnostic assays for a plurality of enteroviruses.

Antibodies raised against the peptides of the invention are useful in therapy or diagnosis. In particular, the antibodies may be used in a diagnostic assay to detect EV71 infection in a patient. Suitable immuno-assays making use of the antibodies of the invention will be apparent to the skilled person, based on conventional techniques such as ELISA.

The peptides of the invention have been selected to result in viral neutralisation and to eliminate immuno-dominant non-protective epitopes. The peptides are therefore very effective in eliciting a specific immune response to infection by EV71 and are of therapeutic benefit.

The peptides of the invention are preferably capable of raising neutralising antibodies against EV71 infection. The term "neutralising antibody" refers to an antibody that reduces or removes the ability of a virus to infect a host. Determining the neutralising effectiveness of the peptides may be carried out using the method disclosed in Gomes et a/., Mem. Inst. Oswaldo Cruz, Rio de Janeiro, 2002; 97(1): 47- 49, the content of which is incorporated herein by reference.

The peptides of the present invention may also be used in screening assays to identify molecules which may have an antiviral activity. The assay may be performed by contacting the potential antimicrobial molecule with a peptide of the invention, and determining whether the molecule interacts with the peptide. In a preferred embodiment, a peptide of the invention is used to screen for monoclonal antibodies that bind to the peptide. Methods for producing and selecting monoclonal antibodies are standard in the art and will be apparent to the skilled person.

Suitable molecules that can be screened for antiviral activity include organic chemicals, peptides or nucleic acids. Affinity between the molecule and the peptide may be detected using conventional techniques. For example, the peptide may be immobilised on a support surface and the molecule brought into contact with the peptide and binding detected. The molecule may be labelled, for example with a radiolabel or fluorophore, to permit detection of the binding reaction. Alternatively, the molecule may be immobilised on a support and the peptide brought into contact with the molecule. Unbound peptide can be washed away and the binding interaction determined either on the basis of a label attached to the peptide, or by contacting the peptide with an antibody that is labelled. This is similar to conventional ELISA reactions. A further alternative is to use the peptide as an inhibitor of a reaction between the molecule and its substrate. Competitive inhibition can therefore be used to determine the strength of the binding reaction.

For the avoidance of doubt, a typical assay to screen for potential antiviral molecule comprises contacting a molecule with a peptide of the invention, followed by detection of the interaction between the molecule and the peptide, and selecting those molecules that bind to the peptide. Preferably, a potential anti-viral molecule binds to the peptide of the invention specifically, as discussed above with reference to antibody binding, with an affinity constant in the micromolar range, for example 50μM or less, preferably 10μM or less. The molecules that bind in this way are potentially anti-viral, and any anti-viral properties can be investigated further. A molecule that is identified by a screening assay as detailed herein is therefore a potential anti-viral molecule.

The peptides of the invention are of general benefit in the treatment or prevention of hand, foot and mouth disease, in both humans and (non-human) animals. While intended primarily as a therapy against infection by EV71 , the peptides may also be of use in treating or preventing infection by Coxsackie virus 16 (CA16).

Claims

1. A peptide comprising the amino acid sequence identified herein as SEQ ID NO: 1 or a homologue thereof with at least 60% sequence similarity, or a fragment thereof capable of eliciting an immunogenic reaction.

2. A peptide according to claim 1 , further comprising an affinity tag.

3. A peptide according to claim 1 or claim 2 which is a fragment of at least 15 amino acids of the SEQ ID NO: 1 peptide.

4. A peptide according to claim 3, comprising at least two fragments of the same or different fragments of the peptide defined as SEQ ID NO: 1.

5. A peptide according to claim 4, wherein the peptide fragments are the same.

6. A peptide according to any preceding claim, linked or conjugated to a heterologous molecule.

7. A peptide according to claim 6, wherein the heterologous molecule is an immunomodulating entity.

9. A peptide according to any preceding claim, for therapeutic use.

10. An immunogenic composition comprising a peptide according to any of claims 1 to 7 and a pharmaceutically acceptable excipient, diluent or adjuvant.

11. A vaccine composition comprising one or more of the peptides according to any of claims 1 to 7, and an adjuvant.

12. Use of a peptide according to any of claims 1 to 7, in the manufacture of a medicament for the treatment or prevention of hand, foot and mouth disease.

13. Use according to claim 11 , wherein the medicament is to treat or prevent infection by enterovirus 71.

14. An antibody raised against a peptide according to any of claims 1 to 7.

15. A cell comprising a peptide according to any of claims 1 to 7.

16. A recombinant vector encoding a peptide as defined in claim 4.

17. The vector defined in claim 15, for therapeutic use.

18. Use of the vector according to claim 15, in the expression of an immunogenic peptide.

19. An assay for screening potential antiviral molecules, comprising: (i) contacting a molecule with a peptide as defined in claim 1 ;

(ii) detecting the interaction between the molecule and the peptide; and (iii) selecting those molecules that bind to the peptide.